RESUMO
The transition towards a sustainable model, particularly the circular economy, emphasizes the importance of redefining waste as a valuable resource, paving the way for innovative upcycling strategies. The olive oil industry, with its significant output of agricultural waste, offers a promising avenue for high-value biomass conversion into useful products through microbial processes. This study focuses on exploring new, high-value applications for olive leaves waste, utilizing a biotechnological approach with Lactobacillus casei for the production of second-generation lactic acid. Contrary to initial expectations, the inherent high polyphenol content and low fermentable glucose levels in olive leaves posed challenges for fermentation. Addressing this, an enzymatic hydrolysis step, following a preliminary extraction process, was implemented to increase glucose availability. Subsequent small-scale fermentation tests were conducted with and without nutrient supplements, identifying the medium that yielded the highest lactic acid production for scale-up. The scaled-up batch fermentation process achieved an enhanced conversion rate (83.58%) and specific productivity (0.26 g/L·h). This research confirms the feasibility of repurposing olive waste leaves for the production of lactic acid, contributing to the advancement of a greener economy through the valorization of agricultural waste. KEY POINTS: ⢠Olive leaves slurry as it did not allow L. casei to ferment. ⢠High concentrations of polyphenols inhibit fermentation of L. casei. ⢠Enzymatic hydrolysis combined to organosolv extraction is the best pretreatment for lactic acid production starting from leaves and olive pruning waste.
Assuntos
Fermentação , Ácido Láctico , Lacticaseibacillus casei , Olea , Azeite de Oliva , Folhas de Planta , Ácido Láctico/metabolismo , Lacticaseibacillus casei/metabolismo , Folhas de Planta/química , Folhas de Planta/metabolismo , Azeite de Oliva/metabolismo , Glucose/metabolismo , Hidrólise , Resíduos Industriais , Polifenóis/metabolismo , BiomassaRESUMO
Biosurfactants are in demand by the global market as natural commodities suitable for incorporation into commercial products or utilization in environmental applications. Fungi are promising producers of these molecules and have garnered interest also for their metabolic capabilities in efficiently utilizing recalcitrant and complex substrates, like hydrocarbons, plastic, etc. Within this framework, biosurfactants produced by two Fusarium solani fungal strains, isolated from plastic waste-contaminated landfill soils, were analyzed. Mycelia of these fungi were grown in the presence of 5% olive oil to drive biosurfactant production. The characterization of the emulsifying and surfactant capacity of these extracts highlighted that two different components are involved. A protein was purified and identified as a CFEM (common in fungal extracellular membrane) containing domain, revealing a good propensity to stabilize emulsions only in its aggregate form. On the other hand, an unidentified cationic smaller molecule exhibits the ability to reduce surface tension. Based on the 3D structural model of the protein, a plausible mechanism for the formation of very stable aggregates, endowed with the emulsifying ability, is proposed. KEY POINTS: ⢠Two Fusarium solani strains are analyzed for their surfactant production. ⢠A cationic surfactant is produced, exhibiting the ability to remarkably reduce surface tension. ⢠An identified protein reveals a good propensity to stabilize emulsions only in its aggregate form.
