Istituto Sperimentale per la Zootecnia

In this study cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two cutinases from T. fusca KW3. The three isolated Thermobifida cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their kcat and Km values on pNP–acetate were in the ranges 2.4–211.9 s–1 and 127–200 μM while on pNP–butyrate they showed kcat and Km values between 5.3 and 195.1 s–1 and between 1483 and 2133 μM. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water contact angle (WCA) decreases. FTIR-ATR analysis revealed an increase in the crystallinity index A1340/A1410 upon enzyme treatment and an increase of the amount of carboxylic and hydroxylic was measured using derivatization with 2-(bromomethyl)naphthalene. Modeling the covalently bound tetrahedral intermediate consisting of cutinase and 3PET indicated that the active site His-209 is in the proximity of the O of the substrate thus allowing hydrolysis. On the other hand, the models indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.

The harmonization of rabbit carcass criteria is a result of an international work. lts task was to specify the main traits to be considerad from the birth of the animal to carcass analysis, to define these traits with enough accuracy and to propose a common terminology. Work on harmonization has been initialized by the Mediterranean Rabbit Group Conference (BLASCO, OUHAYOUN and MASOERO, 1992), completad by several research teams and, finally, discussed during a round table of the Vth World Rabbit Congress (Corvallis, 1992). The proposal described in this work concerns: 1- growth, consumption and breeding measurments, preslaughter handling and slaughter processing, 2- dressing percentage analysis, 3- measurments or prediction of commercial and reference carcass composition. The work represents an official document of the World Rabbit Science Association. A commission has been created to examine the efficiency of the proposed criteria and to modify them according to what the scientific development and the practica! experience or thei use would recommend.

Abstract The in vitro biodegradation of Bombyx mori silk fibroin was studied by incubating fibers and films with proteolytic enzymes (collagenase type F, α‐chymotrypsin type I‐S, protease type XXI), for times ranging from 1 to 17 days. The changes in sample weight and degree of polymerization of silk fibers exposed to proteolytic attack were negligible. However, tensile properties were significantly affected, as shown by the drop of strength and elongation as a function of the degradation time. Upon incubation with proteolytic enzymes, silk films exhibited a noticeable decrease of sample weight and degree of polymerization, the extent of which depended on the type of enzyme, on the enzyme‐to‐substrate ratio, and on the degradation time. Protease was more aggressive than α‐chymotrypsin or collagenase. Film fragments resistant to enzymatic degradation were enriched in glycine and alanine. FT‐IR measurements showed that the degree of crystallinity of biodegraded films increased. Soluble degradation products of silk films consisted of a range of peptides widely differing in size, deriving from the amorphous sequences of the silk fibroin chains. Biodegraded fibers showed an increase of surface roughness, while films displayed surface cracks and cavities with internal voids separated by fiber‐like elements. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 91: 2383–2390, 2004

ABSTRACT Static magnetic fields with intensities starting from 6 gauss (6×10 −4 tesla, T) were found to decrease in an intensity‐dependent fashion, reaching a plateau at 6 × 10 −3 T, the extent of cell death by apoptosis induced by several agents in different human cell systems. This is not due to a change in the mode of cell death (i.e., to necrosis) or to a delay of the process itself; rather, the presence of magnetic fields allows the indefinite survival and replication of the cells hit by apoptogenic agents. The protective effect was found to be mediated by the ability of the fields to enhance Ca 2+ influx from the extracellular medium; accordingly, it was limited to those cell systems where Ca 2+ influx was shown to have an antiapoptotic effect. Magnetic fields thus might interfere with human health by altering/restoring the equilibrium between cell death and proliferation; indeed, the rescue of damaged cells may be the mechanism explaining why magnetic fields that are not mutagenic per se are often able to increase mutation and tumor frequencies.—Fanelli, C., Coppola, S., Barone, R., Colussi, C., Gualandi, G., Volpe, P., Ghibelli, L. Magnetic fields increase cell survival by inhibiting apoptosis via modulation of Ca 2+ influx. FASEB J. 13, 95–102 (1999)

