Effect of Streptomyces roseolus Cell-Free Supernatants on the Fungal Development, Transcriptome, and Aflatoxin B1 Production of Aspergillus flavus.

Crop contamination by aflatoxin B1 (AFB1), an Aspergillus-flavus-produced toxin, is frequently observed in tropical and subtropical regions. This phenomenon is emerging in Europe, most likely as a result of climate change. Alternative methods, such as biocontrol agents (BCAs), are currently being developed to reduce the use of chemicals in the prevention of mycotoxin contamination. Actinobacteria are known to produce many bioactive compounds, and some of them can reduce in vitro AFB1 concentration. In this context, the present study aims to analyze the effect of a cell-free supernatant (CFS) from Streptomyces roseolus culture on the development of A. flavus, as well as on its transcriptome profile using microarray assay and its impact on AFB1 concentration. Results demonstrated that in vitro, the S. roseolus CFS reduced the dry weight and conidiation of A. flavus from 77% and 43%, respectively, and was therefore associated with a reduction in AFB1 concentration reduction to levels under the limit of quantification. The transcriptomic data analysis revealed that 5198 genes were differentially expressed in response to the CFS exposure and among them 5169 were downregulated including most of the genes involved in biosynthetic gene clusters. The aflatoxins' gene cluster was the most downregulated. Other gene clusters, such as the aspergillic acid, aspirochlorine, and ustiloxin B gene clusters, were also downregulated and associated with a variation in their concentration, confirmed by LC-HRMS.

Role of Polyphenols from the Aqueous Extract of Aloysia citrodora in the Inhibition of Aflatoxin B1 Synthesis in Aspergillus flavus.

Aflatoxin B1 (AFB1) is a mycotoxin considered a potent carcinogen for humans that contaminates a wide range of crops. Various strategies have been established to reduce or block the synthesis of AFB1 in food and feed. The use of aqueous extracts derived from plants with high antioxidant activity has been a subject of study in recent years due to their efficacy in inhibiting AFB1. In this study, we assessed the effect of Aloysia citrodora aqueous extract on Aspergillus flavus growth and on AFB1 production. A bio-guided fractionation followed by High Performance Liquid Chromatography (HPLC) and Mass spectrometry analysis of the active fraction were applied to identify the candidate molecules responsible for the dose-effect inhibition of AFB1 synthesis. Our results revealed that polyphenols are the molecules implicated in AFB1 inhibition, achieving almost a total inhibition of the toxin production (99%). We identified luteolin-7-diglucuronide as one of the main constituents in A. citrodora extract, and demonstrated that it is able to inhibit, by itself, AFB1 production by 57%. This is the first study demonstrating the anti-Aflatoxin B1 effect of this molecule, while other polyphenols surely intervene in A. citrodora anti-AFB1 activity.

Preservation of Mimosa tenuiflora Antiaflatoxigenic Activity Using Microencapsulation by Spray-Drying.

Mimosa tenuiflora aqueous extract (MAE) is rich in phenolic compounds. Among them, condensed tannins have been demonstrated to exhibit a strong antioxidant and antiaflatoxin B1 activities in Aspergillus flavus. Since antioxidant capacity can change with time due to environmental interactions, this study aimed to evaluate the ability of encapsulation by spray-drying of Mimosa tenuiflora aqueous extract to preserve their biological activities through storage. A dry formulation may also facilitate transportation and uses. For that, three different wall materials were used and compared for their efficiency. Total phenolic content, antioxidant activity, antifungal and antiaflatoxin activities were measured after the production of the microparticles and after one year of storage at room temperature. These results confirmed that encapsulation by spray-drying using polysaccharide wall materials is able to preserve antiaflatoxin activity of Mimosa tenuiflora extract better than freezing.

Mimosa tenuiflora Aqueous Extract: Role of Condensed Tannins in Anti-Aflatoxin B1 Activity in Aspergillus flavus.

Aflatoxin B1 (AFB1) is a potent carcinogenic mycotoxin that contaminates numerous crops pre- and post-harvest. To protect foods and feeds from such toxins without resorting to pesticides, the use of plant extracts has been increasingly studied. The most interesting candidate plants are those with strong antioxidative activity because oxidation reactions may interfere with AFB1 production. The present study investigates how an aqueous extract of Mimosa tenuiflora bark affects both the growth of Aspergillus flavus and AFB1 production. The results reveal a dose-dependent inhibition of toxin synthesis with no impact on fungal growth. AFB1 inhibition is related to a down-modulation of the cluster genes of the biosynthetic pathway and especially to the two internal regulators aflR and aflS. Its strong anti-oxidative activity also allows the aqueous extract to modulate the expression of genes involved in fungal oxidative-stress response, such as msnA, mtfA, atfA, or sod1. Finally, a bio-guided fractionation of the aqueous extract demonstrates that condensed tannins play a major role in the anti-aflatoxin activity of Mimosa tenuiflora bark.

Microencapsulation of Horseradish (Armoracia rusticana L.) Juice Using Spray-Drying.

