A novel method for quantification of 2-methyl-3-furanthiol and 2-furanmethanethiol in wines made from Vitis vinifera grape varieties

A rapid, easy method has been developed for isolating and quantifying 2-methyl-3-furanthiol (2M3F) in wines. Until now, it was not possible to quantify this highly odoriferous compound, with a smell reminiscent of cooked meat, in wine. The original aspect of this method is the specific release of volatile thiols using a cysteamine solution applied in reverse flow to sample percolation on the basis of a p-hydroxymercuribenzoate (pHMB)-volatile thiol conjugate formed by the direct addition of pHMB to 50 mL of wine. Purification of volatile thiols in wines is much faster and easier than our previous method. This method may also be used to assay 2-furanmethanethiol in wine. This thiol's strong aroma of roasted coffee has been shown to contribute to the "roast coffee" aroma of certain wines. Assaying 2M3F by this method showed that it was present in the wines analyzed (red and white Bordeaux, Loire Valley Sauvignon blanc, white Burgundy, and Champagne) at concentrations up to 100 ng/L, i.e., significantly above the olfactory perception threshold for this compound in model dilute alcohol solution.     

Biologically important thiols in various vegetables and fruits

Biological thiols are important antioxidants, and recent studies showed that their contents vary depending on the groups of foodstuffs. Therefore, we investigated the levels of some biological thiols in various vegetables and fruits by using a sensitive high-performance liquid chromatography (HPLC) technique. Biological thiols measured in some vegetables and fruits include glutathione (L-glutamyl-L-cysteinly glycine, GSH), N-acetylcysteine (NAC), captopril [CAP (C9H15NO3S)], homocysteine (HCYS), cysteine (CYS), and gamma-glutamyl cysteine (GGC). Our results show that biological thiol contents are between 3-349 nM/g wet weight in vegetables and 4-136 nM/g wet weight in fruits. CAP is only found in asparagus (28 nM/g wet weight). Furthermore, none of the biological thiols analyzed were found in cabbages, red grapes, blackberries, apples, and peaches. Therefore, various vegetables and fruits differ significantly in their thiol contents. Oxidation of these important thiols may occur and result in the production of toxic byproducts, if they are exposed to radiation and ozone treatment for sterilization purposes. Further studies should be performed to monitor the levels of these biological thiols.     

Simple multiresidue method for monitoring of trimethoprim and sulfonamide residues in buffalo meat by high-performance liquid chromatography

A simple, specific, and rapid analytical method for the determination of trimethoprim (TMP) and three sulfonamide (SA) antimicrobial drug residues in buffalo meat is developed and validated. This method is based on a solid-phase extraction technique followed by high-performance liquid chromatography (HPLC)-photodiode array (PDA) detection. Target compounds were extracted from the meat by acetonitrile and water, cleaned up on a Bond Elute C 18 cartridge column, and separated on a RP-C 18 column during HPLC analysis. Acetonitrile along with water appears to be an excellent extractant as recovery of the analytes at maximum residues levels (MRLs) in spiked sample was in the range of 75-108%, with coefficient of variations (CVs) ranging between 1.34 and 22%. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.031 and 0.062 microg/g, respectively, for all of the compounds. Intra- and interday assay precisions of the method at 0.125 microg/g concentrations for any drug ranged between 3 and 4%. The linearities of the TMP, sulfadimidine (SDM), sulfadoxine (SDO), and sulfamethoxazole (SMX) were 0.9989, 0.9999, 0.9998, and 0.9997, respectively. For robustness, the analytical method was applied to 122 buffalo meat samples obtained from export meat processing plants.     

