Valorization of date palm (Phoenix dactylifera L.) pruning biomass by co-composting with urban and agri-food sludge.

In the Mediterranean countries, there is an increasing production of date palm wastes (Phoenix dactylifera L.), not only due to the raising production of date palm fruits, but also derived from the maintenance of urban and peri-urban green areas, especially in those affected by red palm weevil (Rhynchophorus ferrugineus). The management of this increasing volume of green wastes usually concludes with a controlled disposal that implies an important loss of resources, in terms of organic matter, nutrients and energy. In addition, the rise of wastewater generation and the incentive of the wastewater treatment processes have derived in an increase of the amount of the sludge produced, which makes difficult its management. This work studies the feasibility of co-composting palm wastes with sludge from the urban and agri-food sectors as alternative treatment to manage these organic waste streams and to obtain added-value compost. For this, four mixtures (P1, P2, P3 and P4) were prepared using as main component palm leave waste (PL) mixed with different types of sludge. In the piles P1, P2 and P3, sewage sludge (SS) was used as co-composting agent, while agri-food sludge (AS) was used in P4. Throughout composting, the thermal profile of the composting piles was assessed, as were physical, chemical, physico-chemical and maturity parameters. In addition, the changes in water-soluble organic matter were assessed using chemical analytical methods and the excitation-emission matrix (EEM) fluorescence spectroscopy. The results obtained showed the viability of the co-composting process to obtain end-products with adequate maturity degree and physical characteristics for their potential use as substrates, except for the salt contents that can limit their use in some agricultural sectors.

Co-composting of distillery wastes with animal manures: carbon and nitrogen transformations in the evaluation of compost stability.

The aim of this work was to study the viability of recycling the solid wastes generated by the winery and distillery industry by means of co-composting with animal manures, as well as to evaluate the quality of the composts obtained. Two piles, using exhausted grape marc and cattle manure or poultry manure, respectively (at ratios, on a fresh weight basis, of 70:30), were composted by the Rutgers static pile composting system. Throughout the composting process, a number of parameters were monitored, such as pH, electrical conductivity, organic matter, water-soluble carbon, water-soluble polyphenols, different forms of nitrogen (organic nitrogen, ammonium and nitrate) and humification indices (humification ratio, humification index, percentage of humic acid-like C, polymerisation ratio and cation exchange capacity), as well as the germination index. Organic matter losses followed first-order kinetics equation in both piles, the highest organic matter mineralisation rate being observed with exhausted grape marc and cow manure. On the other hand, the mixture with the lowest C/N ratio, using exhausted grape marc and poultry manure, showed the highest initial ammonium contents, probably due to the higher and more labile N content of poultry manure. The increase in the cation exchange capacity revealed the organic matter humification during composting. In contrast, other humification parameters, such as the humification ratio and the humification index, did not show the expected evolution and, thus, could not be used to assess compost maturity. Composting produced a degradation of the phytotoxic compounds, such as polyphenols, to give composts without a phytotoxic character. Therefore, composting can be considered as an efficient treatment to recycle this type of wastes, due to composts presented a stable and humified organic matter and without phytotoxic effects, which makes them suitable for their agronomic use.

Salinity, organic content, micronutrients and heavy metals in pig slurries from South-eastern Spain.

The increase in commercial pig production is an opportunity to reuse animal manures in arid and semiarid soils as a source of nutrients and organic matter. However, there are components in pig slurry that are potentially dangerous for the environment. In this study, pig slurries of 36 pig farms in South-eastern Spain were evaluated for salt content (electrical conductivity, chloride and sodium), organic load (BOD5 and COD), micronutrients (Fe, Cu, Mn and Zn), and heavy metals (Cd, Co, Cr, Ni and Pb). Except for electrical conductivity, Cu and Zn, components in pig slurries did not vary considerably between animal production stages, indicating similar management of diverse animal types. Assuming an application rate based on the maximum input of nitrogen from animal manure (210 kg total N/ha/yr), the estimates for soil annual load of Cl and Na, 415 kg/ha, could be a significant salinisation risk. Cu and Zn seemed to be the metals that could be accumulated most in soils where application of pig slurries is common (4 and 15 kg/ha/yr, respectively). The estimated heavy metal (Cd, Co, Cr, Ni and Pb) input to soils would be 260 g/ha/yr, with a relative contribution of Cr>Ni>Pb>Co>Cd.

