Characterization by gas chromatography/mass spectrometry of sterols in saccharomyces cerevisiae during autolysis.

Yeast autolysis affects membrane stability and induces a release of vacuolar enzymes into the cell cytoplasm. Consecutively, it was important to study the evolution of sterol content in Saccharomycescerevisiae for a fourteen day period of accelerated autolysis. Unesterified and esterified sterols were analyzed both in the biomass and in the autolysis medium. Ten sterols were identified by gas chromatography/mass spectrometry. A second group of six sterols was separated and partially characterized. Among the first group of 10 sterols, a dehydroergosterol was identified as ergosta-5, 7,9(11),22-tetraen-3beta-ol, not yet charaterized in S. cerevisiae. Yeast autolysis induced a decrease of esterified sterol content, especially first intermediates in the sequence of the ergosterol biosynthesis, as zymosterol. In contrast, the yeast autolysis resulted in the release of a low quantity of sterols into the medium. At the end of the fourteenth day of autolysis, 0.015% of the total sterol content of the initial biomass was found in the medium.     

Impact of oxygen consumption by yeast lees on the autolysis phenomenon during simulation of wine aging on lees

Potential oxygen consumption by lees, more precisely by nonviable yeasts, during wine aging was recently described. Additionally, yeast autolysis is described as the main mechanism of degradation of lees during wine aging. Thus, to understand the effect of oxygen consumption by yeast lees during wine aging, an accelerated wine aging methodology was tested. Wine aging in the presence of yeast lees was studied both in the presence and in the absence of oxygen. Different markers of yeast autolysis were followed to find a relationship between oxygen consumption by yeast lees and changes in the final wine composition after aging. No differences for compounds tested were found in the wine and in the lees except among sterol compounds in lees: in the presence of oxygen, the concentration of ergosterol in lees was significantly lower than that in the absence of oxygen. It was hypothesized that ergosterol could be oxidized under the influence of oxygen, but none of the known products of ergosterol oxidation were recovered in the corresponding yeast lees. In addition, the decrease of ergosterol content in yeast lees cannot account for the total amount of oxygen consumed by yeast lees during such wine aging.     

Lipid [corrected] classes, fatty acids, and sterols in seafood from Gilbert Bay, southern labrador

Seafood from Gilbert Bay, southern Labrador, was sampled for lipid classes, fatty acid, and sterol composition. Gilbert Bay is a proposed Marine Protected Area, and the composition of seafood from this region is interesting from both human health and ecological perspectives. Analyses included four species of bivalves and flesh and liver samples from four fish species. Lipids from a locally isolated population of northern cod (Gadus morhua) were also compared to lipids from other cod populations. Lipid classes were analyzed by Chromarod/Iatroscan TLC-FID, fatty acids by GC, and sterols by GC-MS. Three cod populations had similar levels of total lipid per wet weight (0.6%) with triacylglycerols (TAG), sterols, and phospholipids comprising on average 13, 11, and 51%, respectively, of their total lipids. Fatty fish such as capelin and herring contained on average 8.4% lipid with 86% present as TAG. Fish livers from cod and herring showed opposite trends, with cod having elevated lipid (27%) and TAG (63%) and herring containing only 3.8% lipid and 20% TAG. Shellfish averaged 0.6% lipid; however, significant lipid class differences existed among species. Fatty acid analysis showed few significant differences in cod populations with on average 57% polyunsaturated fatty acids (PUFA), 18% monounsaturated fatty acids (MUFA), and 24% saturated fatty acids (SFA). Cod livers had lower PUFA (34%) and elevated MUFA (44%) relative to flesh. Bivalves averaged 25% SFA, 18% MUFA, and 57% PUFA, whereas scallop adductor muscle had the highest PUFA levels (63%). Bivalves contained 20 different sterols with cholesterol present as the major sterol (19-39%). trans-22-Dehydrocholesterol, brassicasterol, 24-methylenecholesterol, and campesterol individually accounted for >10% in at least one species. High levels of PUFA and non-cholesterol sterols observed in Gilbert Bay seafood demonstrate their positive attributes for human nutrition.     