Concentrations of low and high molecular weight thiols in wheat dough as affected by different concentrations of ascorbic acid

Different amounts of ascorbic acid (AA) were added to flour, and the concentrations of low and high molecular weight thiols in the dough were determined. For the determination of the low molecular weight thiols, glutathione, cysteine, and the corresponding disulfides, an isotope dilution assay with a (14)C-labeled internal standard was used. For the determination of the high molecular weight thiols, a method was developed that involved derivatization of dough with Ellman's reagent, removal of excess reagent by dialysis, micro-Osborne fractionation, release of the label by reduction, and determination of reduced Ellman's reagent by reversed-phase high-performance liquid chromatography. Mixing of flour without AA led to a decrease of the glutathione and an increase of the cysteine concentration. Addition of AA reduced the concentration of both thiols to a minimum when 125 mg of AA/kg of flour was applied. Furthermore, the concentrations of high molecular weight thiols in the glutenins of flours from different wheat cultivars were determined. The values ranged from 5.6 to 8.2 micromol/kg of protein and showed a correlation between flour quality and SH concentration. On addition of AA and mixing of a dough, the concentrations of the protein thiols in the glutenins isolated from the dough increased to a maximum when 100 mg of AA/kg of flour was added. Higher concentrations of AA led to a decrease of the SH concentration. The last results are not in accordance with previously published data or with current hypotheses about the mechanism of the AA improver action.     

Formaldehyde quantitation in air samples by thiazolidine derivatization: factors affecting analysis

A new method for the determination of trace levels of formaldehyde in air was developed and validated. The method is based on the reaction of formaldehyde with cysteamine to form thiazolidine. Air samples containing trace levels of formaldehyde were prepared from paraformaldehyde. The percent yield of formaldehyde from paraformaldehyde was 85.1 +/- 1.14%. Air samples were bubbled into an aqueous cysteamine trap. Thiazolidine formed from formaldehyde and cysteamine in the trap was determined by gas chromatography with a fused silica capillary column and a nitrogen-phosphorus detector (NPD). The lowest detection level for thiazolidine was 17.2 pg, equivalent to 5.80 pg formaldehyde. The recovery efficiency of trace gas phase formaldehyde in air was greater than 90%. Formaldehyde levels in ambient laboratory air were 48.9-56.2 ppb (v/v).     

高压/酶解/闪式高速提取对金华火腿骨呈味物质的影响

以金华火腿骨为研究对象,研究不同提取方法对其呈味物质释放的影响。通过感官评价及可溶性糖、有机酸、5′-核苷酸和游离氨基酸的测定分析不同处理组间样品的差异,同时采用偏最小二乘回归法（partial least squares regression,PLSR）对提取物感官和呈味物质间进行相关性分析。感官评价发现,高压蒸煮的样品的鲜味、咸味和可接受度最高,其协同滋味的综合评分最佳;呈味物质分析发现,原液中所有呈味物质含量都最低,高压-复配酶解处理的样品中可溶性糖总量最高,高达132.68 mg/100 g;经过酶解处理样品有机酸含量显著增加（P<0.05）,达到3 733.32 mg/100 g;样品经不同处理后,5′-核苷酸含量呈现显著性增加（P<0.05）,总含量最高的是经过高压蒸煮处理的样品,质量分数高达1.24 mg/100 g,是原液和其他处理组样品的2.38～12.4倍;与样品原液相比,游离氨基酸总量都显著增加（P<0.05）,高压蒸煮处理的样品中含量最高,总质量分数为642.44 mg/100 g。借助偏最小二乘回归方法对样品的感官属性及呈味成分进行相关性分析,发现样品间具有显著性差异（P<0.05）,不同的处理方式所释放的呈味物质分布规律显著不同,高压蒸煮的样品与咸味和鲜味具有显著相关性（P<0.05）;酶解处理的样品与酸味和甜味有较强相关性,其他组样品与感官与呈味属性相关性不强。结论：不同处理方式的金华火腿骨具有不同的风味,其风味特征与多种物质有关,该研究结果为火腿骨的深度开发提供了理论指导,提高金华火腿骨的附加值。     