Agrochemical characterisation of the solid by-products and residues from the winery and distillery industry.

The winery and distillery industry produces a great quantity of residues, whose management and disposal are environmental problems due to their seasonal character and some polluting characteristics. The main solid by-products and residues generated are grape stalk, grape pomace or marc, wine lee, exhausted grape marc and winery sludge. In this study, 87 samples of winery and distillery residues were collected from different Spanish wineries and distilleries. Electrical conductivity, pH, total organic matter, organic carbon, polyphenols and contents of plant nutrients and heavy metals were determined. The purpose of this research was to study the composition of these wastes and to find relationships in order to use easily analysable parameters to estimate their composition. In general, the winery and distillery residues showed low pH (mean values ranged from 3.8 to 6.8) and electrical conductivity values (1.62-6.15 dS m(-1)) and high organic matter (669-920 g kg(-1)) and macronutrient contents, especially in K (11.9-72.8 g kg(-1)). However, a notable polyphenol concentration (1.2-19.0 g kg(-1)) and low micronutrient and heavy metal contents were also observed, some of these properties being incompatible with agricultural requirements. Therefore, conditioning treatments are necessary prior to possible use of these wastes. In all wastes, significant correlations were found between easily determined parameters, such as pH, electrical conductivity and total organic matter, and most of the parameters studied. The regression models obtained are also discussed.

Composting of the invasive species Arundo donax with sewage and agri-food sludge: Agronomic, economic and environmental aspects.

This work evaluates several co-composting scenarios based on the use of Arundo donax biomass (AD) as bulking agent for the co-composting of sewage sludge (MS) and agri-food sludge (AS), to manage these organic wastes and to produce balanced organic fertilizers by optimizing the process. For this, six piles were prepared in commercial composting conditions, using AD in a range of 40%-80% (on a dry weight basis). Physico-chemical and chemical parameters and the thermal behaviour were evaluated during the process, as were the physical and chemical parameters of the final composts. The proportion of AD in the mixtures has a significant effect on the development of the thermophilic stage of composting, showing the piles with higher proportion of AD a quicker organic matter degradation. In addition, the evolution of the thermal indices R1 and R2 was different depending on the origin of the sludge used, indicating an increase in the relative concentration of more recalcitrant materials in the piles prepared with AS. The estimation of the global warming potential showed that the use of higher proportion of AD in the composting mixture may be a strategy to mitigate the emission of greenhouse gases during the composting process. Moreover, the end-products obtained had an additional marketable value, with a balanced nutrient content and a good degree of maturity, which indicates the viability of the composting process as a method for the stabilization of these organic wastes.

Short-term carbon and nitrogen mineralisation in soil amended with winery and distillery organic wastes.

The aim of this work was to study the influence of the organic wastes derived from the winery and distillery industry (grape stalk (GS), grape marc (GM), wine lees (WL) and exhausted grape marc (EG)) and the soil type (clayey-loam (S1), loam (S2) and sandy textured (S3)) on different soil characteristics, especially the carbon and nitrogen mineralisation. The evolution of C mineralisation fitted a first-order kinetic for all amended soils. An initial increase was observed in the specific respiration (qCO(2)) at the beginning of the experiment. However, afterwards, the evolution in the qCO(2) was to tend towards the values of the control soil due to the pattern of the soil to recover its initial equilibrium status. The addition of these materials in the soils produced a slight increase of the inorganic nitrogen content, except in the case of GS and EG in most of the studied soils. The use of GS as amendment produced an inhibition in the N mineralisation in the three types of soils studied. Organic matter mineralisation was probably influenced by soil type, the sandy soil favouring more the N and C mineralisation processes than the clayey-loam and loam soils.

Co-composting of distillery and winery wastes with sewage sludge.

