Formation pathways of ethyl esters of branched short-chain fatty acids during wine aging

The particular behavior during wine aging of fermentative branched fatty acid ethyl esters, related to yeast nitrogen metabolism, compared that of their straight-chain analogues, related to yeast lipid metabolism, was first checked in 1-5 year aged Muscadet wines. Quantitative SIDA measurements showed that the levels of the former increased, whereas those of the latter decreased. Then, three hypothetical pathways suggested in the literature to explain these variations of branched esters were investigated. Two Muscadet and Sylvaner wines were spiked with levels of deuterated isobutanoic acid and its ethyl ester, similar to those of their natural analogues, then they were submitted to model aging. Quantitative SIDA measurements on the formation of these natural and labeled ethyl esters from the corresponding acids revealed that the behavior of the natural and labeled compounds were similar. The acid levels were much higher than the ester levels in the initial young wine, and a significant upward trend of their esterification ratios to those of the acid-ester equilibrium was observed with aging. Thus, this equilibrium proved to be the most effective in generating the branched fatty acid ethyl esters during wine aging. In contrast, the formation of these acids by Strecker-type degradation of wine amino acids in the conditions of the model aging or by hydrolysis of their glycoconjugates proved to be ineffective.     

Application of ethyl esters and d3-methyl esters as internal standards for the gas chromatographic quantification of transesterified fatty acid methyl esters in food

Ethyl esters (FAEE) and trideuterium-labeled methyl esters (d3-FAME) of fatty acids were prepared and investigated regarding their suitability as internal standards (IS) for the determination of fatty acids as methyl esters (FAME). On CP-Sil 88, ethyl esters of odd-numbered fatty acids eluted approximately 0.5 min after the respective FAME, and only coelutions with minor FAME were observed. Depending on the problem, one or even many FAEE can be added as IS for the quantification of FAME by both GC-FID and GC-MS. By contrast, d3-FAME coeluted with FAME on the polar GC column, and the use of the former as IS requires application of GC-MS. In the SIM mode, m/z 77 and 90 are suggested for d3-methyl esters of saturated fatty acids, whereas m/z 88 and 101 are recommended for ethyl esters of saturated fatty acids. These m/z values give either no or very low response for FAME and can thus be used for the analysis of FAME in food by GC-MS in the SIM mode. Fatty acids in sunflower oil and mozzarella cheese were quantified using five saturated FAEE as IS. Gravimetric studies showed that the transesterification procedure could be carried out without of loss of fatty acids. GC-EI/MS full scan analysis was suitable for the quantitative determination of all unsaturated fatty acids in both food samples, whereas GC-EI/MS in the SIM mode was particularly valuable for quantifying minor fatty acids. The novel GC-EI/MS/SIM method using fatty acid ethyl esters as internal standards can be used to quantify individual fatty acids only, that is, without determination of all fatty acids (the common 100% method), although this is present. This was demonstrated by the exclusive quantification of selected fatty acids including methyl-branched fatty acids, erucic acid (18:1n-9trans), and polyunsaturated fatty acids in cod liver oil and goat's milk fat.     

Sweet-like off-flavor in Aglianico del Vulture wine: ethyl phenylacetate as the mainly involved compound

Interest in high-quality and peculiar products is a recent trend in the enological field; for this reason, production of wines from autochthonous vine varieties is requested by consumers. Aglianico wine from the Italian region "Basilicata" is an example of a promising product strictly connected to the territory; nevertheless, it is affected by a frequent sweet-like off-flavor. In this study the compositional cause of this off-flavor was investigated by SPME-GC-olfactometry, SPME-GC-MS, and sensory tests. Ethyl phenylacetate (EPhA) was found to be the compound mainly responsible, and its sensory threshold was determined near 73 microg/L; products with the odorant concentration near and up to these values were always recognized as significantly different from the other wines and were often far from wine technical pleasantness; besides EPhA gave to the wines a strong honey-like character. Some preliminary hypotheses about its mechanism of formation (shikimate pathway) are presented in this study: these hypotheses could explain the correlation between EPhA and volatile phenols that was found by both sensory tests and GC quantitative analysis of wines affected by different levels of defect.     