基于酶抑制法的自助式农药残留检测平台建立

以金华火腿骨为研究对象,研究不同提取方法对其呈味物质释放的影响。通过感官评价及可溶性糖、有机酸、5''-核苷酸和游离氨基酸的测定分析不同处理组间样品的差异,同时采用偏最小二乘回归法（partial least squares regression,PLSR）对提取物感官和呈味物质间进行相关性分析。感官评价发现,高压蒸煮的样品的鲜味、咸味和可接受度最高,其协同滋味的综合评分最佳;呈味物质分析发现,原液中所有呈味物质含量都最低,高压-复配酶解处理的样品中可溶性糖总量最高,高达132.68 mg/100 g;经过酶解处理样品有机酸含量显著增加（P<0.05）,达到3 733.32 mg/100 g;样品经不同处理后,5''-核苷酸含量呈现显著性增加（P<0.05）,总含量最高的是经过高压蒸煮处理的样品,质量分数高达1.24 mg/100 g,是原液和其他处理组样品的2.38～12.4倍;与样品原液相比,游离氨基酸总量都显著增加（P<0.05）,高压蒸煮处理的样品中含量最高,总质量分数为642.44 mg/100 g。借助偏最小二乘回归方法对样品的感官属性及呈味成分进行相关性分析,发现样品间具有显著性差异（P<0.05）,不同的处理方式所释放的呈味物质分布规律显著不同,高压蒸煮的样品与咸味和鲜味具有显著相关性（P<0.05）;酶解处理的样品与酸味和甜味有较强相关性,其他组样品与感官与呈味属性相关性不强。结论：不同处理方式的金华火腿骨具有不同的风味,其风味特征与多种物质有关,该研究结果为火腿骨的深度开发提供了理论指导,提高金华火腿骨的附加值。     

Effect of lipid composition on meat-like model systems containing cysteine, ribose, and polyunsaturated fatty acids

This paper compares the volatile constituents of model systems containing the important meat aroma precursors cysteine and ribose, with and without either methyl linoleate, an n-6 fatty acid, or methyl alpha-linolenate, an n-3 acid, both of which are present in meat. Many of the volatile compounds formed from the reaction between cysteine and ribose were not formed, or formed in lower amounts, when lipid was present. This may be due to the reaction between hydrogen sulfide, formed from the breakdown of cysteine, and lipid degradation products. In addition, cysteine and ribose modified lipid oxidation pathways, so that alcohols and alkylfurans were formed rather than saturated and unsaturated aldehydes. Several volatile compounds, which have been found at elevated levels in cooked meat from animals fed supplements high in n-3 acids, were formed when methyl alpha-linolenate reacted with cysteine and ribose. The possible effects of increasing the n-3 content of meat upon flavor formation during cooking are discussed.     

Effects of various cooking processes on the concentrations of arsenic, cadmium, mercury, and lead in foods

The effects of cooking processes commonly used by the population of Catalonia (Spain) on total arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb) concentrations in various foodstuffs were investigated. All food samples were randomly acquired in local markets, big supermarkets, and grocery stores of Reus (Catalonia). Foods included fish (sardine, hake, and tuna), meat (veal steak, loin of pork, breast and thigh of chicken, and steak and rib of lamb), string bean, potato, rice, and olive oil. For each food item, two composite samples were prepared for metal analyses, whose levels in raw and cooked (fried, grilled, roasted, and boiled) samples were determined by inductively coupled plasma-mass spectrometry (ICP-MS). The highest concentrations of As, Hg, and Pb (raw and cooked samples) were mainly found in fish, with a clear tendency, in general, to increase metal concentrations after cooking. However, in these samples, Cd levels were very close to their detection limit. In turn, the concentrations of metals in raw and cooked meat samples were detected in all samples (As) or only in a very few samples (Cd, Hg, and Pb). A similar finding corresponded to string beans, rice, and olive oil, while in potatoes, Hg could not be detected and Pb only was detected in the raw samples. In summary, the results of the present study show that, in general terms, the cooking process is only of a very limited value as a means of reducing metal concentrations. This hypothetical reduction depends upon cooking conditions (time, temperature, and medium of cooking).     

Inhibition of lipid oxidation in pork bundles processing by superheated steam frying

The effect of superheated steam treatment on the oxidative stability of lipids in packaged Zousoon (pork bundles) was investigated. The aroma quality of Zousoon samples was evaluated by sensory analysis and chromatographic analysis of volatiles. Results of this study indicated that oxidation of lipids occurred in pan-fried Zousoon after prolonged storage. Significant amounts of highly volatile compounds such as formaldehyde, acetaldehyde, acetone, and hexanal in Zousoon were identified by a modified method of cysteamine derivatization followed by gas chromatography-mass spectrometry (GC-MS) analysis. Superheated steam was found to be effective in suppressing lipid oxidation in canned Zousoon as compared with Zousoon fried by the conventional method in a frying pan. The superheated steam-fried samples had relatively low thiobarbituric acid reactive substance (TBARS) and peroxide (POV) values before and after storage, whereas samples prepared by pan frying had relatively high TBARS and POV values before and after storage. Superheated steam-fried Zousoon had superior lipid stability to that prepared by the conventional pan-frying method.     