The winery and distillery wastes (grape stalk and marc (GS and GM, respectively), wine lees (WL) and exhausted grape marc (EGM)) are produced in great amounts in the Mediterranean countries, where their treatment and disposal are becoming an important environmental problem, mainly due to their seasonal character and some characteristics that make their management difficult and which are not optimised yet. Composting is a treatment widely used for organic wastes, which could be a feasible option to treat and recycle the winery and distillery wastes. In this experiment, two different piles (pile 1 and 2) were prepared with mixtures of GS, GM, EG and sewage sludge (SS) and composted in a pilot plant by the Rutgers static pile composting system. Initially, GS, GM and EGM were mixed, the pile 1 being watered with fresh collected vinasse (V). After 17 days, SS was added to both piles as a nitrogen and microorganisms source. During composting, the evolution of temperature, pH, electrical conductivity, total organic C, total N, humic acid-like C and fulvic acid-like C contents, C/N ratio, cation exchange capacity and germination index of the mixtures were studied. The addition of V in pile 1 produced higher values of temperature, a greater degradation of the total organic C, higher electrical conductivity values and similar pH values and total N contents than in pile 2. The addition of this effluent also increased the cation exchange capacity and produced a longer persistence of phytotoxicity. However, both piles showed a stabilised organic matter and a reduction of the phytotoxicity at the end of the composting process.

Development of organic fertilizers from food market waste and urban gardening by composting in Ecuador.

Currently, the management of urban waste streams in developing countries is not optimized yet, and in many cases these wastes are disposed untreated in open dumps. This fact causes serious environmental and health problems due to the presence of contaminants and pathogens. Frequently, the use of specific low-cost strategies reduces the total amount of wastes. These strategies are mainly associated to the identification, separate collection and composting of specific organic waste streams, such as vegetable and fruit refuses from food markets and urban gardening activities. Concretely, in the Chimborazo Region (Ecuador), more than 80% of municipal solid waste is dumped into environment due to the lack of an efficient waste management strategy. Therefore, the aim of this study was to develop a demonstration project at field scale in this region to evaluate the feasibility of implanting the composting technology not only for the management of the organic waste fluxes from food market and gardening activities to be scaled-up in other developing regions, but also to obtain an end-product with a commercial value as organic fertilizer. Three co-composting mixtures were prepared using market wastes mixed with pruning of trees and ornamental palms as bulking agents. Two piles were created using different proportions of market waste and prunings of trees and ornamental palms: pile 1 (50:33:17) with a C/N ratio 25; pile 2: (60:30:10) with C/N ratio 24 and pile 3 (75:0:25) with C/N ratio 33), prepared with market waste and prunings of ornamental palm. Throughout the process, the temperature of the mixtures was monitored and organic matter evolution was determined using thermogravimetric and chemical techniques. Additionally, physico-chemical, chemical and agronomic parameters were determined to evaluate compost quality. The results obtained indicated that all the piles showed a suitable development of the composting process, with a significant organic matter decomposition, reached in a shorter period of time in pile 3. At the end of the process, all the composts showed absence of phytotoxicity and suitable agronomic properties for their use as organic fertilizers. This reflects the viability of the proposed alternative to be scaled-up in developing areas, not only to manage and recycle urban waste fluxes, but also to obtain organic fertilizers, including added value in economic terms related to nutrient contents.

Use of composted sewage sludge in growth media for broccoli.

In this study, the use of composted sewage sludge (CSS) as a binary component with peat (P) in growth media for a horticultural crop, broccoli (Brassica oleracea var. Botryti cv. Marathon), was evaluated. Four treatments were established, based on the addition of increasing quantities of composted sewage sludge to peat (0%, 15%, 30% and 50%, v/v). Physical, physico-chemical and chemical analyses of the different mixtures of CSS and P were made. Plant growth, biomass production and macronutrient (N, P, K, Ca, Mg), micronutrient (Fe, Cu, Mn, Zn) and heavy metal (Pb, Ni, Cd, Cr) contents of plants were determined. The addition of CSS to P increased plant nutrient and heavy metal contents of plants and electrical conductivity (EC) and bulk density values of the substrates. The use of CSS did not affect the germination rate, even at 50% compost. For broccoli growth, the highest yield was obtained with the medium prepared by mixing the peat with 30% of compost; however, the mixture with the most sewage sludge compost (50%) had the greatest contents of macro and micronutrients.

