Statistical Optimization and Partial Characterization of Xylanases Produced by Streptomyces sp. S1M3I Using Olive Pomace as a Fermentation Substrate.

Xylanase production by Streptomyces sp. S1M3I was optimized by response surface methodology (RSM), followed by a partial characterization of these enzymes. Olive pomace was used as a substrate for growing Streptomyces sp. S1M3I in submerged fermentation. Effects of incubation time, pH, temperature, carbon source, nitrogen source, and inoculum size on xylanase production were studied, through the one-factor-at-a-time method. Then, a 33-factorial experimental design with RSM and the Box-Behnken design was investigated for the major influence factors. Maximum xylanase production (11.28 U/mL) was obtained when the strain was grown in mineral medium supplemented with 3% (w/v) of olive pomace powder and 0.3% (w/v) of ammonium sulfate, at a pH 7.4 and an incubation temperature of 40 °C. The xylanases in the supernatant degraded all tested substrates, with higher activity for the low-viscosity wheat arabinoxylan substrate. Two xylanases with close molecular masses were detected by zymogram analysis: Xyl-1 and Xyl-2 with molecular masses of 24.14 kDa and 27 kDa, respectively. The optimization of enzyme production parameters of Streptomyces sp. S1M3I and the characterization of these enzymes are prerequisites to enhancing xylanase production yield, which is crucial for further biotechnological processes.

Biotechnological potential of Zymotis-2 bioreactor for the cultivation of filamentous fungi.

BACKGROUND/AIM: A new prototype of Solid-State Fermentation Bioreactor, namely "Zymotis-2 ", was developed to produce fungal spores. MAIN METHODS AND MAJOR RESULTS: A fermentation process for fungal spores, and hydrolase enzymes (endo and exoglucanases, amylases) production by Trichoderma asperellum DWG3, Aspergillus niger G131 and Beauveria bassiana was scaled-up from flasks and glass Raimbault column packed with 20 g of solid substrates (dry weight) to 5 kg of solid substrate by using the new Zymotis-2 bioreactor. Fungi strains growth using a mix of vine shoots, wheat bran, and olive pomace was tested under similar experimental conditions in Zymotis-2 bioreactor, column bioreactor and flasks in a parallel fermentation system. Overall, significant spore production on Zymotis-2 bioreactor was obtained, achieving 22.01 ± 0.01×109 spores/g DM 16.30 ± 0.07 × 109 spores/g DM, and 3.30 ± 0.07 × 109 spores/g DM for B. bassiana, T. asperellum DWG3, and A. niger G131, respectively. Forced aeration increased the endoglucanases, exoglucanases and amylases activities for T. asperellum DWG3 but B. bassiana and A. niger G131 were affected negatively by the aerated process, showing the lowest enzyme activities. CONCLUSIONS AND IMPLICATIONS: In conclusion, a high yield of spores was obtained at 137 h of cultivation time, confirming the validity of the new Zymotis-2 bioreactor to produce virulent spores at low cost by T. asperellum, B. bassiana and A. niger G131.

Authentication and Chemometric Discrimination of Six Greek PDO Table Olive Varieties through Morphological Characteristics of Their Stones.

Table olives, the number one consumed fermented food in Europe, are widely consumed as they contain many valuable ingredients for health. It is also a food which may be the subject of adulteration, as many different olive varieties with different geographical origin, exist all over the word. In the present study, the image analysis of stones of six main Greek protected designation of origin (PDO) table olive varieties was performed for the control of their authentication and discrimination, with cv. Prasines Chalkidikis, cv. Kalamata Olive, cv. Konservolia Stylidas, cv. Konservolia Amfissis, cv. Throuba Thassos and cv. Throuba Chios being the studied olive varieties. Orthogonal partial least square discriminant analysis (OPLS-DA) was used for discrimination and classification of the six Greek table olive varieties. With a 98.33% of varietal discrimination, the OPLS-DA model proved to be an efficient tool to authentify table olive varieties from their morphological characteristics.

Chemometric Discrimination of the Geographical Origin of Three Greek Cultivars of Olive Oils by Stable Isotope Ratio Analysis.

