Survival of the functional yeast Kluyveromyces marxianus B0399 in fermented milk with added sorbic acid.

In this study, the survival of the functional yeast Kluyveromyces marxianus B0399 in an industrially produced fermented milk was evaluated. In particular, the yeast viability was assessed throughout the entire shelf-life of the product (30 d) to ensure the presence of the effective yeast dose (20 million viable cells for each serving of 125 g) while avoiding, by sorbic acid addition, yeast growth, which could affect product quality and stability. To find the best combination of yeast and sorbic acid concentration, 13 different combinations were tested, and then 2 of them were chosen for industrial production. In production at lower concentrations (30 million viable cells, 150 mg/kg of sorbic acid) the effective dose was maintained only at 4 and 6°C, whereas at higher dosages (70 million viable cells, 250 mg/kg of sorbic acid) the effect of temperature was less evident. In all the trials, the concentration of sorbic acid was not affected by microbial metabolism and remained stable throughout the entire shelf-life.

Evaluation of different extraction approaches for the determination of phenolic compounds and their metabolites in plasma by nanoLC-ESI-TOF-MS.

Sample preparation is an important step for the determination of phenolic compounds in biological samples. Different extraction methods have been tested to determine phenolic compounds and their metabolites in plasma by nano-liquid chromatography coupled to electrospray ionisation-time-of-flight mass spectrometry (nanoLC-ESI-TOF-MS). The sample treatment optimisation was performed using commercial foetal bovine serum spiked with representative phenolic standards, namely naringenin, luteolin, verbascoside, apigenin, rutin, syringic acid and catechin. Different protein-precipitation conditions were evaluated as well as enzymatic digestion with trypsin and solid-phase extraction using different phases such as C-18, ABN and ENV+, working at different pH values. The optimum extraction procedure consisted of a previous protein-precipitation step using HCl 200 mmol/L in methanol for 2.5 h at 50 °C followed by a solid-phase extraction using C-18 cartridges at pH 2.5. This procedure was finally applied to the plasma of rats overfed with a phenolic-rich Lippia citriodora extract. These samples were analysed by nanoLC-ESI-TOF-MS, enabling the identification of five compounds previously found in the administered L. citriodora extract and one metabolite.

Classification of Pecorino cheeses using electronic nose combined with artificial neural network and comparison with GC-MS analysis of volatile compounds.

An electronic nose based on an array of 6 metal oxide semiconductor sensors was used, jointly with artificial neural network (ANN) method, to classify Pecorino cheeses according to their ripening time and manufacturing techniques. For this purpose different pre-treatments of electronic nose signals have been tested. In particular, four different features extraction algorithms were compared with a principal component analysis (PCA) using to reduce the dimensionality of data set (data consisted of 900 data points per sensor). All the ANN models (with different pre-treatment data) have different capability to predict the Pecorino cheeses categories. In particular, PCA show better results (classification performance: 100%; RMSE: 0.024) in comparison with other pre-treatment systems.

Classification of Pecorino cheeses produced in Italy according to their ripening time and manufacturing technique using Fourier transform infrared spectroscopy.

Fourier-transform infrared spectroscopy, followed by linear discriminant analysis of the spectral data, was used to classify Italian Pecorino cheeses according to their ripening time and manufacturing technique. The Fourier transform infrared spectra of the cheeses were divided into 18 regions and the normalized absorbance peak areas within these regions were used as predictors. Linear discriminant analysis models were constructed to classify Pecorino cheeses according to different ripening stages (hard and semi-hard) or according to their manufacturing technique (fossa and nonfossa cheeses). An excellent resolution was achieved according to both ripening time and manufacturing technique. Also, a final linear discriminant analysis model considering the 3 categories (hard nonfossa, hard fossa, and semi-hard nonfossa) was constructed. A good resolution among the 3 categories was obtained.

High-performance liquid chromatography separation and light-scattering detection of phospholipids from cooked beef.

A sensitive high-performance liquid chromatography (HPLC) method for the separation and quantitative analysis of major phospholipids (PLs) in biological systems is described. PLs were purified by solid-phase extraction with an amino (NH2) phase. Separation of PLs was carried out on an HPLC silica gel column, with a mobile phase consisting of chloroform, methanol and ammonium hydroxide, and detection was performed with a light-scattering evaporative detector. HPLC analysis of PLs extracted from ground beef cooked under different conditions and capillary gas chromatography of the fatty acid methyl esters showed that cooking treatments did not have a significant effect on the PL composition and fatty acid contents of the single PLs in ground beef.

Pressurized liquid extraction of lipids for the determination of oxysterols in egg-containing food.