Characterisation of the organic matter pool in manures.

In this research, different types of animal manure were evaluated with respect to organic matter (OM), total organic carbon (C(ot)), total N (N(t)), C(ot)/N(t) ratio, water-soluble organic carbon (C(w)), organic N (N(org)), carbohydrates, C(w)/N(org) ratio, humic acid-like carbon (C(ha)), fulvic acid-like carbon (C(fa)), humification index ((C(ha)/C(ot))x100) (HI) and the C(ha)/C(fa) and NH(4)(+)-N/NO(3)(-)-N ratios. In comparison with the limits set by the Spanish legislation for organic fertilisers, most of the manures had high OM contents, moderate N(org) concentrations (except in the case of the chicken and pig manures where this parameter was high) and C(ot)/N(t) ratios above the value stated in the legislation. The study of the different fractions of organic matter showed that the horse, pig and rabbit manures had the greatest content of C(ot). However, the fraction of easily-biodegradable organic compounds (C(w)) was significantly higher in the horse, goat and chicken manures. The study also showed that, in most cases, the percentage of fulvic acid-like C was greater than that of the humic acid-like C, indicating that the organic matter of these wastes is not completely humified. Values of HI ((C(ha)/C(ot))x100) and C(ha)/C(fa) ratio in the studied manures were not significantly different. Regarding the parameters related to the organic matter stability such as C(w), carbohydrates and the C(ot)/N(t), C(w)/N(org) and NH(4)(+)-N/NO(3)(-)-N ratios, it has been determined that the organic matter of these materials was not completely stabilised. The heterogeneity in OM composition of the studied manures did not allow the formulation of simple equations for evaluation of the composition of these wastes from easily-determined parameters.

Co phytoavailability for tomato in amended calcareous soils.

A plot study was conducted to assess changes in Co phytoavailability for a tomato cultivar grown on an agricultural soil (a Calcic Petrocalcid) amended with sewage sludge, under controlled conditions in South-eastern Spain. The experiment consisted of three main treatment blocks: (A) without organic fertilisation, (B) with addition of 60 tha(-1) and (C) 120 tha(-1) of sewage sludge. For each block (A, B, and C), four levels of Co (0, 50, 100 and 200 mgkg(-1)) were added, as CoCl2. Diethylenetriaminepentaacetic acid, DTPA (0.005 M plus triethanolamine), ammonium acetate (1 N at pH 7), and water extractable fractions of the soils were analysed for all the plots. The time dependent Co accumulation in different parts (roots, stems, leaves, and fruits) of the tomato plants was studied. Soil Co seemed to be mainly in non-available forms, according to the low concentrations found in the water and ammonium acetate extracts, compared to DTPA. The gradient of Co accumulation in tomato plants was root > leaf > stem + branches > fruit, with a concentration in the edible parts ranging between 4 and 25 mg kg(-1). The organic amendment enhanced the plant extraction of Co, this effect being more significant with time. Plant extraction efficiency decreased with increasing Co concentration in the soils. Co in fruit showed the best correlation with all the Co extraction pools in the soil.

Estimation of nutrient values of pig slurries in Southeast Spain using easily determined properties.