Α stable isotope ratio mass spectrometer was used for stable isotope ratio (i.e., δ13C, δ18O, and δ2H) measurements, achieving geographical discrimination using orthogonal projections to latent structures discriminant analysis. A total of 100 Greek monovarietal olive oil samples from three different olive cultivars (cv. Koroneiki, cv. Lianolia Kerkyras, and cv. Maurolia), derived from Central Greece and Peloponnese, were collected during the 2019-2020 harvest year aiming to investigate the effect of botanical and geographical origin on their discrimination through isotopic data. The selection of these samples was made from traditionally olive-growing areas in which no significant research has been done so far. Samples were discriminated mainly by olive cultivar and, partially, by geographical origin, which is congruent with other authors. Based on this model, correct recognition of 93.75% in the training samples and correct prediction of 100% in the test set were achieved. The overall correct classification of the model was 91%. The predictability based on the externally validated method of discrimination was good (Q2 (cum) = 0.681) and illustrated that δ18O and δ2H were the most important isotope markers for the discrimination of olive oil samples. The authenticity of olive oil based on the examined olive varieties can be determined using this technique.

Statistical approach to evaluate effect of temperature and moisture content on the production of antioxidant naphtho-gamma-pyrones and hydroxycinnamic acids by Aspergillus tubingensis in solid-state fermentation.

Non-ochratoxigenic Aspergillus tubingensis G131 is a filamentous fungus that can produce naphtho-gamma-pyrones (NγPs), polyketide pigments that exhibit interesting antioxidant properties. This study aims to investigate the effect of two critical parameters, temperature and moisture content on the fungus grown in solid-state fermentation using agricultural by-products (vine shoots and wheat bran) as sole medium. From the kinetic productions of secondary metabolites NγPs (asperpyrone E, dianhydroaurasperone C, fonsecin, fonsecin B and ustilaginoidin A), alkaloids (nigragilin and aspernigrin A), degradation products from the solid medium (ß-D-glucose, p-coumaric acid and trans-ethyl ferulate), ergosterol and conidia obtained for different temperatures and moisture contents, a principal component analysis (PCA) was carried out to highlight the production patterns of these compounds. This approach allowed us to determine that fonsecin, the compound of higher interest-exhibiting the most interesting antiradical potential-is particularly more produced at 25 °C and 66% of moisture content. This study underlines the importance of temperature and moisture content on naphtho-gamma-pyrones and hydroxycinnamic acid production using solid-state fermentation and contributes to the development of agroindustrial by-product valorization.

Naphtho-Gamma-Pyrones Produced by Aspergillus tubingensis G131: New Source of Natural Nontoxic Antioxidants.

Seven naphtho-gamma-pyrones (NγPs), including asperpyrone E, aurasperone A, dianhydroaurasperone C, fonsecin, fonsecinone A, fonsecin B, and ustilaginoidin A, were isolated from Aspergillus tubingensis G131, a non-toxigenic strain. The radical scavenging activity of these NγPs was evaluated using ABTS assay. The Trolox equivalent antioxidant capacity on the seven isolated NγPs ranged from 2.4 to 14.6 µmol L-1. The toxicity and ability of the NγPs to prevent H2O2-mediated cell death were evaluated using normal/not cancerous cells (CHO cells). This cell-based assay showed that NγPs: (1) Are not toxic or weakly toxic towards cells and (2) are able to protect cells from oxidant injuries with an IC50 on H2O2-mediated cell death ranging from 2.25 to 1800 µmol mL-1. Our data show that A. tubingensis G131 strain is able to produce various NγPs possessing strong antioxidant activities and low toxicities, making this strain a good candidate for antioxidant applications in food and cosmetic industries.

Elucidation of functional chemical groups responsible of compost phytotoxicity using solid-state 13C NMR spectroscopy under different initial C/N ratios.

More than 1 million tons of fresh organic wastes is produced in the Souss-Massa region in Morocco. Tomato organic residues represent more than 25% of the total organic wastes and are deposited in uncontrolled landfills. Thus, composting can represent a valuable and pertinent solution to this environmental problem. The objectives of this experiment are to identify the potential functional groups responsible for compost phytotoxicity and to determine the optimum initial carbon to nitrogen ratio (C/N) for maximum recovery of tomato residues. The experiment consisted of the variation of the initial C/N ratios (25, 30, 35, and 40) using mixtures of different raw materials (tomato residues, melon residues, olive mill pomace, and sheep manure). Physicochemical parameters (pH, electrical conductivity, C/N ratio, and humic acid/fulvic acid ratio) were determined and spectroscopic analyses (UV-vis and NMR-13C) were performed during the composting process along with quality parameters (germination and phytotoxicity tests) at the end. The results showed that the compost with the initial C/N ratio of 35 is the most humified with the least phytotoxic effect. The germination and phytotoxicity tests were negatively correlated with the methoxyl/N-alkyl-C ratio and O-alkyl-C. These two functional groups are probably the origin of phytotoxicity expression in compost quality tests. Thus, a simple and precise quality test could be performed to evaluate directly the phytotoxicity and maturity of compost.

