Identification of markers of thermal processing ("roasting") in aqueous extracts of Coffea arabica L. seeds through NMR fingerprinting and chemometrics.

Roasting of Coffea arabica L. seeds gives rise to chemical reactions that produce more than 800 compounds, some being responsible for the desired organoleptic properties for which the beverage called "coffee" is known. In the industry, the "roasting profile," that is, the times and temperatures applied, is key to influence the composition of roasted coffee beans and the flavour of the beverage made from them. The impact of roasting on the chemical composition of coffee has been the subject of numerous studies, including by nuclear magnetic resonance (NMR) spectroscopy. However, the roasting equipment and profiles applied in these studies are often far from real industrial conditions. In this work, the effects of two critical technological parameters of the roasting process, namely, the "development time" (the period of time after the "first crack," a characteristic noise due to seed disruption) and the final roasting temperature on coffee extracts, were investigated. Seeds were roasted at pilot scale according to 13 industrial roasting profiles and extracted in D2 O. The extracts were analysed by 1 H NMR experiments. The NMR spectra were compared using (a) quantitative analysis of main signals by successive orders of magnitude and (b) chemometric tools (principal component analysis, partial least squares and sparse-orthogonal partial least squares analysis). This allowed to identify compounds, which may serve as markers of roasting and showed that changes in chemical composition can be detected even for slight change in final temperature (~1°C) or in total roasting time (~25 s).

Analysis of sitosteryl oleate esters in phytosterols esters enriched foods by HPLC-ESI-MS(2.).

Phytosteryl esters (PE)-enriched spreads are marketed for eating and cooking purposes. Temperature and also light exposure are the major factors leading to the formation of PE oxides in food matrix. In this study a high-speed HPLC-MS(2) method was developed to analyze the major PE present in PE-enriched spreads: sitosteryl oleate (SO) and its oxidation products, by using synthesized compounds as standards. This analytical method was used to quantify seven SO oxides formed in PE-enriched spreads after heating at different temperatures for varying time periods and after prolonged exposure to sunlight. Quantification of remaining native SO was also performed after these different treatments. It was found that under specific heating conditions the decrease of the SO amount was much more important compared to the formation of SO oxides showing that many other products are formed. In contrast to heating, sunlight radiation did not result in the degradation of SO and very few oxides were formed.

Dietary walnut oil modulates liver steatosis in the obese Zucker rat.

PURPOSE: Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. We aimed to clarify the impact of dietary walnut oil versus animal fat on hepatic steatosis, representing the initial step of multistage pathogenesis of NAFLD, in Zucker obese rats. METHODS: Zucker lean ad libitum (a.l.), Zucker obese a.l. or Zucker obese pair fed (p.f.) to the lean received isocaloric diets containing 8% walnut oil (W8), W14 or 14% lard (L14) (n = 10/group). Body weight, clinical serology, liver weight, lipid content and fatty acid composition and hepatic lipid metabolism-related transcripts were evaluated. RESULTS: Compared to lean, Zucker obese a.l. and p.f. showed hepatic triacylglyceride (TAG) accumulation. In Zucker obese p.f., W14 compared to W8 and L14 reduced liver lipids, TAG as well as hepatic omega-6 (n-6)/n-3 ratio and SCD activity index [(C18:0 + C18:1)/C18:0 ratio] paralleled by decreased lipoprotein lipase mRNA in obese p.f. and elevated microsomal triglyceride transfer protein mRNA in lean and obese. Further, W14 elevated the fasting blood TAG and reduced cholesterol levels in obese. CONCLUSIONS: In our model, consumption of W14 inhibited hepatic lipid accumulation along with modulated hepatic gene expression implicated in hepatic fatty acid influx or lipoprotein assembly. These results provide first indication that dietary lipids from walnut oil are modulators of hepatic steatosis as the initial step of progressive NAFLD pathogenesis.

Effects of industrial processing on folate content in green vegetables.

Folates are described to be sensitive to different physical parameters such as heat, light, pH and leaching. Most studies on folates degradation during processing or cooking treatments were carried out on model solutions or vegetables only with thermal treatments. Our aim was to identify which steps were involved in folates loss in industrial processing chains, and which mechanisms were underlying these losses. For this, the folates contents were monitored along an industrial canning chain of green beans and along an industrial freezing chain of spinach. Folates contents decreased significantly by 25% during the washing step for spinach in the freezing process, and by 30% in the green beans canning process after sterilisation, with 20% of the initial amount being transferred into the covering liquid. The main mechanism involved in folate loss during both canning green beans and freezing spinach was leaching. Limiting the contact between vegetables and water or using steaming seems to be an adequate measure to limit folates losses during processing.