Hydroxycinnamic acids and ferulic acid dehydrodimers in barley and processed barley

Hydroxycinnamic acid content and ferulic acid dehydrodimer content were determined in 11 barley varieties after alkaline hydrolysis. Ferulic acid (FA) was the most abundant hydroxycinnamate with concentrations ranging from 359 to 624 microg/g dry weight. p-Coumaric acid (PCA) levels ranged from 79 to 260 microg/g dry weight, and caffeic acid was present at concentrations of <19 microg/g dry weight. Among the ferulic acid dehydrodimers that were identified, 8-O-4'-diFA was the most abundant (73-118 microg/g dry weight), followed by 5,5'-diFA (26-47 microg/g dry weight), the 8,5'-diFA benzofuran form (22-45 microg/g dry weight), and the 8,5'-diFA open form (10-23 microg/g dry weight). Significant variations (p < 0.05) among the different barley varieties were observed for all the compounds that were quantified. Barley grains were mechanically fractionated into three fractions: F1, fraction consisting mainly of the husk and outer layers; F2, intermediate fraction; and F3, fraction consisting mainly of the endosperm. Fraction F1 contained the highest concentration for ferulic acid (from 77.7 to 82.3% of the total amount in barley grain), p-coumaric acid (from 78.0 to 86.3%), and ferulic acid dehydrodimers (from 79.2 to 86.8%). Lower contents were found in fraction F2, whereas fraction F3 exhibited the lowest percentages (from 1.2 to 1.9% for ferulic acid, from 0.9 to 1.7% for p-coumaric acid, and <0.02% for ferulic acid dehydrodimers). The solid barley residue from the brewing process (brewer's spent grain) was approximately 5-fold richer in ferulic acid, p-coumaric acid, and ferulic acid dehydrodimers than barley grains.     

"Untypical aging off-flavor" in wine: synthesis of potential degradation compounds of indole-3-acetic acid and kynurenine and their evaluation as precursors of 2-aminoacetophenone

Kynurenine (1) and indole-3-acetic acid (2) are considered as potential precursors of 2-aminoacetophenone (3), which is regarded to be the aroma impact compound causing an "untypical aging off-flavor" (UTA) in Vitis vinifera wines. The mechanism of the formation of 3 was studied using model fermentation and model sulfuration media spiked with 1 or 2 as potential precursors. Possible degradation products such as kynurenamine (4) and kynurenic acid (5), or skatole (6), 2-oxoskatole (7), 2-formamidoacetophenone (8), 2-oxindole-3-acetic acid (9), and 3-(2-formylaminophenyl)-3-oxopropionic acid (10) were evaluated by HPLC-UV of the fermentation and sulfuration media and comparison with synthesized 7, 8, 9, and 10. The synthesis of the possible precursor 4-(2-aminophenyl)-2,4-dioxobutanoic acid (11), a proposed metabolite of 1 failed because a spontaneous cyclization yields 5 and N-oxo-kynurenic acid (12), but not 11. It could be shown that the formation of 3 is triggered by an oxidative degradation of 2 after sulfuration with potassium bisulfite via the intermediates 10 and 8. However, no formation of 3 occurred during sulfuration of a model wine spiked with 1 or during fermentation of a model must spiked with 1 or 2.     