Selective Isolation and Screening of Actinobacteria Strains Producing Lignocellulolytic Enzymes Using Olive Pomace as Substrate.

BACKGROUND: Olive pomace, as the main by-product of the olive oil industry, is recently recycled as fermentation substrate for enzyme production. OBJECTIVES: Actinobacteria isolates were separated from an Algerian soil under olive pomace cultivation and were evaluated for their lignocellulolytic enzymes production. MATERIALS AND METHODS: Isolates of Actinobacteria were separated from soils around oil mills using four isolation media, among them three were enriched by olive pomace. The isolates were screened for their cellulolytic, xylanolytic and ligninolytic activities. Isolates with potential of producing lignocellulose-degrading enzymes were selected under submerged fermentation based olive pomace. RESULTS: Ninety isolates of Actinobacteria were separated from soil samples. M3 medium (raw pomace autoclaved alone) was the best isolation medium (68 strains), whereas, the soil from oil mill with continuous system (S1) led to separation of 52 strains. Among the 90 isolates, 82 were shown promising enzyme activity, 19 isolates were presented the largest zone diameter (<30 mm). S1M3I and S1M3II isolates were exhibited the highest values. CONCLUSIONS: Olive pomace with medium low cost and high titers of enzymes can be valorized by culture of Actinobacteria to produce lignocellulolytic enzymes for industrial applications.

Challenges and opportunities of the bio-pesticides production by solid-state fermentation: filamentous fungi as a model.

In recent years, production and use of bio-pesticides have increasing and replacing some synthetic chemical pesticides applied to food commodities. In this review, biological control is focused as an alternative, to some synthetic chemical treatments that cause environmental, human health, and food quality risks. In addition, several phytopathogenic microorganisms have developed resistance to some of these synthetic chemicals and become more difficult to control. Worldwide, the bio-pesticides market is growing annually at a rate of 44% in North America, 20% in Europe and Oceania, 10% in Latin and South American countries and 6% in Asia. Use of agro-industrial wastes and solid-state fermentation (SSF) technology offers an alternative to bio-pesticide production with advantages versus conventional submerged fermentations, as reduced cost and energy consumption, low production of residual water and high stability products. In this review, recent data about state of art regarding bio-pesticides production under SSF on agroindustrial wastes will be discussed. SSF can be defined as a microbial process that generally occurs on solid material in the absence of free water. This material has the ability to absorb water with or without soluble nutrients, since the substrate must have water to support the microorganism's growth and metabolism. Changes in water content are analyzed in order to select the conditions for a future process, where water stress can be combined with the best spore production conditions, obtaining in this way an inexpensive biotechnological option for modern agriculture in developing countries.

Commercial Biological Control Agents Targeted Against Plant-Parasitic Root-knot Nematodes

Root-knot nematodes are microscopic round worms, which cause severe agricultural losses. Their attacks affect the productivity by reducing the amount and the caliber of the fruits. Chemical control is widely used, but biological control appears to be a better solution, mainly using microorganisms to reduce the quantity of pests infecting crops. Biological control is developing gradually, and with time, more products are being marketed worldwide. They can be formulated with bacteria, viruses or with filamentous fungi, which can destroy and feed on phytoparasitic nematodes. To be used by the farmers, biopesticides must be legalized by the states, which has led to the establishment of a legal framework for their use, devised by various governmental organizations.

Do spawn storage conditions influence the colonization capacity of a wheat-straw-based substrate by Agaricus subrufescens?

Storage conditions of the spawn of edible fungi are of major importance to facilitate the production of mushrooms. Here, standard storage conditions at 10°C or 15°C were used and the potential of colonization of standard European compost by the tropical species Agaricus subrufescens was assessed during the spawn running phase. Two lignocellulolytic activities, laccase and CMC-cellulase, were enhanced after storage compared to control as well as substrate transformation, as described by the aromaticity ratio and a humification ratio calculated from NMR data. This result indicates that mycelium growth probably occurred during storage at 10 or 15°C, leading to a larger amount of biomass in the inoculum. Moreover, the microbial functional diversity of the substrate was favored, showing that the electivity of the substrate was maintained. Thus, these findings indicate that recommendations for the mushroom producers can be established for A. subrufescens cultivation under European standard conditions.

Initial proteome analysis of caffeine-induced proteins in Aspergillus tamarii using two-dimensional fluorescence difference gel electrophoresis.