Freeze-drying of enzymes in case of water-binding and non-water-binding substrates.

Enzymes typically have a critical instability, which dominates both formulation and process development. In this paper, the ability to preserve the enzyme activity during freeze-drying was investigated for both water-binding and non-water-binding substrates. For this purpose, acid phosphatase was used as model protein. In addition, a procedure for the fast development of a freeze-drying cycle is shown. For the secondary drying part, the effect of processing temperature and time on enzyme activity was investigated. The enzyme activity decreased continuously during secondary drying, with a dramatic drop associated with processing temperatures higher than 293 K. Besides product temperature, the residual moisture level and water mobility are also important. As the residual moisture level and water mobility depend on the product formulation, the stabilizing effect against the enzyme deactivation was studied for a number of formulations which contain different additives, that is, sucrose, lactose, mannitol, and poly-vinylpyrrolidone, with a dramatic activity loss associated with crystallizing excipients. This study also confirmed that not all water-binding substrates can successfully protect the enzyme against deactivation. In fact, water-binding substrates containing reducing sugars (lactose) showed the highest loss of activity.

Investigation of biomarkers of bile tolerance in Lactobacillus casei using comparative proteomics.

The identification of cell determinants involved in probiotic features is a challenge in current probiotic research. In this work, markers of bile tolerance in Lactobacillus casei were investigated using comparative proteomics. Six L. casei strains were classified on the basis of their ability to grow in the presence of bile salts in vitro. Constitutive differences between whole cell proteomes of the most tolerant strain (L. casei Rosell-215), the most sensitive one (L. casei ATCC 334), and a moderately tolerant strain (L. casei DN-114 001) were investigated. The ascertained subproteome was further studied for the six strains in both standard and bile stressing conditions. Focus was on proteins whose expression levels were correlated with observed levels of bile tolerance in vitro, particularly those previously reported to be involved in the bile tolerance process of lactobacilli. Analysis revealed that 12 proteins involved in membrane modification (NagA, NagB, and RmlC), cell protection and detoxification (ClpL and OpuA), as well as central metabolism (Eno, GndA, Pgm, Pta, Pyk, Rp1l, and ThRS) were likely to be key determinants of bile tolerance in L. casei and may serve as potential biomarkers for phenotyping or screening purposes. The approach used enabled the correlation of expression levels of particular proteins with a specific probiotic trait.

Comparative proteomic analysis of Lactobacillus plantarum for the identification of key proteins in bile tolerance.

BACKGROUND: Lactic acid bacteria are commonly marketed as probiotics based on their putative or proven health-promoting effects. These effects are known to be strain specific but the underlying molecular mechanisms remain poorly understood. Therefore, unravelling the determinants behind probiotic features is of particular interest since it would help select strains that stand the best chance of success in clinical trials. Bile tolerance is one of the most crucial properties as it determines the ability of bacteria to survive in the small intestine, and consequently their capacity to play their functional role as probiotics. In this context, the objective of this study was to investigate the natural protein diversity within the Lactobacillus plantarum species with relation to bile tolerance, using comparative proteomics. RESULTS: Bile tolerance properties of nine L. plantarum strains were studied in vitro. Three of them presenting different bile tolerance levels were selected for comparative proteomic analysis: L. plantarum 299 V (resistant), L. plantarum LC 804 (intermediate) and L. plantarum LC 56 (sensitive). Qualitative and quantitative differences in proteomes were analyzed using two-dimensional electrophoresis (2-DE), tryptic digestion, liquid chromatography-mass spectrometry analysis and database search for protein identification. Among the proteins correlated with differences in the 2-DE patterns of the bacterial strains, 15 have previously been reported to be involved in bile tolerance processes. The effect of a bile exposure on these patterns was investigated, which led to the identification of six proteins that may be key in the bile salt response and adaptation in L. plantarum: two glutathione reductases involved in protection against oxidative injury caused by bile salts, a cyclopropane-fatty-acyl-phospholipid synthase implicated in maintenance of cell envelope integrity, a bile salt hydrolase, an ABC transporter and a F0F1-ATP synthase which participate in the active removal of bile-related stress factors. CONCLUSIONS: These results showed that comparative proteomic analysis can help understand the differential bacterial properties of lactobacilli. In the field of probiotic studies, characteristic proteomic profiles can be identified for individual properties that may serve as bacterial biomarkers for the preliminary selection of strains with the best probiotic potential.