Survey of persistent organochlorine contaminants (PCBs, PCDD/Fs, and PAHs), heavy metals (Cu, Cd, Zn, Pb, and Hg), and arsenic in food samples from Huelva (Spain): levels and health implications

Concentrations of PCBs, PCDDs, and PCDFs, heavy metals (Cu, Cd, Zn, Pb, and Hg), and arsenic have been determined in a great variety of food samples purchased in different markets across the city of Huelva, located in southwestern Spain and under strong industrial activity. All samples analyzed presented concentrations below the maximum allowed by the European Community regarding PCDD/Fs, with the exception of samples within the meat group. An estimation of the daily intake resulted in 1.15 pg of WHO(PCDD/Fs)-TEQ/kg of body weight/day for a 70 kg person and 2.63 pg of WHO-TEQ/kg of body weight/day when PCBs were included, therefore accounting for a similar or even higher percentage than PCDD/Fs and showing the importance of their inclusion in monitoring studies. Meat and meat products, together with vegetable oils and dairy products, were the major food groups contributing to the estimated daily intake. For heavy metals and arsenic, the concentrations found were under the value proposed by European regulations, and estimated daily intakes were well below those proposed by the WHO for all metals investigated. PAHs have been analyzed in food samples from marine origin, values ranging from 8.22 to 71.4 ng/g of fresh weight. Pyrene was the most abundant compound, accounting for >80% in the samples investigated. The most carcinogenic PAHs, such as benzo[a]pyrene and dibenz[a,h]anthracene, were in all cases below the limits of detection. Therefore, the samples analyzed in this survey can be considered as safe with regard to the levels obtained and the in-force legislation.     

Determination of sulfur amino acids and tryptophan in foods and food and feed ingredients: collaborative study

Samples of 4 foods, 1 animal feed, isolated soy protein, and beta-lactoglobulin were analyzed by 9 laboratories to determine concentrations of cysteine as cysteic acid, methionine as methionine sulfone, and tryptophan. Sulfur amino acids were determined by AOAC method 43.A08-43.A13 for food and feed ingredients, in which samples are oxidized with performic acid before protein hydrolysis with 6N HCl. Tryptophan was determined after protein hydrolysis with 4.2N NaOH. In both methods, free amino acids were separated by ion-exchange or reverse-phase chromatography. Each laboratory was provided with detailed methods and with sealed vials containing solutions of standards. Samples were analyzed in duplicate, and variation between laboratories was determined. Coefficients of variation between laboratories for the 6 samples ranged from 5.50 to 11.8% for methionine as methionine sulfoxide, 8.59 to 17.3% for cysteine as cysteic acid, and 3.87 to 16.1% for tryptophan. Amino acid recoveries were determined by analysis of beta-lactoglobulin and were based on expected levels of each amino acid obtained from amino acid sequence data. The mean recovery of cysteine was 97% with a range of 88-119%. For methionine, mean recovery was 98% (range 89-115%) and for tryptophan, 85% (range 59-102%). Method 43.A08-43.A13 for food and feed ingredients has been adopted official first action for determination of cysteine and methionine in processed foods. The alkaline hydrolysis method has been adopted official first action for determination of tryptophan in foods and food and feed ingredients.     