Abstract Structural changes of native and regenerated silk fibroin membranes were induced by immersion in water‐methanol solutions and examined as a function of immersion time and methanol concentration. X‐ray diffractometry and infrared spectroscopy data showed that transition from random coil to β‐sheet structure occurred favorably when both native and regenerated silk fibroin membranes were immersed in water‐methanol solutions, regardless of the different immersion time. Only native silk membrane, treated for 2 min with pure methanol, maintained its original amorphous structure, as demonstrated by differential scanning calorimetric (DSC) curves. The degree of displacement, measured by thermomechanical analysis (TMA), was much greater for regenerated than for native silk fibroin membranes. SDS‐PAGE pattern showed that native silk fibroin has a molecular weight of 350, while the regenerated sample is formed by a large number of polypeptides in the range of 200‐50 KD. The amount of acidic and basic amino acids decreased slightly in regenerated silk fibroin. Physical properties of silk membranes treated with water‐methanol solutions are discussed in terms of membrane structure, treatment conditions, and chemical structure of starting material. © 1994 John Wiley & Sons, Inc.

We studied the effect of salinity on amino acid, proline and glycine betaine accumulation in leaves of different stages of development in durum wheat under high and low nitrogen supply. Our results suggest that protective compounds against salt stress are accumulated in all leaves. The major metabolites are glycine betaine, which preferentially accumulates in younger tissues, and proline, which is found predominantly in older tissues. Proline tended to accumulate early, at the onset of the stress, while glycine betaine accumulation was observed during prolonged stress. Nitrate reductase (NR) and glutamate synthase (GOGAT) are positively correlated with these compatible solutes: proline is associated with NR in the oldest leaves of high-nitrate plants and glycine betaine is associated with GOGAT in the youngest leaves of both low- and high-nitrate plants. In high-nitrate conditions proline accounts for more than 39% of the osmotic adjustment in the cytoplasmic compartments of old leaves. Its nitrogen-dependent accumulation may offer an important advantage in that it can be metabolised to allow reallocation of energy, carbon and nitrogen from the older leaves to the younger tissues. The contribution of glycine betaine is higher in young leaves and is independent of nitrogen nutrition.

From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p-nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET-hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2-hydroxyethyl benzoate (HEB), and mono-(2-hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET-hydrolase was isolated from non-denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC-MS/MS analysis. BsEstB was expressed in Escherichia coli with C-terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p-nitrophenyl acetate and 108 U/mg on p-nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half-life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2-hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The kcat values decreased with increasing complexity of the substrate from 6 and 8 (s-1) for p-nitrophenyl-acetate (4NPA) and p-nitrophenyl-butyrate (4NPB), respectively, to 0.14 (s-1) for bis(2-hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water-contact angle (WCA) from 68.2°±1.7° to 62.6°±1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2-(bromomethyl)naphthalene.

Abstract The olive fruit is essentially constituted by the epicarp, or skin, mesocarp, or pulp, and the endocarp, or stone, and the latter includes the seed. Table olives (also called eating olives) are prepared from healthy, specifically cultivated, olive varieties picked at the right maturation stage and whose quality, after appropriate processing, corresponds to that of an edible well preserved product. The most important industrial preparations of table olives are the Spanish (or Sevillian) for green olives, the Californian for oxidised black olives, and the Greek for naturally black olives. In the Spanish and Californian procedures, olives are treated with a diluted aqueous NaOH solution, that brings about several changes in the susceptible classes of compounds in the fruit. Note, however, that the composition of the triglycerides remain unaffected by these procedures. After the treatment the olives are rinsed to remove the alkali, and the fruit is then left to ferment in brine for several months. The production of naturally black olives in brine, according to the Greek traditional method, is a simple, natural process which does not use chemicals. The main phenols in the olive are oleuropein, hydroxytyrosol (β(3‐4‐dihydroxyphenyl)ethanol), tyrosol (β(4‐hydroxyphenyl) ethanol) and verbascoside; the major polycyclic triterpenes are the oleanolic and maslinic acids. The NaOH treatment hydrolysed oleuropein into β(3‐4‐dihydroxyphenyl) ethanol, oleoside 11‐methylester and oleoside. These last glucosides and the oleuropein itself, in acidic medium, yielded glucose and a number of compounds called, collectively, oleuropein aglycones. During the fermentation process phenols diffuse from the pulp into the brine. Also, even during the alkaline treatment, there is the rapid diffusion of the pentacyclic oleanolic and maslinic acids salts into the brine.