Enterocin 96, a novel class II bacteriocin produced by Enterococcus faecalis WHE 96, isolated from Munster cheese.

Enterococcus faecalis WHE 96, a strain isolated from soft cheese based on its anti-Listeria activity, produced a 5,494-Da bacteriocin that was purified to homogeneity by ultrafiltration and cation-exchange and reversed-phase chromatographies. The amino acid sequence of this bacteriocin, named enterocin 96, was determined by Edman degradation, and its structural gene was sequenced, revealing a double-glycine leader peptide. After a comparison with other bacteriocins, it was shown that enterocin 96 was a new class II bacteriocin that showed very little similarity with known structures. Enterocin 96 was indeed a new bacteriocin belonging to class II bacteriocins. The activity spectrum of enterocin 96 covered a wide range of bacteria, with strong activity against most gram-positive strains but very little or no activity against gram-negative strains.

Effects of oxidation on the hydrolysis by cholesterol esterase of sitosteryl esters as compared to a cholesteryl ester.

Phytosteryl esters (PE) are used as ingredients in functional food to decrease plasma concentration of low density lipoprotein-cholesterol (LDL-C). Effective impairment of cholesterol absorption by PE suggests that these esters are hydrolyzed by the pancreatic cholesterol esterase (CEase, EC 3.1.1.13) and the liberated sterol may interfere with cholesterol reducing its intestinal absorption. PE-enriched foods are marketed for cooking purposes, and temperature is one of the most important factors leading to the formation of oxidation products. Very little is known about the outcome of PE oxides during the digestive process. A new analytical method based on mass spectrometric detection directly after enzymatic reaction was developed to determine in vitro the activity of CEase on PE and their oxides present in functional food. Using this method, we identified a new inhibitor of CEase: sitosteryl 9,10-dihydroxystearate, which behaves as a non-competitive inhibitor of the hydrolysis of cholesteryl oleate and sitosteryl oleate.

2-DE and MS analysis of key proteins in the adhesion of Lactobacillus plantarum, a first step toward early selection of probiotics based on bacterial biomarkers.

The identification of cell components involved in probiotic activities is a challenge in current probiotic research. In this work, a new approach based on proteomics as an analytical tool for the identification of characteristic protein profiles related to adhesion to mucin as a model probiotic property was used. Three Lactobacillus plantarum strains with different adhesion rates were used for proteomic analysis: L. plantarum WHE 92 (15.9%), L. plantarum 299 v (9.1%) and L. plantarum CECT 4185 (1.4%). Cell wall extracts were subjected to proteomic analysis of differential protein expression using 2-DE, tryptic digestion, chip-LC-QTOF mass analysis and protein identification using database search. Several proteins, previously reported to be involved in bacterial adhesion: elongation factor EF-Tu, GroEL chaperonin, molecular chaperone DnaK and glyceraldehyde-3-phosphate dehydrogenase were found to be overexpressed in the cell wall proteome of the highly adhesive strain L. plantarum WHE 92. The overexpression of two spots containing GroES co-chaperonin in the most adhesive strain also suggested the involvement of this protein in the adhesion process. The association of proteomic profiles and proteins with particular probiotic properties opens the way for the use of such profiles and proteins as bacterial biomarkers for the properties of bacteria but probably also for their potential health effects.

Smearing of soft cheese with Enterococcus faecium WHE 81, a multi-bacteriocin producer, against Listeria monocytogenes.

Enterococcus faecium WHE 81, a multi-bacteriocin producer, was tested for its antimicrobial activity on Listeria monocytogenes in Munster cheese, a red smear soft cheese. The naturally delayed and superficial contamination of this type of cheese allowed the use of E. faecium WHE 81 at the beginning of the ripening as a surface culture. A brine solution inoculated at 10(5)CFU of E. faecium WHE 81 per mL was sprayed on the cheese surface during the first smearing operation. On day 7, smearing of cheese samples with a brine solution at 10(2)CFU of L. monocytogenes per mL yielded initial cell counts of approximately 50 CFU g(-1) of the pathogen on the cheese surface. Although, in some instances, L. monocytogenes could survive (<50 CFU g(-1)) in the presence of E. faecium WHE 81, it was unable to initiate growth. In control samples however, L. monocytogenes counts often exceeded 10(4) CFU g(-1). In other respects, E. faecium WHE 81, which naturally existed in Munster cheese, did not adversely impact on the ripening process.

Synthesis of highly pure oxyphytosterols and (oxy)phytosterol esters. Part II. (Oxy)-sitosterol esters derived from oleic acid and from 9,10-dihydroxystearic acid [1].