Comparison of components released by fermented or active dried yeasts after aging on lees in a model wine

Comparison of different components released during autolysis with fermented or active dried yeast, which has never been reported previously, is related in this paper. Three autolysates were elaborated with Saccharomyces cerevisiae in a model wine (pH 3.5) at 30 or 18 degrees C. Composition of the autolysate appears to depend on both the growth medium and the physiological state of the yeast. The autolysate obtained from active dried yeast presents a higher total nitrogen concentration (a factor of 1.5-1.9 for the fermented yeast autolysate), a greater proportion of free amino acids (42 vs 16-25%), the lowest proportion of oligopeptides (25 vs 31-37%) and polypeptides (27 vs 45-34%), and an absence of nucleosides. Distribution of amino acids in peptides and proteins is relatively homogeneous in the autolysates but shows significant differences in free amino acids. Parietal macromolecules (mannoproteins and glucans) are present in greater quantities in autolysates elaborated from fermented yeast. The influence of the different fractions of these autolysates (<0.5, 0.5-1, 1-10, and >10 kDa) on bacterial growth has been investigated.     

Synthesis and analysis of symmetrical and nonsymmetrical disaturated/monounsaturated triacylglycerols

Symmetrical disaturated triacylglycerols of the structure SUS, where S is stearic acid (18:0) and U is an unsaturated fatty acid, either oleic (O; 9cis-18:1), linoleic (L; 9cis,12cis-18:2), or linolenic (Ln; 9cis,12cis,15cis-18:3), are important components providing functionality to interesterified fat blends and structurally modified oils. Nonsymmetrical triacylglycerols of the structure SSU can significantly change melting point and solid fat content profiles. To characterize the physical properties of pure and symmetrical and nonsymmetrical triacylglycerol mixtures, the same reaction sequence has been used to prepare multigram quantities of triacylglycerols SUS and SSU. Tristearin was converted to a mixture of mono-, di-, and triacylglycerols, and the 1,3- and 1,2-diacylglycerol fraction was isolated by silica column chromatography. The 1,3-diacylglycerols were removed by crystallization from acetone and esterified with the appropriate fatty acid to form the symmetrical triacylglycerols with >99% SUS structure. The more difficult to obtain 1,2-diacylglycerols were prepared by esterification of the enriched 1,2-diacylglycerol fraction (80-86% 1,2-diacylglycerols) remaining after removal of much of the 1,3-isomer by crystallization, but silver resin or silver nitrate impregnated silica gel chromatography was required to isolate the nonsymmetrical triacylglycerols. SSL and SSLn were prepared in purities of >98% by this procedure, but not SSO. Silver ion HPLC was found to be as accurate as, and more rapid than, lipolysis/gas chromatography for the determination of the isomeric purities of the synthesized triacylglycerols.     

Simultaneous analysis of free phytosterols/phytostanols and intact phytosteryl/phytostanyl fatty acid and phenolic acid esters in cereals

An approach based on solid-phase extraction for the effective separation of free phytosterols/phytostanols and phytosteryl/phytostanyl fatty acid and phenolic acid esters from cereal lipids was developed. The ester conjugates were analyzed in their intact form by means of capillary gas chromatography. Besides free sterols and stanols, up to 33 different fatty acid and phenolic acid esters were identified in four different cereal grains via gas chromatography-mass spectrometry. The majority (52-57%) of the sterols and stanols were present as fatty acid esters. The highest levels of all three sterol and stanol classes based on dry matter of ground kernels were determined in corn, whereas the oil extract of rye was 1.7 and 1.6 times richer in fatty acid esters and free sterols/stanols than the corn oil. The results showed that there are considerable differences in the sterols/stanols and their ester profiles and contents obtained from corn compared to rye, wheat, and spelt. The proposed method is useful for the quantification of a wide range of free phytosterols/phytostanols and intact phytosteryl/phytostanyl esters to characterize different types of grain.     

Consumption of a novel dietary formula of plant sterol esters of canola oil fatty acids, in a canola oil matrix containing 1,3-diacylglycerol, reduces oxidative stress in atherosclerotic apolipoprotein E-deficient mice

The antiatherogenic properties of a novel dietary formula (PS-CO) of plant sterol esters of fatty acids, produced by enzymatic interesterification of plant sterols with canola oil (CO), in a CO matrix containing 1,3-diacylglycerol, were evaluated in apolipoprotein E-deficient mice. PS-CO consumption strongly tended to lower total plasma cholesterol levels by 21%, compared to the placebo group. Blood triglycerides were reduced by 38% and 36% compared to CO and placebo-fed mice, respectively. Serum lipid peroxide levels were lowered following PS-CO administration by 62% and 63%, compared to CO and placebo administration, respectively. Unlike CO supplementation, PS-CO consumption preserved serum paraoxonase (PON1) activity. Mouse peritoneal macrophages from PS-CO-fed mice exhibited reduced cellular uptake of oxidized-LDL compared to those from placebo-fed mice and demonstrated a tendency toward a decreased capability to release superoxide anions. These findings indicate that PS-CO supplementation is beneficial in reducing serum lipid levels, and serum and macrophage oxidative stress, thus contributing to the reduction in atherogenic risk factors.     