Oxidation and hydrolysis determination of sulfur amino acids in food and feed ingredients: collaborative study

Samples of 6 food and feed ingredients and a purified protein, beta-lactoglobulin, were analyzed by 7 laboratories to determine the concentrations of cysteine as cysteic acid and methionine as methionine sulfone. Samples were oxidized by reaction with performic acid before hydrolysis with 6N HCl. The free amino acids were then separated and measured by ion-exchange chromatography on dedicated amino acid analyzers. Each laboratory was provided with a detailed method as well as sealed vials containing solutions of standards. For the determination of cysteine as cysteic acid, the coefficients of variation between laboratories for duplicate samples ranged from 7.13 to 10.8% for the 6 ingredients. For the determination of methionine as methionine sulfone, the coefficients of variation between laboratories for duplicate samples ranged from 1.18 to 12.8% for the 6 ingredients. Cysteine and methionine recoveries were determined by analysis of beta-lactoglobulin and were based on expected levels of each amino acid from amino acid sequence data. The mean recovery of cysteine was 95% with a range of 91-101%. The mean recovery of methionine was 101% with a range of 98-106%. This method has been adopted official first action.     

Occurrence of 6-methoxymellein in fresh and processed carrots and relevant effect of storage and processing

The occurrence of 6-methoxymellein (6-MM) in fresh and conventionally processed carrot products (for a total of 176 samples) marketed in European locations and the effect of Alternaria spp. infection and storage conditions on 6-MM accumulation were investigated. 6-MM was found in 78% of tested samples with levels ranging from 0.02 to 76.00 microg/g, with only 1 of 79 fresh carrots exceeding the "just noticeable difference" level for 6-MM. Storage of carrots at 1 degree C was suitable to maintain low levels of 6-MM for a period of at least 17 weeks. No effect of Alternaria spp. infection was observed on 6-MM occurrence. The fate of 6-MM during carrot juice processing was also investigated by using different enzyme formulations for maceration and blanching procedures. Levels of 6-MM in blanched carrots obtained by boiling water or steam treatment were reduced by 69 or 33%, respectively, as compared to fresh carrots. No decrease in 6-MM levels was observed after maceration with pectinolytic enzyme preparations (Rapidase Carrot Juice and Ultrazym AFP-L). A reduction of 6-MM by 85 or 94% was obtained after the entire cycle of carrot juice processing, depending on the blanching procedure used.     

Liquid chromatographic determination of chloramphenicol residues in meat: interlaboratory study

A liquid chromatographic (LC) method for the determination of chloramphenicol (CAP) residues in meat at the 10 microgram/kg level was tested in an interlaboratory study. The method used, based on aqueous extraction and sample cleanup with a cartridge containing Extrelut, was published earlier. A prestudy to familiarize collaborators with the method was performed before the actual interlaboratory precision study. The meat samples used in the precision study were prepared by diluting dosed chicken and pig muscle tissues with blank tissues from other species. Fourteen laboratories received 20 meat samples; 13 laboratories actually participated in the study. Two blank samples and 2 positive samples each of pig, calf, chicken, lamb, and cow meat were tested. The chloramphenicol concentrations in the positive samples ranged from 6.5 to 21 micrograms/kg. The overall mean reproducibility coefficient of variation was 17.9% after the results per laboratory were corrected for the mean recovery obtained within each sample series. The overall mean recovery was 55.1% with a coefficient of variation of 18.0% at the 10 micrograms/kg level. The limit of detection, based on chromatograms of blank samples, was estimated to be 1.5 micrograms/kg of chloramphenicol. No false positives or false negatives were observed in the concentration range tested; only 2 false positive results above the detection limit (1.7 and 6 micrograms/kg) on a total number of 60 blank analyses (3.3%) were observed.     

Cholesterol oxidation in meat products and its regulation by supplementation of sodium nitrite and apple polyphenol before processing

The levels of cholesterol oxidation derivatives (OxChol) in eight commercial species of meat products were examined. These products contained more than 1 mg/100 g of OxChol, and 7beta-hydroxycholesterol + 5beta-epoxycholesterol (111-1092 microg/100 g), 5alpha-epoxycholesterol (80-712 microg/100 g), cholestanetriol (0-368 microg/100 g), and 7-ketocholesterol (708-1204 microg/100 g) were detected. To know the interaction of sodium nitrite supplementation against cholesterol oxidation in meat products, sausage was produced with or without varying levels of sodium nitrite and stored in the refrigerator for 15 days. As a result, cholesterol oxidation in sausage was inhibited by addition of sodium nitrite in a dose-dependent manner. This observation may be associated with inactivation of O(2)(-) radical and stabilization of polyunsaturated fatty acids (PUFAs). In fact, the levels of OxChol in sausage increased, accompanying the decrease of coexisting linoleic acid when sodium nitrite was not added to sausage meat. Thus, cholesterol oxidation in meat products seems to be considarably promoted by the oxidation of coexisting PUFAs. On the other hand, additive apple polyphenol also inhibited linoleic acid oxidation in sausage and then suppressed cholesterol oxidation through its radical scavenging effects. Therefore, apple polyphenol, having a large amount of an oligomer of catechin, may interfere with cholesterol oxidation in meat processing or storage of meat products through its antioxidative action and be useful as a new antioxitant for meat products when it is added to the original meat before processing.     

