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The application of laccases for industrial use is restricted by biochemical properties and availability. Recombinant enzymes frequently show molecular properties different of the wild enzymes. Ideally the biodegradation of complex mixtures of phenols by laccases must use the more efficient enzymes.
Under the stress produced by mixtures of aromatic compounds (as OMW, or wastes from the textile industry, for example) many fungus respond producing specific laccase to decrease the stressing conditions by oxidizing phenols.
These enzymes have been purified and characterized in Coriolopsis polyzona cultures containing OMW. The organism and their laccases are available for the investigation of industrial applications.
Triacetyhydroxytyrosol was very efficiently produced as a highly pure stabilized antioxidant compound by a short treatment of olive mill waste water (OMWW) organic extracts, rich in hydroxytyrosol, with an acetylating mixture composed of HClO4-SiO2 and Ac2O (Chakborti and Gulhane reaction), in mild and safe conditions. A successive single step of middle pressure liquid chromatography
(MPLC) purification of the reaction product was performed, with an overall yield of 35.6%.
The purified natural triacetylhydroxytyrosol confirmed the same strong protective effects against the oxidative stress in human cells as the corresponding synthetic compound, likely because of the biochemical activation of the acetyl derivative into the active parent hydroxytyrosol by esterases.
The synthetic triacetylhydroxytyrosol was prepared by reaction of synthetic hydroxytyrosol in presence of pyridine and acetic anhydride. Hydroxytyrosol was in turn prepared by reduction of 3,4-hydroxyphenilacetic acid.
Experiments performed under laboratory conditions evaluated the effect of OMW on chemical and biochemical properties of soil. A soil from Morocco 4 was used to simulate conditions likely occurring in that Country. The changes of the activities of several enzymes involved in the cycles of main nutrients, soil respiration, and microbial biomass-C and -N of the soil, amended with OMW at two different rates (equivalent to 40 and 80 m3 ha-1) were determined at different incubation times. An unamended soil was used as a control. The residual phytotoxicity of treated and untreated soil was also evaluated by performing germination tests using soil as germination medium.
The obtained results indicated that several concurring activating and/or inhibitory effects occurred (increased enzyme production, presence of inhibitory OMW ions and/or OMW compounds, and/or their metabolites, release of soluble ions from soil components etc.). Furthermore, transformation processes of OMW phenols catalysed by Mn and Fe soil components took place and decreased the phytotoxicity of soil. As regards chemical and physical properties no significant differences of the physical-chemical properties of the soil were observed at the two OMW/soil ratios. As regards biochemical properties the enzyme activities of C cycle, dehydrogenase and beta-glucosidase, N cycle, urease and nitrate reductase, P cycle, phosphatase, S cycle, arylsulphatase, and aspecific hydrolases, fluorescein diacetate hydrolase, varied widely in soil samples with and without OMW treatment and with time.
Experiments were performed on the effect on the same soil properties after the application of OMW before and after a detoxifying treatment able to reduce consistently their phenolic content. The obtained results indicated that the enzymatic activities in soil with detoxified OMW (i.e. with a lower amounts of monomeric phenols) are generally lower than those of the soil added with OMW without any previous treatment, except for FDA-hydrolase. When detoxified OMW was used, the majority of parameters showed a consistent increase after 7-14 days from treatment followed by a gradual decrease. After 56 days from treatment, some parameters reached the initial level (for example the biomass carbon) and other ones stabilized at higher activity levels (for example the biomass nitrogen).
As regards germination tests soil phytotoxicity suddenly increased immediately after the addition of the highest OMW amount, whereas only 20% reduction of germination occurred with the lowest OMW rate. However, a reduction of phytotoxicity was observed with increasing the incubation time. An interesting result was obtained with OMW after the removal of monomeric phenols. When treated OMW were used, the restoration of a non-phytotoxicity environment is reached in few days and it was complete; in contrast, when untreated OMW were used, the reduction of phytotoxicity was very slow and never was complete.
These results obviously suggest that the use of OMW without monomeric phenols is less harmful on soil properties than untreated OMW.
It has been found that Olive Oil Mill Waste Waters (OMW) have systematically a higher differences in terms of electric conductivity, proteins, soluble and bound phenolics and all of them exhibit a certain toxicity with regard to Zea mays germination at lab level. These results indicated that it will be difficult to have an universal strategy for the treatment of OMW by biological and/or chemical protocols leading to their detoxification and re-use in agriculture.
Several agronomical experiments have been conducted with crude OMW in greenhouse and in field taking into account their impact on the soil and on the growth and the biomass allocation of several species such as date palm, maize, atriplex and wheat.
When a moderate volumes of OMW were sprayed once a week or several weeks before sowing, no phytotoxicity has been revealed. In contrario, a positive effect has been detected concerning the biomass allocation in the case of the four species (maize, date palm, atriplex and wheat) that we have selected as the most suitable crops to be fertirrigated by OMW.