The contents of available nutrients in pig slurries are not easy to quantify in situ without laboratory facilities, but chemical analyses using standard laboratory methods also take time and are costly and not practical for most farms. Thus, when animal slurries are applied to land, their fertiliser potential is often unknown. In addition, in the last years, the changes in the management of industrial piggeries has changed the nature of the pig slurries vg. decrease of the dry matter content, and consequently the methods and equations used for estimating the nutrient contents in these residues must be checked. In our study, slurry samples were collected from the storage tanks of 36 commercial farms in Southeast Spain. Samples were analysed for pH, electrical conductivity (EC), redox potential (RP), specific density (D), total solids (TS), sedimentable solids (SS), biological oxygen demand (BOD(5)), chemical oxygen demand (COD), total nitrogen (TKN), ammonium nitrogen (AN), organic nitrogen (ON), and total contents of phosphorus, potassium, calcium and magnesium. Relationships between major nutrient levels of pig slurries and a range of physical and chemical properties were investigated. We also analysed the variability of pig slurries according to the production stage. TKN, AN and K were closely related to EC. The P content in slurries was related more closely to solids-derived parameters such as D. The use of multiple properties to estimate nutrient contents in pig slurries, especially for AN and K, seemed unnecessary due to the limited improvement achieved with an additional property. Therefore, electrical conductivity seemed to be the most appropriate single, easily determined parameter for estimation of total and ammonium nitrogen and potassium in pig slurries, with more than 83% of the variance explained. P seemed to be the worst key nutrient for estimation using any easily determined parameter.

Uses of winery and distillery effluents in agriculture: characterisation of nutrient and hazardous components.

Twenty-one samples of winery and distillery effluents were collected from different Spanish winery and distillery industries. Electrical conductivity, pH, redox potential, density, organic charge (chemical oxygen demand, biological oxygen demand, total, volatile and suspended solids, oxidisable organic C and polyphenols) and contents of plant nutrients and heavy metals were analysed. The aim of this work was to study the composition of these effluents and to find relationships which would make it possible to use easily determined parameters to estimate their composition. The winery wastewater (WW) and vinasse (V) showed an acidic pH, a high organic load and notable polyphenol, macronutrient, micronutrient and heavy metal contents. Some of these properties are not compatible with agricultural requirements; therefore, conditioning treatment of these liquid wastes is necessary to produce a safe, stable and easily manageable end product. Generally, in both effluent types, significant correlations were found between easily analysable parameters, such as suspended, volatile and total solids, pH, electrical conductivity, density and redox potential, and most of the parameters studied. The linear regression equations obtained permitted an immediate characterisation of the WW and V samples using these parameters.

Chemical, Thermal and Spectroscopic Methods to Assess Biodegradation of Winery-Distillery Wastes during Composting.

The objective of this work was to study the co-composting process of wastes from the winery and distillery industry with animal manures, using the classical chemical methods traditionally used in composting studies together with advanced instrumental methods (thermal analysis, FT-IR and CPMAS 13C NMR techniques), to evaluate the development of the process and the quality of the end-products obtained. For this, three piles were elaborated by the turning composting system, using as raw materials winery-distillery wastes (grape marc and exhausted grape marc) and animal manures (cattle manure and poultry manure). The classical analytical methods showed a suitable development of the process in all the piles, but these techniques were ineffective to study the humification process during the composting of this type of materials. However, their combination with the advanced instrumental techniques clearly provided more information regarding the turnover of the organic matter pools during the composting process of these materials. Thermal analysis allowed to estimate the degradability of the remaining material and to assess qualitatively the rate of OM stabilization and recalcitrant C in the compost samples, based on the energy required to achieve the same mass losses. FT-IR spectra mainly showed variations between piles and time of sampling in the bands associated to complex organic compounds (mainly at 1420 and 1540 cm-1) and to nitrate and inorganic components (at 875 and 1384 cm-1, respectively), indicating composted material stability and maturity; while CPMAS 13C NMR provided semi-quantitatively partition of C compounds and structures during the process, being especially interesting their variation to evaluate the biotransformation of each C pool, especially in the comparison of recalcitrant C vs labile C pools, such as Alkyl /O-Alkyl ratio.

Tomatinase from Fusarium oxysporum f. sp. lycopersici defines a new class of saponinases.