ANTIOXIDANT DEFENSE MECHANISM IN PIGEON PEA UNDER COBALT STRESS

Influence of excess cobalt (Co; 10 to 400 μM Co) on growth, biomass, Co accumulation, photosynthetic pigments, lipid peroxidation, proline, non-protein thiols and cysteine contents as well as activities of anti-oxidative enzymes was studied in pigeon pea (Cajanus cajan Mill). In pigeon pea leaves decreased concentrations of chlorophyll and carotenoids on exposure to excess Co was associated with decrease activity of catalase and super oxide dismutase and suggest antiperoxidative nature of excess Co. However, a marked increase in the activities of ascorbate peroxidase and peroxidase and enhanced levels of cysteine, non-protein thiols, and proline are suggestive of induction of antioxidants in excess Co. The threshold of toxicity (10% growth reduction) and toxicity (33% growth reduction) values of Co in pigeon pea were 75 and 160 μg g^?1in leaves, 42 and 180 μg g^?1in stem and 50 and 340 μg g^?1Co in roots, respectively.     

Identification and quantitation of 3-S-cysteinylglycinehexan-1-ol (Cysgly-3-MH) in Sauvignon blanc grape juice by HPLC-MS/MS

Precursors to varietal wine thiols are a key area of grape and wine research. Several such precursors, in the form of odorless conjugates, have been closely studied in recent years. A new conjugate has now been identified as 3-S-cysteinylglycinehexan-1-ol (Cysgly-3-MH), being the dipeptide intermediate between cysteine and glutathione precursors of tropical thiol 3-mercaptohexan-1-ol (3-MH). Authentic Cysgly-3-MH was produced via enzymatic transformation of the glutathione conjugate and used to verify the presence of both diastereomers of Cysgly-3-MH in Sauvignon blanc juice extracts. Cysgly-3-MH was added into our HPLC-MS/MS precursor method, and the validated method was used to quantify this new analyte in a selection of Sauvignon blanc juice extracts. Cysgly-3-MH was found in the highest concentrations (10-28.5 μg/L combined diastereomer total) in extracts from berries that had been machine-harvested and transported for 800 km in 12 h. This dipeptide conjugate was much less abundant than the glutathione and cysteine conjugates in the samples studied. On the basis of the results, the new cysteinylglycine conjugate of 3-MH seemingly has a short existence as an intermediate precursor, which may explain why it has not been identified as a natural juice component until now.     

Enzymatic method with polyphenol oxidase for the determination of cysteine and N-acetylcysteine

Thiols, such as cysteine and N-acetylcysteine, are included in many pharmaceutical products for their mucolytic properties. The method described here uses mushroom polyphenol oxidase (PPO) to determine two thiols and consists of measuring the lag period in the formation of the product generated as PPO acts on o-diphenol in the presence of a thiol. In the experimental conditions, o-quinone is formed enzymatically and then reacts stoichiometrically with the thiol, originating the corresponding thiol-diphenol adduct, which does not absorb visible light. Once the thiol has been used up, the o-quinone can be observed in the medium. It must be borne in mind that the inhibition of PPO is practically null at low concentrations of thiol, and the only effect observed is the formation of the thiol-diphenol adduct. In the following, an exact kinetic method capable of rapidly and accurately assaying thiols with PPO and o-diphenol is optimized and is shown to be a straightforward way of calculating thiol concentration. The method has been successfully applied to the determination of cysteine in model solutions and of N-acetylcysteine in pharmaceutical products.