A 6-wk trial was performed with 40 late-lactation Comisana ewes, which were either exposed to or protected from solar radiation and fed either in the morning (EXPM, PROM) or afternoon (EXPA, PROA) during summer in a Mediterranean climate. Behavioral traits of ewes were recorded once per week from 0800 to 2000 h. Rectal temperature (RT) and respiration rate (RR) were measured twice weekly at 1430 h. The phytohemagglutinin (PHA) skin test was performed to induce nonspecific delayed-type hypersensitivity at d 10, 20, and 32 of the experiment. Jugular blood samples were taken from ewes at the beginning and at d 21 and 42 of the experiment. Ewe milk yield was recorded daily. Individual milk samples were analyzed weekly for milk composition, coagulating properties, somatic cell count (SCC) and polymorphonuclear neutrophil leukocyte counts (PMNLC) and every 2 wk for bacteriological characteristics. Solar radiation and the interaction of solar radiation x time of feeding had significant effects on rectal temperatures. EXPM ewes had higher rectal temperatures than EXPA ewes, which in turn exhibited higher RT compared with PROM and PROA ewes. EXP groups also had significantly higher respiration rates than PRO groups. Immune response was lower in EXPM ewes at d 10 and in EXPM, EXPA, and PROM animals at d 20 compared with PROA ewes. Exposure to solar radiation resulted in decreased plasma concentrations of alanine amino-transferase, alkaline phosphatase, potassium, and magnesium, as well as in increased levels of nonesterified fatty acids and aspartate amino-transferase. Milk yield and composition were not changed by exposure to solar radiation and time of feeding, but the EXPM treatment resulted in lower yields of casein and fat and reduced clot firmness compared with the three other treatments. Milk SCC was similar across treatments, but PMNLC was higher in EXPM than in PROM and PROA milk. EXPM animals also had the greatest amounts of total and fecal coliforms and of Pseudomonadaceae as well as the highest number of mastitis related pathogens in their milk. Results suggest that provision of shaded areas can play a major role in helping lactating ewes to minimize the adverse effects of high ambient temperatures on thermal balance and energy and mineral metabolism. Changing the time of feeding to late afternoon may be beneficial to exposed ewes in lowering their heat loads during the warmest hours of the day, thereby reducing the detrimental impact of thermal stress on immune function and udder health.

To understand mechanisms of osmoprotection, the composition of sugars and related compounds were analyzed in extracts of fully hydrated and desiccated leaves of the desiccation‐tolerant resurrection plant Myrothamnus flabellifolia . During the dehydration process the concentrations of fructose and glucose decrease, whereas sucrose, arbutin and glucopyranosyl‐β‐glycerol increase. The substances were identified by GC‐MS and NMR‐analyses. This is the first report of large amounts of glucopyranosyl‐β‐glycerol in higher plants which may act as an osmoprotectant. Significant levels of the nonreducing sugar trehalose were present in all samples tested.

ABSTRACT Pasta yellowness is affected by different factors, the most important of which are intrinsic to the quality of semolina (natural carotenoid pigments, protein, ash, and lipoxygenase [LOX] activity) and processing conditions. Because all the parameters involved in pasta color are under the control of varietal and environmental factors, the role of the genotype, environment, and the interaction between genotype and environment on color expression were studied. Although the analysis of variance showed the genotype‐by‐environment interaction to be significant, a nonorthogonal analysis attributed a higher weight to genotype on parameters directly involved in color expression: β‐carotene content, yellow index, and LOX activity. Furthermore, the loss of pigments and yellow index after milling and processing was evaluated and correlated with all the parameters involved in the determination of final pasta color. The phase mainly responsible for pigment loss was pasta processing. A decrease of 16.3% in semolina β‐carotene content during pasta processing versus a 7.9% loss during milling was determined. The isoenzymatic forms LOX‐2 and LOX‐3, active at the pH of dough, were responsible for the loss of color in pasta products. Simple correlations and the linear multiple regression corroborated this finding. Hydroperoxidation activity at pH 6.6, bleaching activity, and ash content were responsible for 87% ( R 2 adjusted) of total variance, with each variable accounting for 57, 61, and 22% of the variation, respectively. This confirms that LOX activity is the main factor involved in the loss of color, while a secondary and lesser role can be seen for ash content. Therefore, a high pigment content, located in the interior of the whole grain, and a lower LOX activity in semolina must be the selection characteristics by which breeding programs obtain a bright yellow pasta.