Formation of micella containing solubilized sterols during rehydration of active dry yeasts improves their fermenting capacity

During their rehydration in aqueous media, active dry yeasts (ADY) may be supplemented with inactive yeasts, yeast derivatives, or other optional complementary nutrients to improve their fermentation capacity. We found that yeast sterols solubilized in situ during ADY rehydration were particularly efficient for stimulating the fermenting capacity of ADY. Spontaneous solubilization of sterols during rehydration occurred by the formation of micelles by membrane phospholipids and specific cell wall polysaccharides and sterols, both compounds being provided by inactive dry yeasts (IDY). These micelles contained a specific distribution of the initial sterols from the inactive yeasts. Above a concentration of 100 mg L(-1) in the rehydration medium, these micelles acted as emulsifiers. Their critical micellar concentration (cmc) was found to be about 4 g L(-1). During rehydration, purified micelles, at a concentration near the cmc, were able to interact quickly with yeast cell membranes by modifying the yeast plasma membrane order [monitored by steady-state fluorescence anisotropy of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluenesulfonate (TMA-DPH) probe] and by increasing the sterol contents of ADY. Such an enrichment of ADY by very low concentrations of solubilized sterols was very efficient for the completion of fermentations. This is useful when musts are limited in available phytosterols or when micro-oxygenation is not desirable during fermentation.     

Effect of different lysis treatments on the characteristics of yeast derivatives for winemaking

The effects of three preparation techniques on the oenological properties of a yeast autolysate were investigated: enzymatic autolysis, thermolysis, and the combination of a slow freezing-defrosting and mechanical disruption were carried out on a commercial formulation of active dry yeasts (Saccharomyces cerevisiae). The powders obtained by freeze drying, were characterized: volatile compounds were analyzed by SPME-GC with mass spectrometric (MS) and olfactometric detection (O); the release of colloids in winelike solution was studied by SDS-PAGE and size exclusion chromatography (SEC). Finally, the effects of the powders addition on the aroma composition of a white wine were investigated by SPME-GC-MS, SPME-GC-O, and sensory evaluation. The products obtained were quite different from each other. In particular, enzymatic autolysis led to higher contents of nonglycosilated soluble proteins in the powders and determined a higher retention of wine aroma compounds. On the contrary, thermal autolysate was richer in glycoproteins, and it was able to increase the wine aroma intensity; nevertheless, in the wines treated with such preparation, a slight yeastlike olfactory note was perceived.     

Characterization of the nitrogen compounds released during yeast autolysis in a model wine system

The nitrogen composition of wines aged with yeast for a long period of time, as in the case of sparkling wines, depends on the composition of the base wine and on the compounds released by the yeast. In this paper, the release of the different classes of nitrogen compounds during autolysis of one of the strains of yeast used in the manufacture of sparkling wines has been studied. The yeast, Saccharomyces bayanus, was suspended in a model wine buffer, pH 3.0 and 10% ethanol, and incubated at 30 degrees C. Samples of the autolysate were taken after 4, 24, 48, 72, 168, and 360 h of autolysis. An electrophoretic and chromatographic study was conducted of the proteins, peptides with molecular weights higher and lower than 700 Da, and amino acids released during the autolysis. Using SDS-PAGE, it was observed that it was predominantly polypeptides with molecular weights lower than 10 000 that were released. Through HPLC of the fraction lower than 10 000 Da, it was observed that it is polypeptides with molecular weights of between 10 000 and 700 Da that are released first and that these later break up to give rise to peptides with molecular weights lower than 700 Da, which in turn break down into amino acids. This indicates that the nature of the nitrogen compounds present in wines aged with yeast depends on the aging time, being less polymerized as the aging time increases.     

Neutral Lipids in Free,Bound, and Starch Lipid Extracts of Flours,Sourdough, and Portuguese Sourdough Bread Determined by NP‐HPLC‐ELSD