Several efficient synthetic routes giving readily access to (oxy)-sitosterol esters and (oxy)-cholesterol esters derived respectively from oleic acid and from 9,10-dihydroxystearic acid were developed for the first time. This approach allowed that sufficient amounts of the latter were available in order to carry out further biological studies.

Synthesis of highly pure oxyphytosterols and (oxy)phytosterol esters Part I. Regioselective hydrogenation of stigmasterol: an easy access to oxyphytosterols.

The synthesis of several oxyphytosterols is described starting from stigmasterol, the key step being the regioselective hydrogenation of the 22-23 double bond of the latter.

Mitochondrial perturbation, oxidative stress and lysosomal destabilization are involved in 7beta-hydroxysitosterol and 7beta-hydroxycholesterol triggered apoptosis in human colon cancer cells.

We reported previously that 7beta-hydroxysitosterol and 7beta-hydroxycholesterol induced apoptosis in Caco-2 cells. Apoptosis caused by 7beta-hydroxysitosterol but not by 7beta-hydroxycholesterol was related to a caspase-dependent process. In the present report, we compared the effects of both compounds on mitochondria integrity and on various modulators of apoptosis. When Caco-2 cells were exposed to both hydroxysterols, no changes in Bcl-2 and Bax expressions were detected indicating a Bcl-2/Bax-independent cell death pathway, whereas loss of mitochondrial membrane potential and cytochrome c release were observed. Endonuclease G expression and enhanced production of reactive oxygen species were detected in 7beta-hydroxycholesterol treated cells, but not with 7beta-hydroxysitosterol. Loss of mitochondrial membrane potential and cell death produced by both hydroxysterols were prevented by vitamin C. Lysosomal membrane integrity was altered with both hydroxysterols, but 7beta-hydroxysitosterol was significantly more active on than 7beta-hydroxycholesterol. Both hydroxysterols induced apoptosis by mitochondrial membrane permeabilization. However, 7beta-hydroxycholesterol exhibited a specific enhancement of oxidative stress and of endonuclease G expression despite its closely related chemical structure with 7beta-hydroxysitosterol. The two hydroxysterols exhibit different lipophilic properties which may explain their different biological effects.

Perturbation of polyamine metabolism and its relation to cell death in human colon cancer cells treated by 7beta-hydroxycholesterol and 7beta-hydroxysitosterol.

7beta-OHsitosterol and 7beta-OHcholesterol are natural compounds of plant and animal cells with high structural similarity. Recently it was reported that both compounds induced apoptosis on human colon cancer cells by targeting different signalling pathways. Our study aimed at comparing their effects on polyamine metabolism and its relation to apoptosis. When human colon cancer cells were exposed to 7beta-OHsitosterol and to 7beta-OHcholesterol at concentrations inhibiting growth by the same degree, both compounds caused a reduction of polyamine biosynthetic enzyme activity, of the polyamine pools, and an increase of N1-acetylspermidine concentration indicating the enhancement of polyamine catabolism. Exogenous putrescine did not prevent cell death caused by 7beta-OHsitosterol, whereas 7beta-OHcholesterol-induced apoptosis was inhibited. MDL 72527, an inhibitor of polyamine oxidase, an enzyme of the polyamine catabolic pathway, potentiated the antiproliferative effects of 7beta-OHcholesterol by increasing the N1-acetylspermidine pool and enhanced the accumulation of apoptotic cells. In contrast, MDL 72527 did not change the apoptosis rate and the N1-acetylspermidine content in cells treated with 7beta-OHsitosterol. These data indicate that polyamine metabolic perturbations triggered by 7beta-OHcholesterol but not by 7beta-OHsitosterol are related to cell death.

Quantitative analysis of beta-sitosterol oxides induced in vegetable oils by natural sunlight, artificially generated light, and irradiation.

UV radiation is able to induce lipid peroxidation. Photooxidation-induced beta-sitosterol oxides were monitored in four vegetable oils exposed to sunlight for 10, 20, and 30 days during May 2005 (northeastern France), exposed to artificial light generated by a high-pressure Hg lamp for 21, 42, and 63 h at room temperature, and exposed to a 10 MeV electron beam at 0.93, 2.69, and 9.30 kGy at 8 degrees C. Quantification was performed by capillary gas chromatography-mass spectrometry according to the total ion current mode and using a reconstructed ion trace chromatogram with specific ion fragments. Sunlight induced the formation of higher amounts of oxides than UV light, while no significant oxidizing effect was observed with electron beam irradiation. However, data suggested that the amount of the main oxides formed was strongly dependent on the dose rate (length of exposure). Accordingly, shorter but more intense treatments had lower oxidizing effects.