Plants produce a variety of secondary metabolites, many of which have antifungal activity. Saponins are plant glycosides that may provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici and other tomato pathogens produce extracellular enzymes known as tomatinases, which deglycosylate alpha-tomatine to yield less toxic derivatives. We have cloned and characterized the cDNA and genomic DNA encoding tomatinase from the vascular pathogen of tomato F. oxysporum f. sp. lycopersici. This gene encodes a protein (FoTom1) with no amino acid sequence homology to any previously described saponinase, including tomatinase from Septoria lycopersici. Although FoTom1 is related to family 10 glycosyl hydrolases, which include mainly xylanases, it has no detectable xylanase activity. We have overexpressed and purified the protein with a bacterial heterologous system. The purified enzyme is active and cleaves alpha-tomatine into the less toxic compounds tomatidine and lycotetraose. Tomatinase from F. oxysporum f. sp. lycopersici is encoded by a single gene whose expression is induced by alpha-tomatine. This expression is fully repressed in the presence of glucose, which is consistent with the presence of two putative CREA binding sites in the promoter region of the tomatinase gene. The tomatinase gene is expressed in planta in both roots and stems throughout the entire disease cycle of F. oxysporum f. sp. lycopersici.

Nitrogen mineralisation potential in calcareous soils amended with sewage sludge.

Mineralisation of organic N is an important consideration when determining the annual amount of sewage sludge to be applied to agricultural soils. The mineralisation of sludge organic N was studied in two different textured soils (clayey and sandy soil) treated with aerobic and anaerobic sludge at two different rates (30 and 50 g sludge kg(-1) soil). The mineralisation of sludge organic N was determined during 20 weeks incubation period by analysis of inorganic N produced by a non-leached procedure. Sludge organic N mineralisation was influenced by soil type, organic N mineralisation being greater in the sandy soil (from 30% to 41%) than in the clayey soil (from 13% to 24%). Mineralisation rates decreased rapidly the first two weeks, followed by a slower decrease with time. Although total mineralisation increased with sludge addition rate, net mineralisation decreased with sludge addition rate, probably due to denitrificaton losses. The aerobically treated sludge gave higher mineralisation rates than the anaerobically treated one. The values of N0 and k for treated soils varied depending on the type of sludge and soil.

The potential of near infrared reflectance spectroscopy (NIRS) for the estimation of agroindustrial compost quality.

Composting is an environmentally friendly alternative for the recycling of organic wastes and its use is increasing in recent years. An exhaustive monitoring of the composting process and of the final compost characteristics is necessary to certify that the values of compost characteristics are within the limits established by the legislation in order to obtain a safe and marketable product. The analysis of these parameters on each composting batch in the commercial composting plant is time-consuming and expensive. So, their estimation in the composting facilities based on the use of near infrared reflectance spectroscopy (NIRS) could be an interesting approach in order to monitor compost quality. In this study, more than 300 samples from 20 different composting procedures were used to calibrate and validate the NIRS estimation of compost properties (pH, electrical conductivity (EC), total organic matter (TOM), total organic carbon (TOC), total nitrogen (TN) and C/N ratio, macronutrient contents (N, P, K) and potentially pollutant element concentrations (Fe, Cu, Mn and Zn)). The composts used were elaborated using different organic wastes from agroindustrial activities (GS: grape stalk; EGM: exhausted grape marc; GM: grape marc; V: vinasse; CJW: citrus juice waste; Alpeorujo: olive-oil waste; AS: almond skin; EP: exhausted peat; TSW: tomato soup waste; SMS: spent mushroom substrate) co-composted with manures (CM: cattle manure; PM: poultry manure) or urban wastes (SS: sewage sludge) The estimation results showed that the NIRS technique needs to be fitted to each element and property, using specific spectrum transformations, in order to achieve an acceptable accuracy in the prediction. However, excellent prediction results were obtained for TOM and TOC, successful calibrations for pH, EC, Fe and Mn, and moderately successful estimations for TN, C/N ratio, P, K, Cu and Zn.

Pantothenate synthetase from Fusarium oxysporum f. sp. lycopersici is induced by alpha-tomatine.