A detailed analysis was developed, focused on the neutral lipids (NL) in free (FL), bound (BL), and starch lipid (SL) extracts of maize and rye flours, sourdough, and broa (a traditional bread manufactured in Portugal). Selective sequential extraction of said lipids with hexane at 20°C, water‐saturated n‐butanol at 20°C, and n‐propanol‐water (3:1, v/v) at 100°C was performed to clean the lipid extracts from extraneous impurities, and isolation thereof from glyco‐ and phospholipids was by solid phase extraction of NL; these classes were then quantitatively assayed by HPLC, using evaporative light scattering detection, with calibration curves prepared with standard mixtures of NL. The BL and SL contents in the original flours increased and that of FL decreased throughout the fermentation and baking processes. The dominant NL class was not the same in all lipid extracts; the highest concentrations of triacylglycerols and the lowest concentrations of free fatty acids were detected in FL—with the former accounting for 82, 76, and 71% of the total FL in flours, sourdough, and bread, respectively. Triacylglycerols and free fatty acids also accounted for the highest concentrations found in BL: these, together with diacylglycerols, contributed up to 84% of the total neutral BL. High levels of free fatty acids and low levels of the remaining NL classes were typically found in SL: free fatty acids, triacylglycerols, sterol esters, and diacylglycerols accounted for ≈90% of the total SL.     

Acylglycerol and fatty acid components of pulp,seed, and whole olive fruit oils. Their use to characterize fruit variety by chemometrics

The contents of triacylglycerols and diacylglycerols in three kinds of olive fruit oils (pulp, seed, and whole fruit) were determined. The fatty acid composition and the quality ratios 1,2-diacylglycerols/1,3-diacylglycerols and 1,2-diacylglycerols/total diacylglycerols were also assessed. Seven major Italian olive varieties were considered. Results of univariate statistical analyses indicated that the above analytical parameters (glyceridic ratios excepted) were effective in discriminating between pulp and seed oils. The seed oil fraction did not determine any change in the glyceridic indices and the acylglycerol or fatty acid composition concerning the whole fruit oil (mixture of pulp and seed oil fractions), the weight (%) of seed ( approximately 2%) being by far lower than the weight (%) of pulp ( approximately 85%) (fruit weight basis). Based on the data of triacylglycerol or fatty acid composition, and using appropriate parametric or nonparametric multivariate statistics, the genetic origins (olive variety) of the three fruit oil kinds were characterized.     

Sulfometuron resistance as a genetic marker for yeast populations in wine fermentations

Winemaking with selected yeasts requires simple and cheap techniques to monitor the yeast population dynamics. We obtained new sulfometuron (smr) resistant mutants, easy to detect by replica-plate assay, from selected wine yeasts. The mutations were dominant and were located at the ilv2 locus that encodes for acetolactate synthase enzyme. The mutants were genetically stable and maintained the killer phenotype of the parent yeast strain. They were genetically improved by elimination of recessive growth-retarding alleles followed by spore clone selection according to the must fermentation kinetics and the organoleptic quality of the wine. Some mutants were tested in industrial winemaking and were easily monitored during must fermentation using a simple plate assay. They accounted for more than 95% of the total yeasts in the must, and the resulting wine had as good a quality as those made with standard commercial wine yeasts.     

Phenolic compounds and browning in sherry wines subjected to oxidative and biological aging

The composition in hydroxybenzoic and hydroxycinnamic acids, hydroxycinnamic esters, tyrosol, syringaldehyde, and flavan-3-ol derivatives of three different types of sherry wine obtained by aging of the same starting wine under different conditions was studied. So-called "fino" wine was obtained by biological aging under flor yeasts, "oloroso" wine by oxidative aging, and "amontillado" wine by a first stage of biological aging followed by a second oxidative step. On the basis of the results, the wines subjected to oxidative aging exhibited higher phenol contents, in addition to scarcely polar compounds absorbing at 420 nm that were absent in the wines obtained by biological aging. Taking into account that flavan-3-ol derivatives play an important role in wine browning, a model catechin solution was inoculated with flor yeast which, contrary to the findings of other authors in the absence of yeasts, formed no colored compounds. This different behavior may account for the resistance to browning of pale sherry wines in the presence of flor yeasts.     

Rhodamine-pink as a genetic marker for yeast populations in wine fermentation

Winemaking with selected yeasts requires simple techniques to monitor the inoculated yeast. New high-concentration rhodamine-resistant mutants and low-concentration rhodamine-pink mutants, easy to detect by replica-plate assay, were obtained from selected wine yeasts. The rhodamine-pink mutations were dominant and were located at the pdr5 locus that encodes for the Pdr5 ATP-binding cassette multidrug resistance transporter. The mutants were genetically stable but had lost the killer phenotype of the parent yeast strain. They were genetically improved by elimination of recessive growth-retarding alleles followed by crossing with selected killer wine yeasts. Several spore-clones were selected according to their must fermentation kinetics and the organoleptic quality of the wine. Some spore-clones were tested in industrial winemaking, and they were easily monitored during must fermentation using a simple color-plate assay. They accounted for >96% of the total yeasts in the must, and the resulting wine had as good a quality as those made with standard commercial wine yeasts. The rhodamine-pink yeasts may also be detected by direct seeding onto rhodamine agar or by observation under fluorescence microscopy. These possibilities greatly reduce the time of analysis and make the monitoring procedure for rhodamine-pink yeasts faster, easier, and cheaper than for the genetically marked wine yeasts obtained previously.     