脂肪酸甲酯生物柴油改善低硫柴油的润滑性能

生物柴油可作为改善低硫柴油润滑性能的天然添加剂。该文将豆蔻酸甲酯(C14:0)、棕榈酸甲酯(C16:0)、硬脂酸甲酯(C18:0)、油酸甲酯(C18:1)、亚油酸甲酯(C18:2)、亚麻酸甲酯(C18:3)、蓖麻醇酸甲酯(C18:1 OH)及蓖麻油甲酯和餐饮废油甲酯按照0.5%、1.0%、1.5%和3.0%的体积分数添加到低硫柴油中,在高频往复试验机(high-frequency reciprocating rig,HFRR)上进行润滑性能测试,探究脂肪酸甲酯的碳链长度、不饱和度及含羟基等结构特征对润滑性能的影响。结果表明,长碳链脂肪酸甲酯一般比短链润滑效果好;碳链长度为十八的脂肪酸酯中,不饱和程度即碳碳双键数目越高则润滑性能越好;而在相同碳链长度和不饱和度条件下,含羟基的蓖麻醇酸甲酯的润滑改善效果优于油酸甲酯。由多种脂肪酸酯构成的混合物生物柴油的润滑性能要优于某单一的纯脂肪酸甲酯。在低硫柴油中,当某饱和脂肪酸甲酯的体积分数比例达3.0%时,或不饱和酯的体积分数达到1.5%时,或生物柴油的体积分数达1.0%时,可使低硫柴油的润滑性能指标满足相关标准。研究脂肪酸甲酯的各种结构特征对其润滑性能的影响及作用机制,有助于筛选合适的生物柴油组分及其添加浓度作为低硫柴油的润滑添加剂。     

Multielement composition of wines and their precursors including provenance soil and their potentialities as fingerprints of wine origin

The influence of the provenance soil and vinification process on the wine multielemental composition was investigated. For this purpose, two different vineyards from the Douro wine district, Portugal, were selected. Monovarietal grapes from a 10 year old vineyard were used to produce a red table wine, in a very modern winery. Polyvarietal grapes from a 60-70 year old vineyard were used to produce a red fortified wine, similar to Port, through a traditional vinification process. The multielement compositions (Al, As, B, Ba, Be, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ga, Hf, Li, Mn, Mo, Nb, Ni, Pb, Rb, Sb, Sc, Sr, Ti, Th, Tl, U, V, W, Y, Zn, Zr, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu) of soil, grape juices (prepared in the laboratory), and samples collected in the different steps of each winemaking process were measured. Inductively coupled plasma mass spectrometry was used, after suitable pretreatment of the samples (by UV irradiation for liquid samples and high-pressure microwave digestion for soil). Both vinification processes influenced the multielement composition of the wines. Most of the elements presented similar or even lower concentrations in the wine as compared to that observed in the respective grape juice, probably as a result of precipitation or coprecipitation with suspended particles during fermentation and/or wine aging. Evidence of effective contamination during grape pressing, fermentation, and/or fining of wines (depending on the element) was observed for Cd, Cr, Cu, Fe, Ni, Pb, V, and Zn in the fortified wine and Al, Cr, Fe, Ni, Pb, and V in the table wine. Nevertheless, significant correlations were obtained between the multielement composition of the wine and the respective grape juice (R = 0.997 and 0.979 for the fortified and table wines, respectively, n = 31, P < 0.01), as well as between that in the wine (median of the two studied wines) and the provenance soil (R = 0.994, n = 19, P < 0.01), for the set of elements determined in common in the different types of samples. These results are promising concerning the usefulness of the elemental patterns of both soil and wine as fingerprints of the origin of the studied wines. Nevertheless, more wines from the same and other wine districts must be studied in order to consolidate this conclusion. The multielement compositions of the studied wines were compared with those of wines of different characteristics and origins, as well as with the respective legal threshold limit values, when available. Relatively low metal levels, below their threshold limit values, were found in all cases.     

Ability of possible DMS precursors to release DMS during wine aging and in the conditions of heat-alkaline treatment

The origin of dimethyl sulfide (DMS) produced during wine aging was examined through different assays. The production of DMS during the model aging of a wine and the concomitant decrease of residual potential DMS (PDMS), as DMS released by heat-alkaline treatment in 0.5 M sodium hydroxide at 100 degrees C for 1 h, were demonstrated. Then, dimethyl sulfoxide (DMSO), methionine sulfoxide (MSO), S-methylmethionine (SMM), and dimethylsulfonium propanoic acid (DMSPA), reported previously as possible DMS precursors, were investigated for their ability to be DMS precursors in wine in the conditions of this model aging and of the heat-alkaline treatment. The results showed that DMSO, MSO, and DMSPA could hardly be DMS precursors in the conditions used, whereas SMM appeared to be a good candidate. Finally, the use of [(2)H(6)]-DMSPA as an internal standard for PDMS determination was proposed, because it provided better reproducibility than [(2)H(6)]-DMS used as an external standard.     