Future climate changes are expected to increase risks of drought, which already represent the most common stress factor for stable barley ( Hordeum vulgare L.) production in Mediterranean areas. It is important, therefore, to evaluate if there are needs of specific adaptive measures in selecting genotypes for these more stressful environments. Our objective was to study diversity of yield performance under rainfed (R) and irrigated (I) conditions in 89 barley genotypes of different origin, growth habit, and year of release, representing a sample of cultivars grown in Europe. The experiment was conducted at Foggia (southern Italy) for 3 yr. For each trial, a water stress index (WSI) was calculated on the basis of the daily potential and actual evapotranspiration in the growing season, estimated by Thornthwaite's method. The WSI explained most of the variation in yield ( R 2 = 0.89**) among years and treatments. We examined, using the yield vs. WSI regression, the behavior of a given genotype across trials. The intercept and slope values were used as measures of yield potential and adaptability under drought, respectively. Several cultivars showing high yield potential and minimal genotype × environment (GE) interaction were identified. Notably, they were characterized by a high slope of the yield vs. WSI regression. Furthermore, within the range of water stress here examined, high yield potential played a preeminent role in the performance of these barley genotypes. This explains why, in this specific context, a selection based on minimum yield decrease under stress with respect to favorable conditions failed to identify the best genotypes.

Near-infrared (NIR) spectroscopy is a practical spectroscopic procedure for the detection of organic compounds in matter. It is particularly useful because of its nondestructiveness, accuracy, rapid response, and easy operation. This work assesses the applicability of NIR for the rapid identification of micotoxigenic fungi and their toxic metabolites produced in naturally and artificially contaminated products. Two hundred and eighty maize samples were collected both from naturally contaminated maize crops grown in 16 areas in north-central Italy and from ears artificially inoculated with Fusarium verticillioides. All samples were analyzed for fungi infection, ergosterol, and fumonisin B1 content. The results obtained indicated that NIR could accurately predict the incidence of kernels infected by fungi, and by F. verticillioides in particular, as well as the quantity of ergosterol and fumonisin B1 in the meal. The statistics of the calibration and of the cross-validation for mold infection and for ergosterol and fumonisin B1 contents were significant. The best predictive ability for the percentage of global fungal infection and F. verticillioides was obtained using a calibration model utilizing maize kernels (r2 = 0.75 and SECV = 7.43) and maize meals (r2 = 0.79 and SECV = 10.95), respectively. This predictive performance was confirmed by the scatter plot of measured F. verticillioides infection versus NIR-predicted values in maize kernel samples (r2 = 0.80). The NIR methodology can be applied for monitoring mold contamination in postharvest maize, in particular F. verticilliodes and fumonisin presence, to distinguish contaminated lots from clean ones, and to avoid cross-contamination with other material during storage and may become a powerful tool for monitoring the safety of the food supply.

The authentication of extra virgin olive oil and its adulteration with lower-priced oils are serious problems in the olive oil industry. In addition to the obvious effect on producer profits, adulteration can also cause severe health and safety problems. A number of techniques, including chromatographic and spectroscopic methods, have recently been employed to assess the purity of olive oils. In this study Raman spectroscopy together with multivariate and evolutionary computational-based methods have been employed to assess the ability of Raman spectroscopy to discriminate between chemically very closely related oils. Additionally, the levels of hazelnut oils used to adulterate extra virgin olive oil were successfully quantified using partial least squares and genetic programming. Keywords: Raman spectroscopy; olive oil; hazelnut oil; adulteration; quantification; principal component analysis; partial least-squares regression; genetic programming