The steroidal glycoalkaloid alpha-tomatine which is present in tomato (Lycopersicum sculentum) is assumed to protect the plant against phytopathogenic fungi. We have isolated a gene from the fungal pathogen Fusarium oxysporum f. sp. lycopersici that is induced by this glycoalkaloid. This gene, designated panC, encodes a predicted protein with a molecular mass of 41 kDa that shows a high degree of sequence similarity to pantothenate synthetases from yeast, plants and bacteria. Recombinant PanC protein from F. oxysporum has been over-expressed in Escherichia coli and purified to homogeneity. It shows pantothenate synthetase activity in the presence of D-pantoate, beta-alanine and ATP. The panC gene from F. oxysporum functionally complements an E. coli panC mutant, demonstrating that the PanC protein functions in vivo as a pantothenate synthetase. Southern analysis of F. oxysporum genomic DNA from other formae speciales indicates that there is a single copy of the pantothenate syntethase gene in this fungus. The presence of a STRE consensus sequence (CCCCT) in the promoter region of the gene suggests that the induction of panC may be part of a cellular stress response triggered by alpha-tomatine.

Purification and characterization of tomatinase from Fusarium oxysporum f. sp. lycopersici.

The antifungal compound alpha-tomatine, present in tomato plants, has been reported to provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici, a tomato pathogen, produces an extracellular enzyme inducible by alpha-tomatine. This enzyme, known as tomatinase, catalyzes the hydrolysis of alpha-tomatine into its nonfungitoxic forms, tomatidine and beta-lycotetraose. The maximal tomatinase activity in the fungal culture medium was observed after 48 h of incubation of germinated conidia at an alpha-tomatine concentration of 20 micrograms/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 35,000, and the enzyme was then purified to electrophoretic homogeneity by a procedure that includes preparative isoelectric focusing and preparative gel electrophoresis as main steps. The purification procedure had a yield of 18%, and the protein was purified about 40-fold. Tomatinase was found to be a monomer of 50 kDa by both native gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The analytical isoelectric focusing of the native tomatinase showed at least five isoforms with pIs ranging from 4.8 to 5.8. Treatment with N-glycosidase F gave a single protein band of 45 kDa, indicating that the 50-kDa protein was N glycosylated. Tomatinase activity was optimum at 45 to 50 degrees C and at pH 5.5 to 7. The enzyme was stable at acidic pH and temperatures below 50 degrees C. The enzyme had no apparent requirement for cofactors, although Co2+ and Mn2+ produced a slight stimulating effect on tomatinase activity. Kinetic experiments at 30 degrees C gave a K(m) of 1.1 mM for alpha-tomatine and a Vmax of 118 mumol/min/mg. An activation energy of 88 kJ/mol was calculated.

Effects of dietary fats (fish, olive and high-oleic-acid sunflower oils) on lipid composition and antioxidant enzymes in rat liver.

The effects of two oleic-acid-rich diets (containing olive oil, OO, and high-oleic-acid sunflower oil, HOSO) on plasma and liver lipid composition detoxification enzyme activities, were compared with those of a fish-oil (FO) diet and a control diet. Compared with the control diet, plasma and hepatic total triacylglycerol concentrations were increased in the animals fed on the HOSO and OO diets and decreased in those fed on the FO diet. The animals fed on FO showed the highest level of cholesterol in the liver and had lower plasma cholesterol concentrations when compared with those fed on the two oleic-acid-rich diets. In comparison with the animals fed on the diets enriched in oleic acid, the FO group showed higher hepatic levels of polyunsaturated fatty acids of the n-3 series and lower levels of fatty acids of the n-6 series. Livers of FO-fed rats, compared with those of OO- and HOSO-fed rats showed: (1) significantly higher activities of catalase (EC 1.11.1.6) glutathione peroxidase (EC 1.11.1.9) and Cu/Zn superoxide dismutase (EC 1.15.1.1); (2) no differences in the NADPH-cytochrome c reductase (EC 1.6.99.3) activity. The HOSO diet had a similar effect on liver antioxidant enzyme activities as the OO diet. In conclusion, it appears that changes in the liver fatty acid composition due mainly to n-3 lipids may enhance the efficiency of the antioxidant defence system. The two monounsaturated fatty acids oils studied (OO and HOSO), with the same high content of oleic acid but different contents of natural antioxidants, had similar effects on the antioxidant enzyme activities measured.