Gas-liquid chromatographic determination of dehydroacetic acid in squash and wine

A gas-liquid chromatographic procedure has been developed for the determination of the preservative dehydroacetic acid (DHA) in frozen cut squash and white wine. The cleanup procedure uses the acidic character of DHA to enhance separation from interfering substances. Recoveries were 93-104% from squash fortified at 30, 65, and 130 ppm and 96-99% from wine fortified at 50, 100, and 200 ppm. The method can be used to establish that the amount of DHA used in squash does not exceed the permissible level of 65 ppm.     

Enzymatic-ultraviolet determination of L-citric acid in wine: collaborative study

A collaborative study was carried out on an enzymatic method for the determination of L-citric acid in wine, using the enzymes citrate lyase, malate dehydrogenase, and lactate dehydrogenase and the coenzyme nicotinamide-adenine dinucleotide phosphate. The study was performed by 18 laboratories using 4 blind duplicates of commercial wine. The method is simple and shows good precision. Coefficients of variation (CV) for reproducibility ranged from 1.8 to 3.4%; CVs for repeatability ranged from 0.76 to 2.62%. Analysts are cautioned to check the linear absorbance response of their spectrophotometers when performing this assay and also to take care in pipetting the relatively small volumes used in this procedure. The method has been adopted official first action.     

Steryl phenolic acid esters in cereals and their milling fractions

The steryl ferulate contents of rye and wheat grains and their milling fractions were analyzed using a reversed-phase high-performance liquid chromatographic (HPLC) method. HPLC-mass spectrometry was used for identification. In addition, steryl ferulates of some selected milling byproducts were determined. The total steryl ferulate contents of rye and wheat grains were 6.0 and 6.3 mg/100 g, respectively. Uneven distribution of steryl ferulates in the grains led to considerable differences in the milling products; their steryl ferulate contents ranged from trace amounts in flours with low ash content to 20 and 34 mg/100 g in rye and wheat brans, respectively. Campestanyl ferulate and sitostanyl ferulate were the main components, followed by campesteryl ferulate and sitosteryl ferulate, whereas sitosterol was the main component in total sterols. Among the other samples, a byproduct of rice milling (pearling dust) was the best source of steryl ferulates, its total steryl ferulate content being 119 mg/100 g, whereas no measurable amounts of steryl ferulates were measured in oat bran or pearling dust of barley. The results indicated that rye and wheat and especially their bran fractions are comparable to corn as steryl ferulate sources.     

2-chloroethyl fatty acid esters as indicators of 2-chloroethanol in black walnuts, seasoning mixes, and spices

Residues of 2-chloroethyl fatty acid esters (CEEs) and 2-chloroethanol (ECH), by-products of ethylene oxide fumigation, were determined in black walnuts, seasoning mixes, and spices. Extracts containing ECH and CEE were cleaned up by previously described procedures, and residue levels were quantitatively determined using a gas chromatograph equipped with a halogen-selective electrolytic conductivity detector. All food products that contained CEE residues also contained ECH. ECH residues ranged from less than 0.2 to 880 ppm and were less than 0.2-7 times the CEE levels found.     

Defining the ascorbic acid crossover from anti-oxidant to pro-oxidant in a model wine matrix containing (+)-catechin

An examination of the ascorbic acid-induced oxidation of (+)-catechin was carried out. Using varying concentrations of ascorbic acid in a model white winebase, it was observed that there are at least two distinct steps in its oxidation process. The first step involves the formation of species that absorb in the visible region of the spectrum, while the second step generates species of less or no absorbance in the visible region. The first step reaches an absorbance maximum when ascorbic acid is completely oxidized. In winebase solutions containing both ascorbic acid and (+)-catechin, the lag period prior to the onset of (+)-catechin oxidation was dependent on the concentration of ascorbic acid. It was also observed that the end of the lag period corresponds to the complete oxidation of ascorbic acid. Xanthylium cations were identified as a species responsible for the increase in absorbance at 440 nm post lag period. The implication of the results, for establishing a chemical basis to the ascorbic acid crossover from antioxidant to pro-oxidant, is discussed.     