Concerning the impact of OMW on the soil, it has been detected that the effluents lead to a significant increase in salinity expressed by a high electrical conductivity (EC) and hydro-soluble sodium contents depending on the initial volume and the time. A significant increase in organic matter, total nitrogen content, phosphorus, potassium and hydrosoluble phenolics has been also detected.
However, the pH of the Marrakech soil did not show a significant difference compared to the control. This situation puts forward the neutralizing capacity of the calcareous soil with respect to the OMW acidity.
The olive oil market has recently developed, since the Mediterranean diet is widely appreciated throughout the world by consumers more attentive to both health and nutritional aspects of food. The increasing popularity of olive oil has been mainly attributed to its high content of oleic acid, which may affect the plasma lipid/lipoprotein profiles and its richness in phenolic compounds acting as natural antioxidants, which may contribute to the prevention of diseases in human. However, during olive oil extraction the addition of water resulted in the release of the major part of olive polyphénols in the liquid phase (vegetation water) due to their amphiphilic character.
High added value antioxidants present in olive oil are found in olive oil processing by-products at very high concentrations. In particular, olive oil mill wastewaters, which become a major environmental problem in the main olive producing countries of the Mediterranean region, could be considered as a source of natural antioxidants.
Our objective was to develop effective procedures to recover potentially high-added-value phenolic compounds contained in OMW. Biological activities of the major compound and extracts were carried out in vivo and in vitro in order to evaluate their antioxidant and potential application as food additives and for stabilisation of refined husk oil.
A process was developed to extract and purify hydroxytyrosol, one of the major compounds occurring in OMW. Batch optimization experiments showed that ethyl acetate is the most efficient solvent for the recovery of phenolic monomers from OMW. The latter was used with an optimal pH equal to 2. Furthermore, the percentage of each monomer and particularly hydroxytyrosol in the extract was maximum for a solvent ratio and a theoretical extraction stage numbers equal to 2 and 3, respectively.
High yield (85.46%) recovery of hydroxytyrosol was achieved from OMW using a three-staged continuous counter-current liquid-liquid extraction unit. 1.225 g of hydroxytyrosol were extracted per liter of OMW. One gram of hydroxytyrosol per liter of OMW was then purified by means of a chromatographic system which could be adapted to a large scale production process.
The effects of OMW origin (time of olive harvest, process of olive oil extraction) and storage method were studied with regard to the recovered phenolic compound concentration, particularly hydroxytyrosol. OMW storage in the darkness, at 25°C in intermittent agitation and aerobic condition for 3 to 5 months led to an increase in the concentration of hydroxytyrosol.
The antiradical and antioxidant activities of the OMW extract as well as pure phenolic compounds identified in this extract were evaluated. Results showed that pure hydroxytyrosol and 3,4-dihydroxyphenyl acetic acid had the highest radical scavenging effect on 1.1 diphenyl -2 picrylhydrazyl (DPPH) radical and the highest antioxidant activities using the ?-carotene linoleate model system .
The effect of addition of individual phenolic compounds and OMW extract to refined olive and husk oils was compared with that of control, BHA and BHT at 50 °C. 3,4-dihydroxyphenyl acetic acid and hydroxytyrosol have the highest protective effect against oil oxidation The results suggested that hydroxytyrosol and OMW extract possess useful antioxidant properties and may be used as alternative in the search for natural replacement of synthetic antioxidant food additives.
Hypocholesterolemic effects of hydroxytyrosol and OMW extract in rats fed a cholesterol-rich diet were tested. Wistar rats, fed a standard laboratory diet or a cholesterol-rich diet for 16 weeks, were used. Serum lipid levels, as well as thiobarbituric acid reactive substances (TBARS) and superoxide dismutase (SOD) and catalase (CAT) activities in liver were examined. Cholesterol-rich diet induced hypercholesterolemia was manifested in the elevation of serum total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C).
Administration of a low-dose (2.5 mg/kg of body weight) of hydroxytyrosol and high-dose (10 mg/kg of body weight) of OMW extract significantly lowered the serum levels of TC and LDL-C while increasing the serum levels of high density lipoprotein cholesterol (HDL-C). Furthermore, the TBARS contents in liver, heart, kidney and aorta, decreased significantly after oral administration of hydroxytyrosol and OMW extract compared with those of rats fed a cholesterol-rich diet. In addition, OMW phenolics increased CAT and SOD activities in liver. These results suggested that the hypocholesterolemic effect of hydroxytyrosol and OMW extract might be due to their abilities to lower serum TC and LDL-C levels as well as slowing the lipid peroxidation process and enhancing antioxidant enzyme activity.
Basidiomycete laccases are being exploited because of their oxidizing abilities and stability. The production of recomnbinant laccases for industrial application is limited because (between other reasons) of unpredictable changes during laccase production in heterologous hosts.