Horseradish contains many bioactive compounds with antioxidant activity. The current study aimed to evaluate the effect of various wall materials and their ratios on the physical properties and bioactive-compound retention and stability in microencapsulated horseradish leaf and root juices. Horseradish juice was microencapsulated using maltodextrin, maltodextrin/gum Arabic, soy protein isolate, and starch with three different core-to-wall ratios. The total phenolic, total flavonoid, total flavan-3-ol, and total phenolic-acid contents, as well as antioxidant activity, were determined using spectrophotometric methods, whereas individual phenol profiles were determined by high-performance liquid chromatography (HPLC). Multivariate analysis of variance showed that plant material, wall material, and core-to-wall ratio had a significant effect on the bioactive-compound retention and antioxidant-activity preservation. Microcapsules produced from horseradish leaf juice had a significantly higher content of phenolic compounds and antioxidant activity compared to root-juice microcapsules. However, better retention was observed for microencapsulated horseradish root juice. Maltodextrin and maltodextrin/gum Arabic were the most effective wall materials for the retention of bioactive compounds, while they also had a smaller particle size and better solubility. The horseradish-juice microcapsules possess a high content of rutin. The highest stability of bioactive compounds after storage was found at a core-to-wall ratio of 20:80.

Twin-screw extrusion encapsulation of MCT-oil in a maltodextrin matrix using compatibilizing biopolymers.

Carbohydrates are the most recurrent materials employed for active components encapsulation using twin-screw extrusion. However, the influence of process parameters on the properties of the final product remains a challenge. In this paper, special attention was given to the incorporation of a hydrophobic model compound (MCT-oil), in a maltodextrin matrix with a compatibilizing biopolymer. The effects of the extrusion parameters, as well as the influence of different formulations were analyzed. The mild extrusion conditions allowed obtaining blends with acceptable texture and viscosity to enhance the dispersion of the active compound. The encapsulation systems obtained were in a glassy state at room temperature and they remained stable at 60 % RH for a long time. Satisfactory incorporation rates of MCT-oil were found reaching encapsulation efficiencies up to 90 %. These results showed that the chosen compatibilizing agent enhanced the dispersion and stabilization of the MCT-oil within the matrix and significantly improved encapsulation.

Impact of Spray-Drying on Biological Properties of Chitosan Matrices Supplemented with Antioxidant Fungal Extracts for Wine Applications.

Black aspergilli produce many bioactive compounds: enzymes, organic acids, and secondary metabolites. One such fungus, Aspergillus tubingensis G131, isolated from French Mediterranean vineyards, produces secondary metabolites with antioxidant properties that can be extracted with ethanol. In this study, crude antioxidant extracts obtained from A. tubingensis G131 cultures were encapsulated with two types of chitosan matrix. Spray-drying was used to obtain dried particles from a dispersion of fungal crude extracts in a solution of the coating agent chitosan. This process appeared to be an efficient method for obtaining a dry extract with antioxidant activity. Three types of fungal extracts, with different antioxidant capacities, were produced: two different concentrations of crude extract and a semi-purified extract. In this study, the chitosan matrices for encapsulation were chosen on the basis of their antimicrobial activities for wine applications. Classical low molecular weight chitosan was compared with NoBrett Inside® which is already used to prevent the development of Brettanomyces spp. in wine. The objective of this study was to confirm that both antioxidant (fungal extract) and antimicrobial (chitosan) properties were preserved after spray-drying. The combination of these two properties and the powder formulation of this entirely natural product would make it a good alternative to chemicals, such as sulfites, in the food and wine industries.

Spray-Dried Succinylated Soy Protein Microparticles for Oral Ibuprofen Delivery.

The potential value of succinylated soy protein (SPS) as a wall material for the encapsulation of ibuprofen (IBU), a model hydrophobic drug, by spray-drying was investigated. A succinylation rate of 93% was obtained for soy protein isolate, with a molar ratio of 1/1.5 (NH2/succinic anhydride). The solubility profile at 37°C showed that this chemical modification decreased the solubility of the protein below its isoelectric point, whereas solubility increased in alkaline conditions. Various SPS/IBU ratios (90/10, 80/20, and 60/40) were studied and compared with the same ratio of soy protein isolate (SPI/IBU). High encapsulation efficiency was achieved (91-95%). Microparticles were spherical and between 4 and 8 µm in diameter. The spray-drying of protein/IBU solutions appeared to be beneficial, as it resulted in an amorphous solid dispersion of IBU within the microparticles, coupled with an increase in the thermal stability of IBU. In vitro release was evaluated in acidic (pH 1.2 in the presence of pepsin) and neutral (pH 6.8) conditions similar to those in the gastrointestinal (GI) tract. IBU was released significantly more slowly at pH 1.2, for both proteins. However, this slowing was particularly marked for SPS, for which rapid (within 2 h) and complete release was observed at pH 6.8. These results validate the hypothesis that SPS is suitable for use as a coating material for hydrophobic active pharmaceutical ingredients (APIs) due to its pH sensitivity, which should delay IBU release in the gastrointestinal tract.

