Chemical authentication of extra virgin olive oil varieties by supervised chemometric procedures.

This work has focused on discriminating extra virgin olive oils from Sabina (Lazio, Italy) by olive fruit variety (cultivar). A set of oils from five of the most widespread cultivars (Carboncella, Frantoio, Leccino, Moraiolo, and Pendolino) in this geographical area was analyzed for chemical composition using only the Official Analytical Methods, recognized for the quality control and commercial classification of this product. The obtained data set was converted into a computer-compatible format, and principal component analysis (PCA) and a method based on the Fisher F ratio were used to reduce the number of variables without a significant loss of chemical information. Then, to differentiate these samples, two supervised chemometric procedures were applied to process the experimental data: linear discriminant analysis (LDA) and artificial neural network (ANN) using the back-propagation algorithm. It was found that both of these techniques were able to generalize and correctly predict all of the samples in the test set. However, these results were obtained using 10 variables for LDA and 6 (the major fatty acid percentages, determined by a single gas chromatogram) for ANN, which, in this case, appears to provide a better prediction ability and a simpler chemical analysis. Finally, it is pointed out that, to achieve the correct authentication of all samples, the selected training set must be representative of the whole data set.     

Enzyme-mediated solvent extraction of carotenoids from marigold flower (Tagetes erecta)

Marigold flowers are the most important source of carotenoids for application in the food industry. However, the extraction gives almost 50% losses of the carotenoids depending on conditions for silaging, drying, and solvent extraction. In the past decades, macerating enzymes have been successfully applied to improve the extraction yield of valued compounds from natural products. In this work, an alternative extraction process for carotenoids is proposed, consisting of a simultaneous enzymatic treatment and solvent extraction. The proposed process employs milled fresh flowers directly as raw material, eliminating the inefficient silage and drying operations as well as the generation of hard to deal with aqueous effluents present in traditional processes. The process developed was tested at the 80 L scale, where under optimal conditions a carotenoid recovery yield of 97% was obtained.     

Effects of pig slurry application on soils and soil humic acids

The effect of three annually consecutive additions of pig slurry at two rates (90 and 150 m3 x ha(-1) x year(-1) on soils and soil humic acids (HAs) was investigated in a field experiment under semiarid conditions. Soils and pig slurries were analyzed by standard methods. The HAs were isolated from soils and pig slurry by a conventional procedure based on alkaline extraction, acidic precipitation to pH 1, purification by repeated alkaline dissolutions and acidic precipitations, water washing, dialysis, and final freeze-drying. The HAs obtained were analyzed for elemental (C, H, N, S, and O) and acidic functional group (carboxylic and phenolic) composition, and by UV-vis, FT-IR, fluorescence, and ESR spectroscopies. With respect to the control soil, the pig slurry amended soils had greater pH and electrical conductivity, slightly larger total N content, and smaller values of C/N ratio. A decrease of total organic C was observed only in soils amended for 2 and 3 years at the higher slurry rate. With respect to control soil HA, pig slurry HA was characterized by larger contents of S- and N-containing groups, smaller acidic functional group and organic free radical contents, a prevalent aliphatic character, extended molecular heterogeneity, and smaller aromatic polycondensation and humification degrees. Amendment with pig slurry HA determines a number of modifications in soil HAs, including increase of C, S, and COOH contents, C/N ratios, and aliphaticity and decrease of extraction yields and N, O, phenolic OH, and organic free radical contents. These effects are generally more evident after the first year of slurry application and tend to disappear with increasing number of treatments. Most probably, over the years the slightly humified slurry HA is mineralized through extended microbial oxidation, whereas only the most recalcitrant components, such as S-containing, phenolic, and aliphatic structures, are partially accumulated by incorporation into soil HA.     

Subcellular localization and isoenzyme pattern of peroxidase and polyphenol oxidase in beet root (Beta vulgaris L.)

The two enzymes involved in enzymatic browning reactions, polyphenol oxidase (PPO) and peroxidase (PO), have been partially purified and extracted from different fractions of beet root. PPO is mainly located in the membrane fraction, and it was also found in the soluble fraction. In both cases PPO was in its latent state. However, PO activity was higher in the soluble fraction than in the membrane fraction. Nevertheless, the highest values of specific activity for PO were obtained from the solubilized enzyme from acetone powders. Under native isoelectric focusing (IEF), several PPO isoenzymes were present in the pH range of 4.8-5.8. All of these isoenzymes shared a single band with a similar apparent mass under sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PO was also analyzed by IEF, showing a complex isoenzyme pattern in all fractions. The characteristic basic PO isoenzyme of high pI found in both the soluble fraction and the solubilized enzyme from acetone powders was not detected in the membrane fraction. The kinetic characterization of PPO and PO from all fractions was carried out.     