Caffeine is toxic to most microorganisms. However, some filamentous fungi, such as Aspergillus tamarii, are able to metabolize this alkaloid when fed caffeine as the sole nitrogen source. The aim of the present work was to identify intracellular A. tamarii proteins, regulated by caffeine, using fluorescence difference two-dimensional gel electrophoresis. Specific proteins from two culture media of A. tamarii grown either on ammonium sulfate or caffeine as the sole nitrogen source were analysed by mass spectrometry. Thirteen out of a total of 85 differentially expressed spots were identified after database search. Identified up-regulated proteins include phosphoglycerate kinase, malate dehydrogenase, dyp-type peroxidase family protein, heat shock protein, Cu, Zn superoxidase dismutase and xanthine dehydrogenase. Some of the proteins identified in this study are involved in the caffeine degradation pathway as well as in stress response, suggesting that stress proteins could be involved in caffeine metabolism in filamentous fungi.

Robusta coffee beans post-harvest microflora: Lactobacillus plantarum sp. as potential antagonist of Aspergillus carbonarius.

Coffee contamination by ochratoxigenic fungi affects both coffee quality as well as coffee price with harmful consequences on the economy of the coffee exporting countries for whom which is their main source of income. Fungal strains were isolated from coffee beans and identified as black Aspergilli. Ochratoxigenic moulds like Aspergillus carbonarius were screened and selected for detailed studies. Also lactic acid bacteria (LAB) were isolated from silage coffee pulp and their antifungal activity was tested on dual-culture agar plate. Ten of the isolated LAB demonstrated antifungal effect against A. carbonarius. API 50 CH and APIZYM were used to perform phenotypic identification. 16S rDNA sequencing was made to confirm the results.

Gamma irradiation of air-dried olive leaves: Effective decontamination and impact on the antioxidative properties and on phenolic compounds.

Olive leaves are commercialized for their antioxidative value due to their valuable phenolic compounds. The present study aimed to evaluate the effect of gamma irradiation on microbial load, on antioxidative properties and on phenolic compounds of air-dried olive leaves. Irradiation was applied up to 25kGy (5kGy intervals) to powdered and intact samples. Total aerobic bacteria, yeast and mold, and lactic acid bacteria were counted after gamma irradiation. Decontamination was obtained at 20kGy. The radioresistance of microbial population was high with D10 values between 9.74 and 25.12kGy. Besides, gamma irradiation up to 25kGy was found to maintain the antioxidant capacity, molecular mass distribution of polyphenolics, total phenolics, ortho-diphenols, flavonoids, oleuropein, verbascoside and rutin contents. To conclude, the improvement of the microbial quality of air-dried olive leaves, without affecting phenolic composition and antioxidative properties, can be successively achieved by the application of gamma irradiation treatment.

Degradation of procyanidins by Aspergillus fumigatus: identification of a novel aromatic ring cleavage product.

Aspergillus fumigatus was able to grow on apple-purified procyanidins (PCs). PCs concentration decreased 30% over the first 60 h of liquid fermentation. The mean degree of polymerization (DPn) of apple-purified PCs increased from 8 to 15 during the fermentation. A fungal enzyme extract from the liquid fermentation was used to study procyanidin B2 [(-)-epicatechin-(4beta-8)-(-)-epicatechin] degradation. The major degradation product (PB2-X) had a retention time of 10.5 min and a molecular mass at m/z 609. High-performance liquid chromatography/multiple fragment mass spectrometry (HPLC/MS(n)) was used for the structural characterization of PB2-X as well as that of thiolysis-treated PB2-X. Twelve fragment ions at m/z 565, 547, 457, 439 (two fragment ions), 421, 413, 377, 395, 351, 287 and 277 were completely identified. It was therefore deduced that the terminal unit of procyanidin B2 dimer was modified by an oxygenase from A. fumigatus leaving the extension unit intact. In addition, FT-IR analysis confirmed a lactone formation in (-)-epicatechin moiety involved in oxidative degradation. Two reaction schemes were postulated for the interpretation of the results.

Standardisation of methods for assessing mould germination: a workshop report.

The first workshop on predictive mycology was held in Marseille, France, 2--4 February 2005 under the auspices of the French Microbiological society. The purpose of the workshop was to list the different techniques and definitions used by scientists for assessing mould germination and to evaluate the influence of the different techniques on the experimental results. Recommendations were made when a large consensus was obtained. In order to facilitate the study of germination, alternative methods to microscopic examination were examined.

Characterization of filamentous fungi isolated from Moroccan olive and olive cake: toxinogenic potential of Aspergillus strains.