Determination of total lipid and lipid subclasses in meat and meat products

Current interest in physiological and nutritional activities of the sterol, polyunsaturated fatty acid, and polar lipid fractions of meats and other foods indicates that analytical methods for lipids should be evaluated on their ability to recover and quantitate these classes. Current methods of lipid isolation furnish an extract that is dependent on the solvent(s) used, the type of food material, the temperature of extraction, and the relative proportions of the lipid classes present. Extraction with ethers or other relatively nonpolar solvents removes principally the neutral fats and nonpolar lipids. For an approximation of the crude fat content, such extraction is often sufficient, because the nonpolar fraction generally constitutes over 90% of the total lipids present. The polar lipids include the biochemically important (omega-3) and (omega-6) polyunsaturated fatty acid classes; thus, the method of lipid extraction of food products becomes relevant for a more complete and valuable characterization of their nutritional value. The various methods of lipid determination for meat products are examined for their total recovery of these important lipid groups. A sequential extraction in conjunction with subsequent analytical methods is recommended.     

HPLC separation and NMR structural elucidation of sn-1,2-, 2,3-, and 1,3-diacylglycerols from olive oil as naphthylethylurethane derivatives

In this study, sn-1,2-, sn-2,3-, and sn-1,3-diacylglycerols were isolated from olive oil, and their urethane derivatives (urethanes) were prepared. Normal-phase high-performance liquid chromatography (NP-HPLC) separation of the urethane isomers was performed and the separate classes were studied by nuclear magnetic resonance (NMR). The use of 1H NMR and homo- and heteronuclear 2D techniques provided a great amount of information in a very short time, particularly when a high-field NMR instrument (700 MHz) was used. Particularly diagnostic for this kind of compound was the glyceridic moiety that presents typical chemical shifts both for carbon and hydrogen. These studies show the usefulness of NMR spectroscopy to recognize clearly the sn-1,3- and, moreover, sn-1,2- with respect to sn-2,3-diacylglycerols, although very minor differences occur between them.     

Influence of yeast macromolecules on sweetness in dry wines: role of the saccharomyces cerevisiae protein Hsp12

Yeast autolysis during lees contact influences the organoleptic properties of wines especially by increasing their sweet taste. Although observed by winemakers, this phenomenon is poorly explained in enology. Moreover, the compounds responsible for sweetness in wine remain unidentified. This work provides new insights in this way by combining sensorial, biochemical and genetic approaches. First, we verified by sensory analysis that yeast autolysis in red wine has a significant effect on sweetness. Moderate additions of ethanol or glycerol did not have the same effect. Second, a sapid fraction was isolated from lees extracts by successive ultrafiltrations and HPLC purifications. Using nano-LC-MS/MS, peptides released by the yeast heat shock protein Hsp12p were distinctly identified in this sample. Third, we confirmed the sweet contribution of this protein by sensorial comparison of red wines incubated with two kinds of yeast strains: a wild-type strain containing the native Hsp12p and a deletion mutant strain that lacks the Hsp12p protein (Δ°HSP12 strain). Red wines incubated with wild-type strain showed a significantly higher sweetness than control wines incubated with Δ°HSP12 strains. These results demonstrated the contribution of protein Hsp12p in the sweet perception consecutive to yeast autolysis in wine.     

Changes in nitrogen compounds in must and wine during fermentation and biological aging by flor yeasts.

Urea, ammonium, and free amino acid contents were quantified in a must from Vitis vinifera cv. Pedro Ximenez grapes and in fermented wine and after a short aging of this wine by Saccharomyces cerevisiae race capensis yeast under variable oxygen availability conditions. The previous compounds were also determined in a wine in which the nitrogen source was depleted by the same race of flor yeast (old wine) and also following the addition of ammonium ion, L-glutamic acid, and L-proline. Under specific conditions such as low oxygen level and the absence of some nutrients, the yeasts release some amino acids including L-threonine, L-tryptophan, L-cysteine, and L-methionine to the medium. These amino acids must originate primarily in a de novo synthesis from ethanol that regenerates NAD(P)+. On the basis of these results, the yeasts may be able to use amino acids not only as nitrogen sources but also as redox agents to balance the oxidation-reduction potential under conditions of restricted oxygen, when electron transport along the respiratory chain may be hindered or limited.