Soy Protein Microparticles for Enhanced Oral Ibuprofen Delivery: Preparation, Characterization, and In Vitro Release Evaluation.

The objective of this work was to evaluate soy protein isolate (SPI) and acylated soy protein (SPA) as spray-drying encapsulation carriers for oral pharmaceutical applications. SPI acylation was performed by the Schotten-Baumann reaction. SPA, with an acylation rate of 41%, displayed a decrease in solubility in acidic conditions, whereas its solubility was unaffected by basic conditions. The drug encapsulation capacities of both SPI and SPA were tested with ibuprofen (IBU) as a model poorly soluble drug. IBU-SPI and IBU-SPA particles were obtained by spray-drying under eco-friendly conditions. Yields of 70 to 87% and microencapsulation efficiencies exceeding 80% were attained for an IBU content of 20 to 40% w/w, confirming the excellent microencapsulation properties of SPI and the suitability of the chemical modification. The in vitro release kinetics of IBU were studied in simulated gastrointestinal conditions (pH 1.2 and pH 6.8, 37°C). pH-sensitive release patterns were observed, with an optimized low rate of release in simulated gastric fluid for SPA formulations, and a rapid and complete release in simulated intestinal fluid for both formulations, due to the optimal pattern of pH-dependent solubility for SPA and the molecular dispersion of IBU in soy protein. These results demonstrate that SPI and SPA are relevant for the development of pH-sensitive drug delivery systems for the oral route.

Characterization of Non-Derivatized Cellulose Samples by Size Exclusion Chromatography in Tetrabutylammonium Fluoride/Dimethylsulfoxide (TBAF/DMSO).

This paper deals with the use of tetrabutylammonium fluoride/dimethylsulfoxide (TBAF/DMSO) to characterize the molar mass distribution of non-derivatized cellulosic samples by size exclusion chromatography (SEC). Different cellulose samples with various average degree of polymerization (DP) were first solubilized in this solvent system, with increasing TBAF rates, and then analyzed by SEC coupled to a refractive index detector (RID), using DMSO as mobile phase. The Molar Masses (MM) obtained by conventional calibration were then discussed and compared with suppliers' data and MM determined by viscosimetry measurements. By this non-classic method, molar mass of low DP samples (Avicel® and cotton fibers) have been determined. For high DP samples (α-cellulose and Vitacel®), dissolution with TBAF concentration of 10 mg/mL involved elution of cellulose aggregates in the exclusion volume, related to an incomplete dissolution or the dilution of TBAF molecules in elution solvent, preventing the correct evaluation of their molar mass.

Structural modifications of cellulose samples after dissolution into various solvent systems.

This work deals with the modifications resulting from the dissolution of four commercial cellulosic samples, with different crystallinity rates and degrees of polymerization (DPs), in four solvent systems, known and used to dissolve cellulose. The dissolution conditions were optimized for the 16 various systems and followed by turbidity measurements. After regeneration, the samples were analyzed by thermal gravimetric analysis (TGA), scanning electron microscopy (SEM), and X-ray diffractometry (XRD) to study their modification. Viscosimetry measurements were used to evaluate the potential decrease of the DP after dissolution. The observed structural modifications established that, for low DP, all the solvent systems were efficient in dissolving the cellulose without altering the DP, except BMIM [Cl], which provoked a decrease of up to 40 % and a decrease of around 20 % of the degradation temperature (onset temperature, T o). For high molecular weight (MW) celluloses, DMSO/TBAF was the only system to allow a complete dissolution without any molar mass loss and degradation temperature modification.

Influence of DE-value on the physicochemical properties of maltodextrin for melt extrusion processes.

In this study, five different types of maltodextrins (DE-2, DE-6, DE-12, DE-17 and DE-19) were characterized for the physico-chemical properties. TGA, DVS and SEC analyses were carried out and additionally apparent melt-viscosity (in a micro-extruder) and the glass transition temperature (analyzed by DMA) of maltodextrin/plasticizer mixtures were also measured in order to evaluate both the effect of plasticizer nature and content and the effect of the DE-value. For this, three plasticizing agents were compared: water, d-sorbitol and glycerin. The adsorption isotherms showed that depending on the DE-value and the relative humidity they were exposed to, different behavior could be obtained. For example, for relative humidities below 60% RH maltodextrin DE-2 was the least hygroscopic. And on the contrary for relative humidities above 75% RH maltodextrin DE-2 was the most hygroscopic. The rheology measurements showed that the viscosity decreased with the increase of the DE-value and with the plasticizer content, as expected. On the contrary, no direct correlation could be established between the DE-value and the glass transition temperature. These results demonstrated that to predict maltodextrins behavior and to better adapt the process conditions, combined analyses are mandatory as the DE-value alone is not sufficient. The most compelling evidence was obtained by size exclusion chromatography, which pointed out that maltodextrins had a bimodal molecular weight distribution composed of high and low molecular weight oligo-saccharides. Indeed, maltodextrins are highly polydisperse materials (i.e. polydispersity index ranging from 5 to 12) and that should be the reason why such distinct behaviors were observed in some of the physico-chemical analyses that were preformed.