During the 2003 and 2004 olive oil production campaigns in Morocco, 136 samples from spoiled olive and olive cake were analyzed and 285 strains were isolated in pure culture. Strains included 167 mesophilic strains belonging to ten genera: Penicillium, Aspergillus, Geotrichum, Mucor, Rhizopus, Trichoderma, Alternaria, Acremonium, Humicola, Ulocladium as well as 118 thermophilic strains isolated in 2003 and 2004, mainly belonging to six species: Aspergillus fumigatus, Paecilomyces variotii, Mucor pusillus, Thermomyces lanuginosus, Humicola grisea, and Thermoascus aurantiacus. Penicillium and Aspergillus, respectively, 32.3 and 26.9% of total isolates represented the majority of mesophilic fungi isolated. When considering total strains (including thermotolerant strains) Aspergillus were the predominant strains isolated; follow-up studies on mycotoxins therefore focused primarily on aflatoxins (AFs) and ochratoxin A (OTA) from the latter strains. All isolated Aspergillus flavus strains (9) and Aspergillus niger strains (36) were studied in order to evaluate their capacity to produce AFs and OTA, respectively, when grown on starch-based culture media. Seven of the nine tested A. flavus strains isolated from olive and olive cake produced AF B1 at concentrations between 48 and 95 microg/kg of dry rice weight. As for the A. niger strains, 27 of the 36 strains produced OTA.

Development of a bionematicide with Paecilomyces lilacinus to control Meloidogyne incognita.

Root-knot disease caused by Meloidogyne incognita is a matter of grave concern because it affects several economically important crop plants. The use of solid-state fermentation (SSF) may help to elaborate efficient formulations with fungi to be employed in the biologic control of nematodes. Attempts were made to select low-cost substrates for spore production of a strain of Paecilomyces lilacinus with known nematicide capacity. Coffee husks, cassava bagasse, and defatted soybean cake were utilized as substrates, and sugarcane bagasse was used as support. Fermentations were carried out in flasks covered with filter paper at 28 degrees C for 10 d. The products obtained by SSF were evaluated for their nematicide activity in pot experiments containing one seedling of the plant Coleus inoculated with the nematode M. incognita. The plants were evaluated 2 mo after inoculation. Fermented products showed a reduction in the number of nematodes. The best results were obtained with defatted soybean cake, which showed almost 100% reduction in the number of nematodes; the reduction with coffee husk was 80% and with cassava bagasse was about 60%.

Characterization and estimation of proanthocyanidins and other phenolics in coffee pulp (Coffea arabica) by thiolysis-high-performance liquid chromatography.

Fresh and 3-day-old coffee pulp of the Arabica variety were analyzed for polyphenol composition followed by characterization by two different methods. The first method consisted in subjecting coffee pulp powder to direct thiolysis. For the second method, coffee pulp was subjected to successive solvent extractions, followed by thiolysis. Quantification of phenolic compounds was then achieved by high-performance liquid chromatography (HPLC) analysis of thiolysis products. Four major classes of polyphenols were identified: flavan-3-ols (monomers and procyanidins), hydroxycinnamic acids, flavonols, and anthocyanidins. Differences in concentration of procyanidins were observed between fresh and 3-day-old coffee pulp. Constitutive units were mainly epicatechin, representing more than 90% of the proanthocyanidin units, with average degrees of polymerization in the range of 3.8-9.1. Monomer to hexamer units of flavan-3-ols from fresh coffee pulp were separated by normal-phase HPLC. Molecular size of oligomeric proanthocyanidins was obtained by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Results obtained confirm the presence of oligomers of the flavan-3-ol (-)-epicatechin.

Coffee residues as substrates for aroma production by Ceratocystis fimbriata in solid state fermentation

Neste trabalho duas diferentes cepas de Ceratocystis fimbriata foram testadas para a producão de aromas frutais em fermentacão no estado sólido (FES) utilizando como substratos casca e polpa de café, suplementados com glicose. Os experimentos foram realizados em frascos Erlenmeyer de 250 mL. As condicões experimentais foram: umidade inicial de 70%, adicão de 20% de glicose e pH 6,0. Os frascos foram cobertos com gaze e a aeracão ocorreu por difusão passiva. A análise do headspace da cultura foi feita por cromatografia gasosa e 12 compostos foram detectados utilizando a casca de café. A análise respirométrica foi realizada para o acompanhamento do crescimento do microrganismo pela determinacão do dióxido de carbono produzido. A producão de ésteres caracterizou o aroma frutal da cultura. A concentracão máxima de voláteis totais foi alcancada após 72 h de cultivo em casca de café (28 µmol.L-1.g-1). Os principais compostos produzidos foram acetato de etila, etanol e acetaldeído, representando 84,7%, 7,6% and 2,0% dos voláteis totais, respectivamente.