Determination of anthocyanidins in berries and red wine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the determination of anthocyanidins from berries and red wine is described. Delphinidin, cyanidin, petunidin, pelargonidin, peonidin, and malvidin contents of bilberry (Vaccinium myrtillus), black currant (Ribes nigrum), strawberry (Fragaria ananassa cv. Jonsok), and a Cabernet sauvignon (Vitis vinifera) red wine were determined. The aglycon forms of the anthocyanins present in the samples were revealed by acid hydrolysis. A reversed phase analytical column was employed to separate the anthocyanidins before identification by diode array detection. The suitability of the method was tested by determining the recovery (95-102% as aglycons and 69-104% from glycosides) for each anthocyanidin. Method repeatability was tested by charting the total aglycon content of two samples over a period of 14 analyses and determining the coefficients of variation (1.41% for bilberry and 2.56% for in-house reference material). The method developed proved thus to be effective for reliable determination of anthocyanidins from freeze-dried berry samples and red wine. The total anthocyanidin content of the tested samples was as follows: in-house reference material, 447 +/- 8 mg/100 g; strawberry, 23.8 +/- 0.4 mg/100 g; black currant, 135 +/- 3 mg/100 g; bilberry, 360 +/- 3 mg/100 g; and Cabernet sauvignon red wine, 26.1 +/- 0.1 mg/100 mL.     

Evidence of the glycation and denaturation of LTP1 during the malting and brewing process

The influence of malting and brewing processes on the chemical and structural modifications occurring on LTP1 was investigated by mass spectrometry and circular dichroism. Proteins were first purified from malt, and samples were collected at various steps of beer processing performed on two barley cultivars. The levels of LTP1 found in malt were not significantly different from the amounts in barley seed. However, in malt, both LTP1b, a post-translational form of LTP1, and a third isoform named LTP1c were isolated. Moreover, both of these proteins were found to be heterogeneously glycated but still exhibited an alpha-helix structure. Both glycated LTP1 and LTP1b were recovered during mashing. It was also shown that glycated LTP1 was unfolded during heat treatment of wort boiling, which is in agreement with the denatured form previously isolated from beer.     

Formation of Amadori compounds in dehydrated fruits.

The presence of Amadori compounds in commercial dehydrated fruits has been shown through HPLC analysis of the corresponding 2-furoylmethyl-amino acids obtained by acid hydrolysis. Furosine (2-furoylmethyl-lysine) was the main 2-furoylmethyl derivative observed in dried figs and apricot samples, whereas in prunes and dates similar amounts of furosine and 2-furoylmethyl-gamma-aminobutyric acid were detected. A considerable variation of 2-furoylmethyl-amino acid contents among commercial raisin samples was observed. 2-Furoylmethyl-gamma-aminobutyric acid and 2-furoylmethyl-arginine, the most abundant 2-furoylmethyl-amino acids, ranged between 9.9 and 75.8 mg/100 g sample and 10.0 and 62.5 mg/100 g sample, respectively. Most of the Amadori compounds present in raisins seem to have originated during the commercial shelf life period rather than during processing. Determination of 2-furoylmethyl-amino acids could be used as a method of controlling commercial dehydrated fruit and selecting storage conditions.     

Efficient In Vitro Shoot-regeneration Systems in Cassava (Manihot esculenta Crantz)

Two different protocols for in vitro regeneration of cassava using zygotic embryos and nodal axillary meristems have been developed. In both cases, buds were regenerated directly from excised explants without an intervening callus phase after a two-step culture procedure. In cotyledonary explants derived from zygotic embryos, prolific shoot formation occurred within 2—3 weeks on MS medium supplemented with 0.5—5 mg/1 BAP alone or in combination with 0.1 mg/1 NAA. Nodal explants with axillary meristems derived from aseptically grown seedlings or stem cuttings were used to initiate a round compact bulb-like structure on MS medium containing 10 mg/1 BAP. These latter structures, when cultured on MS medium supplemented with 0.1 mg/1 NAA, 1 mg/1 BAP and 0.1 mg/1 GA₃, produced multiple shoots. Somatic embryos isolated at the globular/torpedo stage from zygotic embryo explants were also capable of multiple shoot production on medium with 1 mg/1 BAP. Rooting of regenerated shoots exceeded 95 % in phytohormone-free MS medium. No change in their ploidy levels was observed. Therefore, the protocols developed should be of use in the particle gun and Agrobacterium-mediated genetic transformation of cassava.