The purpose of this study was to investigate the changes induced by a lypolytic enzyme on the surface properties of polyethylene terephthalate (PET). Changes in surface hydrophilicity were monitored by means of water contact angle (WCA) measurements. Fourier Transform Infrared spectroscopy (FTIR) in the Attenuated Total Reflectance mode (ATR) was used to investigate the structural and conformational changes of the ethylene glycol and benzene moieties of PET. Amorphous and crystalline PET membranes were used as substrate. The lipolytic enzyme displayed higher hydrolytic activity towards the amorphous PET substrate, as demonstrated by the decrease of the WCA values. Minor changes were observed on the crystalline PET membrane. The effect of enzyme adhesion was addressed by applying a protease after-treatment which was able to remove the residual enzyme protein adhering to the surface of PET, as demonstrated by the behavior of WCA values. Significant spectral changes were observed by FTIR-ATR analysis in the spectral regions characteristic of the crystalline and amorphous PET domains. The intensity of the crystalline marker bands increased while that of the amorphous ones decreased. Accordingly, the crystallinity indexes calculated as band intensity ratios (1,341/1,410 cm(-1) and 1,120/1,100 cm(-1)) increased. Finally, the free carboxyl groups formed at the surface of PET by enzyme hydrolysis were esterified with a fluorescent alkyl bromide, 2-(bromomethyl)naphthalene (BrNP). WCA measurements confirmed that the reaction proceeded effectively. The fluorescence results indicate that the enzymatically treated PET films are more reactive towards BrNP. FTIR analysis showed that the surface of BrNP-modified PET acquired a more crystalline character.

Fruits from seven different varieties of Olea europaea L., grown in the same environmental conditions, were harvested in two succeeding years at the same ripening degree and immediately processed. The oils obtained were submitted to gas chromatographic determination of the volatile compounds extracted by dynamic headspace technique. The results demonstrated that the accumulation of the different metabolites in the oils obtained from the various cultivars were strictly connected with the varietal parameter because of the enzyme differences genetically determined. This feature made possible the differentiation of the examined cultivars on the basis of the percent of each metabolite from the enzymatic transformation of 13-hydroperoxides of linolenic acid. Oils from Picual and Koroneiki varieties cultivated in Spain and Greece, respectively, showed contents of volatiles very similar to those detected in the oils of the same varieties cultivated in Italy, proving that they were not significantly influenced by the environmental conditions.

A new cutinase from Thermobifida alba (Tha_Cut1) was cloned and characterized for polyethylene terephthalate (PET) hydrolysis. Tha_Cut1 showed a high degree of identity to a T. cellulolysitica cutinase with only four amino acid differences outside the active site area, according to modeling data. Yet, Tha_Cut1 was more active in terms of PET surface hydrolysis leading to considerable improvement in hydrophilicity quantified based on a decrease of the water contact angle from 87.7° to 45.0°. The introduction of new carboxyl groups was confirmed and measured after esterification with the fluorescent reagent alkyl bromide, 2-(bromomethyl) naphthalene (BrNP), resulting in a fluorescence emission intensity increase from 980 to 1420 a.u. On the soluble model substrates p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB), the cutinase showed Km values of 213 and 1933 μM and kcat values of 2.72 and 6.03 s−1 respectively. The substrate specificity was investigated with bis(benzoyloxyethyl)terephthalate (3PET) and Tha_Cut1 was shown to release primarily 2-hydroxyethyl benzoate. This contrasts with the well-studied Humicula insolens cutinase which preferentially liberates terminal benzoic acid from 3PET.

Abstract This study focuses on the conformational characterization of differently processed Bombyx mori silk fibroin samples by Raman spectroscopy. The Raman spectra of silk fibroin film and liquid silk are discussed in comparison with those of the crystalline fractions of Bombyx mori silk fibroin (Cp, chymotryptic precipitate) with Silk I (Silk I‐Cp) and Silk II (Silk II‐Cp) structures. The complete 1800–200 cm −1 Raman spectrum of Silk I‐Cp is reported for the first time. The amide I and amide III modes were found to be scarcely suitable for the spectroscopic characterization of silk fibroin in the Silk I form in the presence of a random coil conformation. Raman marker bands for the Silk I form were identified in other spectral ranges at about 1415, 950, 930, 865, 260 and 230 cm −1 . On the basis of the above findings, the comparison of the Raman spectra of film, liquid silk and Silk I‐Cp in the range 1000–800 cm −1 clearly indicates that in addition to random coil, both film and liquid silk contain local domains of Silk I structure; their amount is higher in liquid silk, as indicated by the relative intensity of the bands at about 950, 930 and 865 cm −1 and by the I 1415 / I 1455 intensity ratio. The assignments of the bands at about 1275 and 1107 cm −1 are also discussed. These bands were previously assigned to the presence of α‐helical conformation in Bombyx mori silk but, from the results reported, they should rather be attributed to the Silk I form. Copyright © 2001 John Wiley & Sons, Ltd.