Separation of Delta5- and Delta7-phytosterols by adsorption chromatography and semipreparative reversed phase high-performance liquid chromatography for quantitative analysis of phytosterols in foods.

A method for the separation, isolation, and identification of phytosterols was developed. A commercial phytosterols mixture, Generol 95S, was fractionated first by adsorption silica gel column chromatography and then separated by means of a semipreparative reverse phase high-performance liquid chromatography fitted with a Polaris C8-A column (250 mm x 10 mm i.d., 5 microm) using isocratic acetonitrile:2-propanol:water (2:1:1, v/v/v) as the mobile phase. Milligram scales of six individual phytosterols, including citrostadienol, campesterol, beta-sitosterol, Delta7-avenasterol, Delta7-campesterol, and Delta7-sitosterol, were obtained. Purities of these isolated sterols were 85-98%. Relative response factors (RRF) of these phytosterols were calculated against cholestanol as an authentic commercial standard. These RRF values were used to quantify by gas chromatography-mass spectrometry (GC-MS) the phytosterols content in a reference material, oils, and chocolates.

DNA comet assay for the detection of time-temperature abuse during the storage of poultry.

Effects of abusive storage conditions on the quality of fresh chicken were studied by detecting DNA damage to breast fillets and liver with the neutral comet assay. Chilled samples were kept at 4 degrees C for prolonged periods, whereas frozen samples were exposed to temperatures of 4 degrees C, representing inadvertent thawing, and 20 degrees C, representing extreme abuse in the distribution chain. Comets' mean tail moment distributions reflected the increasing patterns of DNA damage, but the differences of values between close levels of treatment were sometimes insignificant. The design of the DNA damage index, integrating the distribution of mean tail moments over three trials, provided values significantly different, which allowed a more precise discrimination between samples according to the treatment levels. Considering the background level of DNA damage in control cells, a DNA damage index value of 50 microm was set as a limit for the detection of abusive storage. Temperature abuse could be detected after 7 and 22 h of exposure at 4 degrees C for liver and breast, respectively. These durations were by far shorter (1.5 and 2.5 h, respectively) when the temperature was increased to 20 degrees C. As for chilled storage, its damaging effects could be detected after 1.5 and 2.5 days for liver and breast, respectively. Liver cells were more sensitive to abusive conditions than breast muscle cells. The comet assay's detection limit was applicable to samples that were still considered of good quality with regard to the microbiological shelf life, thereby showing its high sensitivity as a rapid test for assessing the quality of fresh chicken.

Identification and quantitative analysis of beta-sitosterol oxides in vegetable oils by capillary gas chromatography-mass spectrometry.

As vegetable oils and phytosterol-enriched spreads are marketed for frying food or cooking purposes, temperature is one of the most important factors leading to the formation of phytosterol oxides in food matrix. A methodology based on saponification, organic solvent extraction, solid-phase extraction (SPE), followed by mass spectrometric identification and quantitation of beta-sitosterol oxides using capillary gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode was developed and characterized. Relative response factors of six beta-sitosterol oxides, including 7alpha-hydroxy, 7beta-hydroxy, 5,6alpha-epoxy, 5,6beta-epoxy, 7-keto, and 5alpha,6beta-dihydroxysitosterol, were calculated against authentic standards of 19-hydroxycholesterol or cholestanol. Linear calibration data, limit of detection, and sample recoveries during analytical process. Recoveries of these oxidation compounds in spiked samples ranged from 88 to 115%, while relative standard derivation (R.S.D.) values were below 10% in most cases. The analytical method was applied to quantify beta-sitosterol oxides formed in thermal-oxidized vegetable oils which were heated at different temperatures and for varying time periods. Sitosterol oxidation is strikingly higher in sunflower oil relative to olive oil under all conditions of temperature and heating time.

Gram-scale chromatographic purification of beta-sitosterol. Synthesis and characterization of beta-sitosterol oxides.

An effective purification method for beta-sitosterol was developed starting from a commercial source of a phytosterol mixture using preparative adsorption column chromatography. beta-Sitosterol (> or = 95% purity) was obtained on a gram-scale. Thus, the synthesis of six beta-sitosterol oxides, including 7alpha-hydroxy, 7beta-hydroxy, 5,6alpha-epoxy, 5,6beta-epoxy, 7-keto, and 5alpha,6beta-dihydroxysitosterol, were successfully carried out. The spectral characteristics of all the synthetic intermediates and target compounds (approximately 95% purity) were well-documented.