The characterization of Phanerochaete flavido-alba laccase as a member of a new subfamily of multicopper oxidases add new interest to the applied and basic research on laccases. P. flavido-alba ( a basidiomycete) laccase production seems to be regulated by a mechanism(s) more complex than the assumed as general for laccase production in basidiomycetes
As the best know ligninolytic fungi (P. chrysosporium), P. flavido alba is ligninolytic. In addition is the only member of the Phanerochaete genus producing amounts of laccase for applied studies. Now, P. flavido-alba is revealed an excellent model to gain new knowledge in ligninolysis and environmental and industrial applications of these fungus.
The efficiency of a pure commercial laccase and a synthesized birnessite in the detoxification of OMW was evaluated. Experiments were performed on the whole OMW and on the two fractions deriving from the Soxhlet extraction procedure, used for the isolation and preparation of hydroytyrosol. This procedure produces a phenolic extract rich in lipophylic monomeric phenols and an exhausted fraction, rich of all the other OMW components including polymeric phenols, amino acids, proteins and carbohydrates. The phytotoxicity of samples before and after laccase action was assessed by germination test using L. esculentum (tomato) and L. sativum seeds. The bacterial toxicity was determined with B. megaterium. The results clearly indicate that a 48 h treatment with laccase was most efficient in reducing phenol content on crude OMW than on its phenolic extract. A corresponding decrease of phytotoxicity was observed which was much more evident when G.I./phenol index was considered. By contrast no decrease of bacterial toxicity was observed. These results seem to suggest that either some OMW components improve or enhance the activity of laccase or that some phenols, probably present in the phenolic extract at higher concentrations inhibit laccase activity and than other OMW components rather than monomeric phenols are responsible of OMW toxicity.
To deeply understand these behaviour further experiments were performed on the catalytic action of laccase on single phenolic compounds tested alone or in combination of two, three or more phenolic mixtures. According to the obtained results, the phenols were classified in three classes: consistently transformed by laccase, recalcitrant to laccase action and showing UV-Vis spectra with either changed shapes or appearance of new absorbance peaks.
Further comparative experiments performed with three different laccases: a plant laccase from R. vernicifera, and two fungal laccases from C. polyzona and from T. versicolor indicated that the three enzymes had a different kinetic behaviour towards the substrates and were differently affected by their concentrations. Therefore, the choice of the most convenient enzymatic catalyst to apply for the transformation of a such complex phenolic mixture as OMW is a very difficult task, being the responses of the different phenols, presumably present in OMW, so differentiate to the different enzymatic catalysts.
Investigations carried out with birnessite showed a completely different behaviour as compared to laccase activity. The abiotic catalyst was more efficient than laccase in the phenol transformation reactions. Treated samples were also analysed about their antibacterial activity and the obtained results showed a complete disappearance of antibacterial activity after 2 h treatment. Previous experiments indicated that the OMW phytotoxicity was only partially reduced and not completely eliminated by the treatment with laccase or birnessite although the complete removal of monomeric phenols occurred.
The indication that other OMW components are responsible of their phytotoxicity may be drawn. Phytotoxicity tests, performed with crude OMW, OMW treated with two soils differing for their chemical and physical properties for 24 h (an incubation time sufficient to eliminate completely the presence of monomeric phenols detectable to HPLC analysis and corresponding to a decrease of total phenol content of about 30-50%) and OMW-soxhlet extract and its corresponding exhausted fraction indicated a lower toxicity for the OMW after their treatment with only one of the two soils.
However, when the data were elaborated in terms of GI/phenol concentration no significant differences were evident between crude OMW and treated-OMW, containing half content of phenolics. This seems to suggest that other OMW components are responsible of their toxicity or that the used test has a low sensitivity.
The results obtained with OMW and their extracts and exhausted fractions obtained with both tomato and Lepidium sativum seeds still indicated that no a substantial difference in phytotoxicity was evidenced between crude OMW and their fractions by germination tests performed on tomato seed. In contrast, a higher sensitivity of germination tests was observed with Lepidium sativum. Indeed with these seeds a significant difference of toxicity appeared between the extract and exhausted fractions, that was not visible with tomato seeds. Furthermore, it was evident that at the same phenol concentration the exhausted fraction exhibited a lower toxicity than the extract one and the effect was evident also when the GI were normalized by the phenols contained in the two fractions.
Laboratory experimentation has shown that OMW polyphenols can be removed massively by one-day treatment in aerated soil slurry. The removal is mainly due to an abiotic reaction promoted by iron and manganese oxides generally present in the clay fraction of agricultural soils. A modest contribution of biotic mineralization has also been found for less reactive phenols. Such treatment substantially reduces microbial and phyto-toxicity of OMW without destroying the organic load. The resulting wastewater can be used for fertirrigation of poor agricultural soil, by exploiting the organic and mineral content of OMW without the restraints imposed by the presence of polyphenols in non-treated wastewaters. Scale-up of the treatment to the production rate of a small olive-mill and field application of treated OMW are subject of future research.
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