GC-based analysis of plant stanyl fatty acid esters in enriched foods

Approaches for the capillary gas chromatographic (GC) based analysis of intact plant stanyl esters in enriched foods were developed. Reference compounds were synthesized by enzyme-catalyzed transesterifications. Their identities were confirmed by means of mass spectrometry. Using a medium polar trifluoropropylmethyl polysiloxane stationary phase, long-chain plant stanyl esters could be separated according to their stanol moieties and their fatty acid chains. Thermal degradation during GC analysis was compensated by determining response factors; calibrations were performed for ten individual plant stanyl esters. For the analysis of low-fat products (skimmed milk drinking yogurts), the GC separation was combined with a "fast extraction" under acidic conditions. For fat-based foods (margarines), online coupled LC-GC offered an elegant and efficient way to avoid time-consuming sample preparation steps. The robust and rapid methods allow conclusions on both, the stanol profiles and the fatty acid moieties, and thus provide a basis for the authentication of this type of functional food ingredients.     

Ellagic acid and ellagitannins affect on sedimentation in muscadine juice and wine

A mechanism for the formation of water-insoluble sediments in wines and juices made from red and white muscadine grapes (Vitis rotundifolia) was investigated as a function of processing methodology and storage. Sediments are considered quality defects in muscadine grape products, and their presence may influence consumer acceptability and expansion of retail markets. Processing regimes included both hot (70 degrees C) and cold (25 degrees C) press techniques for wine or juice production, and fermentations in contact with grape skins for 3, 5, and 7 days. Relationships between free ellagic acid (FE), total ellagitannins (ET), and total ellagic acid (TE) concentrations were evaluated initially in each product and in sediments that formed during storage for 50 and 120 days at 20 degrees C. Processing techniques influenced initial concentrations of these compounds and the extent of sediment formation. Following storage, juices generally had higher concentrations of FE in sediments compared to wines, but sedimentation was independent of initial FE or TE concentrations. Decreases in ET were observed for hot-pressed juice and skin-fermented wines after storage indicating their hydrolysis during storage and possible contribution to FE in sediments. However, quantitative analysis of the collected sediments revealed that no more than 12% FE by weight was actually present in the sediments, with the remainder consisting of either unidentified compounds or conjugated forms of ellagic acid. This work elucidated a potential mechanism for the presence of FE in muscadine wine and juice sediments through ellagitannin hydrolysis and suggests that sedimentation from mechanisms other than ellagic acid precipitation may also contribute to wine and juice quality.     

Direct methylation procedure for converting fatty amides to fatty acid methyl esters in feed and digesta samples

Two direct methylation procedures often used for the analysis of total fatty acids in biological samples were evaluated for their application to samples containing fatty amides. Methylation of 5 mg of oleamide (cis-9-octadecenamide) in a one-step (methanolic HCl for 2 h at 70 degrees C) or a two-step (sodium methoxide for 10 min at 50 degrees C followed by methanolic HCl for 10 min at 80 degrees C) procedure gave 59 and 16% conversions of oleamide to oleic acid, respectively. Oleic acid recovery from oleamide was increased to 100% when the incubation in methanolic HCl was lengthened to 16 h and increased to 103% when the incubation in methoxide was modified to 24 h at 100 degrees C. However, conversion of oleamide to oleic acid in an animal feed sample was incomplete for the modified (24 h) two-step procedure but complete for the modified (16 h) one-step procedure. Unsaturated fatty amides in feed and digesta samples can be converted to fatty acid methyl esters by incubation in methanolic HCl if the time of exposure to the acid catalyst is extended from 2 to 16 h.     

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.