Pressurized liquid extraction (PLE, ASE) was compared with the Folch procedure (a solid-liquid extraction with chloroform/methanol 2:1, v/v) for the lipid extraction of egg-containing food; the accuracy of PLE for the quantitative determination of oxysterols in whole egg powder was evaluated. Samples of spray-dried whole egg, an Italian vanilla cake (Pandoro) and egg noodles were used. Two different extraction solvents (chloroform/methanol 2:1, v/v, and hexane/isopropanol 3:2, v/v) were tested at different extraction temperatures and pressures (60 degrees C at 15 MPa, 100 degrees C at 15 MPa, 120 degrees C at 20 MPa). No significant differences in the lipid recovery of the egg powder sample using PLE were found. However, PLE of the vanilla cake and egg noodles with the chloroform/methanol mixture was not selective enough and led to the extraction of a non-lipid fraction, including nitrogen-containing compounds. In the same samples, the pressurized hexane/isopropanol mixture gave a better recovery result, comparable to that obtained using the Folch method. Cholesterol oxidation products of the Folch extract and the pressurized liquid extract of spray dried egg powder (obtained with hexane/isopropanol 3:2, v/v, at 60 degrees C and 15 MPa) were determined by gas chromatography. PLE performed under these conditions is suitable to replace the Folch extraction, because the differences between the two methods tested were not statistically significant. Moreover, PLE shows important advantages, since the analysis time was shortened by a factor of 10, the solvent costs were reduced by 80% and the use of chlorinated solvents was avoided.

Effect of different cooking methods on some lipid and protein components of hamburgers.

The effects of different cooking methods on the lipid and protein fractions of hamburger were evaluated. The lipid component was subjected to the following analyses: peroxide value; p-anisidine; total and free fatty acids; cholesterol and its oxidation products (quantified as 7-ketocholesterol). Lysinoalanine (LAL), free amino acids and D-amino acids (D-AA) were also determined in the protein fraction. All results were compared with a raw control. No significant differences were found among the cooking treatments with respect to D-AA and LAL. The degree of proteolysis, lipolysis and lipid oxidation varied depending on the treatment conditions. Regarding cholesterol oxidation, the combination of roasting and microwave heating caused more oxidation than the other treatments. The raw meat, however, showed an advanced degree of oxidation (25.2 ppm of total 7-ketocholesterol/120 g ground meat).

Analysis of phospholipids in cow's milk by high-temperature injection gas chromatography and high-performance liquid chromatography.

An investigation of the high-temperature injection (350 degrees C) gas chromatographic behaviour of standards of various classes of phospholipids has elucidated certain characteristic fragments ("tracers") in the pyrogram of each class. Under these conditions of pyrolysis of the phospholipids, a natural mixture of such substances (extracted and purified from cows' milk) has provided evidence for the same "tracers" as for the standards. Analogous results were obtained with a more representative specimen of cows' milk of various breeds grown in areas of different altitudes. The content of fatty acids in phospholipids, tested on each class (separated by means of radial compression high-performance liquid chromatography from polar lipids of milk) appeared to be relatively similar for all the phospholipid classes, and over half the content consisted of unsaturated fatty acids. The major component was delta 9-octadecenoid (oleic) acid.

Incorporation of cholesterol oxidation products into cell lipids and their influence on the proliferation of cultured cardiomyocytes.

We have investigated the incorporation of cholesterol oxidation products into cardiomyocyte lipids and related this to changes in cell proliferation, evaluated by measuring cellular protein content. Primary cultures of neonatal rat ventricular cells were supplemented with scalar concentrations of several cholesterol oxidation products (cholestan-5 alpha, 6 alpha-epoxy-3 beta-ol, 5 alpha-cholestane-3 beta, 5, 6 beta-triol, 5-cholesten-3 beta, 4 beta-diol, 5-cholesten-3 beta-ol-7-one, and 5-cholesten-3-one). Although all the cholesterol oxidation products were incorporated into the cardiomyocyte lipids when added to the medium at a concentration higher than 0.5 microM, the extent of the incorporation of the different cholesterol oxidation products differed, depending on the concentration in the culture medium and on the chemical structure of the compound. The effects of the cholesterol oxidation products on the cellular protein content were also different: 5 alpha-cholestane-3 beta, 5, 6 beta-triol was shown to be the most potent inhibitor of cell proliferation, followed by cholestan-5 alpha, 6 alpha-epoxy-3 beta-ol, 5-cholesten-3 beta, 4 beta-diol and 5-cholesten-3 beta-ol-7-one. 5-Cholesten-3-one did not affect the cellular protein content. The ability of cholesterol oxidation products to inhibit cell proliferation, and their capacity to increase the permeability of the plasma membrane to calcium, could be deleterious for cardiac cells.

Effect of cholesterol-5 alpha,6 alpha-epoxide supplementation to cultured cardiomyocytes.

In order to evaluate the effect of one of the main oxysterols derived from cholesterol oxidation, cholesterol-5 alpha,6 alpha-epoxide (epox), on cardiac cells, we have supplemented the culture medium of neonatal rat cardiomyocytes with scalar concentrations of epox (0.1-100 microM). While 0.1 microM epox supplementation was ineffective, epox supplementation in the range 1-100 microM determined a reduction in cellular protein level, without affecting cell viability, and a dose-dependent epox incorporation into cardiomyocyte lipids. Furthermore, in the same concentration range of epox supplementation, a gas chromatographic peak unambiguously identified by gas chromatography-mass spectrometry as cholestane-3 beta,5 alpha,6 beta-triol, an hydrolytic metabolite of epox, was detected. The mechanism of cytotoxicity of epox to cardiomyocytes could be due to the insertion of epox itself into cellular lipids, and to its metabolization to the more toxic triol.