Assuntos
Proteínas Fúngicas , Fusarium , Tensoativos , Fusarium/metabolismo , Fusarium/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Tensoativos/metabolismo , Tensoativos/química , Emulsificantes/metabolismo , Emulsificantes/química , Microbiologia do Solo , Emulsões/química , Emulsões/metabolismo , Tensão Superficial , Cisteína/metabolismo , Cisteína/química , Azeite de Oliva/metabolismo , Azeite de Oliva/química , Micélio/metabolismoRESUMO
Lipases play a vital role in various biological processes, from lipid metabolism to industrial applications. However, the ever-evolving challenges and diverse substrates necessitate the continual exploration of novel high-performance lipases. In this study, we employed an in silico mining approach to search for lipases with potential high sn-1,3 selectivity and catalytic activity. The identified novel lipase, PLL, from Paenibacillus larvae subsp. larvae B-3650 exhibited a specific activity of 111.2 ± 5.5â¯U/mg towards the substrate p-nitrophenyl palmitate (pNPP) and 6.9 ± 0.8â¯U/mg towards the substrate olive oil when expressed in Escherichia coli (E. coli). Computational design of cysteine mutations was employed to enhance the catalytic performance of PLL. Superior stability was achieved with the mutant K7C/A386C/H159C/K108C (2M3/2M4), showing an increase in melting temperature (Tm) by 1.9°C, a 2.05-fold prolonged half-life at 45°C, and no decrease in enzyme activity. Another mutant, K7C/A386C/A174C/A243C (2M1/2M3), showed a 4.9-fold enhancement in specific activity without compromising stability. Molecular dynamics simulations were conducted to explore the mechanisms of these two mutants. Mutant 2M3/2M4 forms putative disulfide bonds in the loop region, connecting the N- and C-termini of PLL, thus enhancing overall structural rigidity without impacting catalytic activity. The cysteines introduced in mutant 2M1/2M3 not only form new intramolecular hydrogen bonds but also alter the polarity and volume of the substrate-binding pocket, facilitating the entry of large substrate pNPP. These results highlight an efficient in silico exploration approach for novel lipases, offering a rapid and efficient method for enhancing catalytic performance through rational protein design.
Assuntos
Proteínas de Bactérias , Estabilidade Enzimática , Lipase , Paenibacillus , Lipase/genética , Lipase/metabolismo , Lipase/química , Paenibacillus/enzimologia , Paenibacillus/genética , Especificidade por Substrato , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Simulação por Computador , Engenharia de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimologia , Cinética , Simulação de Dinâmica Molecular , Azeite de Oliva/metabolismo , Azeite de Oliva/química , Mutagênese Sítio-Dirigida , Biocatálise , PalmitatosRESUMO
The olive oil industry recently introduced a novel multi-phase decanter with the "Leopard DMF" series, which gives a by-product called pâté, made up of pulp and olive wastewater with a high content of phenolic substances and without pits. This study aims to create a new culture medium, the Olive Juice Broth (OJB), from DMF pâté, and apply it to select bacteria strains able to survive and degrade the bitter substances normally present in the olive fruit. Thirty-five different bacterial strains of Lactiplantibacillus plantarum from the CREA-IT.PE Collection of Microorganisms were tested. Seven strains characterized by ≥50% growth in OJB (B31, B137, B28, B39, B124, B130, and B51) showed a degradation of the total phenolic content of OJB ≥ 30%. From this set, L. plantarum B51 strain was selected as a starter for table olive production vs. spontaneous fermentation. The selected inoculant effectively reduced the debittering time compared to spontaneous fermentation. Hydroxytyrosol, derived from oleuropein and verbascoside degradation, and tyrosol, derived from ligstroside degradation, were produced faster than during spontaneous fermentation. The OJB medium is confirmed to be useful in selecting bacterial strains resistant to the complex phenolic environment of the olive fruit.
Assuntos
Meios de Cultura , Fermentação , Olea , Fenóis , Olea/microbiologia , Olea/metabolismo , Olea/química , Fenóis/metabolismo , Fenóis/química , Meios de Cultura/química , Lactobacillales/metabolismo , Azeite de Oliva/química , Azeite de Oliva/metabolismo , Álcool Feniletílico/metabolismo , Álcool Feniletílico/química , Álcool Feniletílico/análogos & derivados , Glucosídeos Iridoides/metabolismo , Glucosídeos/metabolismo , Glucosídeos/química , Lactobacillus plantarum/metabolismo , PolifenóisRESUMO
The lipoxygenase pathway has a significant influence on the composition of the volatile components of virgin olive oil (VOO). In this work, the influence of the maturity index (MI) on the activity of the lipoxygenase enzyme (LOX) in the fruits of the autochthonous Dalmatian olive cultivars Oblica, Levantinka and Lastovka was studied. The analysis of the primary oxidation products of linoleic acid in the studied cultivars showed that LOX synthesises a mixture of 9- and 13-hydroperoxides of octadecenoic acid in a ratio of about 1:2, which makes it a non-traditional plant LOX. By processing the fruits of MI~3, we obtained VOOs with the highest concentration of desirable C6 volatile compounds among the cultivars studied. We confirmed a positive correlation between MI, the enzyme activity LOX and the concentration of hexyl acetate and hexanol in cultivars Oblica and Lastovka, while no positive correlation with hexanol was observed in the cultivar Levantinka. A significant negative correlation was found between total phenolic compounds in VOO and LOX enzyme activity, followed by an increase in the MI of fruits. This article contributes to the selection of the optimal harvest time for the production of VOOs with the desired aromatic properties and to the knowledge of the varietal characteristics of VOOs.
Assuntos
Lipoxigenase , Olea , Azeite de Oliva , Compostos Orgânicos Voláteis , Azeite de Oliva/química , Azeite de Oliva/metabolismo , Lipoxigenase/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/química , Olea/metabolismo , Olea/química , Frutas/química , Frutas/metabolismo , Fenóis/metabolismo , Fenóis/análise , Fenóis/química , Ácido Linoleico/metabolismoRESUMO
Chronic inflammation and insulin resistance lead to metabolic syndrome and there is an urgent need to establish effective treatments and prevention methods. Our previous study reported that obese model Zucker (fa/fa) rats fed with ozonated olive oil alleviated fatty liver and liver damage by suppressing inflammatory factors. However, differences among animal species related to the safety and efficacy of ozonated olive oil administration remain unclear. Therefore, this study investigated the effects of oral intake of ozonated olive oil on lipid metabolism in normal mice and mice in the obesity model. C57BL/6J and db/db mice were fed the following AIN-76 diets for four weeks: the mice were either fed a 0.5% olive oil diet (Control diet) or 0.5% ozonated olive oil diet (Oz-Olive diet) in addition to 6.5% corn oil. The results indicated that four weeks of Oz-Olive intake did not adversely affect growth parameters, hepatic lipids or serum parameters in normal C57BL/6J mice. Subsequent treatment of db/db mice with Oz-Olive for four weeks reduced the levels of hepatic triglycerides, serum alkaline phosphatase, and serum insulin. These effects of Oz-Olive administration might be due to suppression of fatty acid synthesis activity and expression of lipogenic genes, as well as suppression of inflammatory gene expression. In conclusion, this study confirmed the safety of Oz-Olive administration in normal mice and its ability to alleviate hepatic steatosis by inhibiting fatty acid synthesis and inflammation in obese mice.
Assuntos
Fígado Gorduroso , Camundongos , Ratos , Animais , Azeite de Oliva/farmacologia , Azeite de Oliva/uso terapêutico , Azeite de Oliva/metabolismo , Camundongos Endogâmicos C57BL , Ratos Zucker , Fígado Gorduroso/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Ácidos Graxos/metabolismo , Inflamação/metabolismo , Camundongos ObesosRESUMO
Olive (Olea europea L.) is one of the oldest and most important fruit tree species cultivated in the Mediterranean region. Various plant tissues, drupes, and olive oil contain several phenolics (including verbascoside, although it is present in the plant at a low level) that are well-known for their highly beneficial effects on human health. An in vitro olive cell suspension culture (cultivar Cellina di Nardò, "CdN") was established, characterized for its growth and morphological features. Furthermore, a vital and relatively uniform population of protoplasts was generated from the olive suspension culture to investigate their cellular characteristics during growth. The polyphenolic extract of the in vitro "CdN" olive cells contained almost exclusively verbascoside, as revealed by the UPLC-ESI-MS analysis. The content of verbascoside reached up to 100 mg/g DW, with an average production rate of approximately 50 mg/g DW over one year of culture. This level of production has not been previously reported in a limited number of previous studies. This remarkable production of verbascoside was associated with an exceptionally high antioxidant capacity. The high level of verbascoside production and purity of the extract make this system a promising tool for secondary metabolite production.