Identification of amino acids in wines by one- and two-dimensional nuclear magnetic resonance spectroscopy

Amino acids are minor compounds in wines, but they have a profound influence on wine quality, and amino acids composition can be used to differentiate wines according to the vine variety, geographical origin, and year of production. The NMR signals of amino acids in NMR spectra are overlapped by the signals of other compounds present and especially by the signals of dominant compounds such as water, ethanol, and glycerol. In this work we used 1D (1)H and (13)C, 2D homonuclear COSY, TOCSY, and 2D heteronuclear HSQC and HMQC pulse sequences, also with an incorporated WET pulse sequence element that allows the simultaneous suppression of several frequencies. Complete (1)H and (13)C NMR assignments for 17 amino acids commonly present in wine and of gamma-aminobutyric acid at pH 3 have been achieved in wine sample of Sauvignon from the Coastal wine-growing region of Slovenia, vintage 1994.     

Study of the compositional changes of mango during ripening by use of nuclear magnetic resonance spectroscopy

Liquid-state NMR spectroscopy was used to follow the compositional changes in mango juice during ripening, whereas MAS and HR-MAS techniques enabled resolved (13)C and (1)H NMR spectra of mango pulps to be recorded. Spectral assignment enabled the identification of several organic acids, amino acids, and other minor components, and the compositional changes upon ripening were followed through the changes in the spectra. In pulps, sucrose was found to predominate over fructose and glucose at most ripening stages, and citric acid content decreased markedly after the initial ripening stages while alanine increased significantly. Other spectral changes reflect the complex biochemistry of mango ripening and enabled the role played by some compounds to be discussed. Some differences observed between the composition of juices and pulps are discussed. This work shows that NMR spectroscopy enables the direct characterization of intact mango pulps, thus allowing the noninvasive study of the overall biochemistry in the whole fruit.     

Nuclear magnetic resonance profiling of wine blends

Nuclear magnetic resonance (NMR) profiling is used for characterization of monocultivar binary wine mixtures. Classification and quantification of the relative amount of wine in the mixture are made in two steps. First, each sample is classified as a mixture of a determined type by solving the appropriate classification problem using NMR profiles. The relative amount of the two corresponding monovarietal wines is then evaluated by multilinear regression of a selected set of NMR variables. Linear discriminant analysis (LDA), used in the classification step, gives a very good separation among the different mixture classes. On the other hand, a single layer artificial neural network, used to solve the multilinear problem, gives the relative amount of wine type in the mixture with a precision of about 10%.     

Identification of anthocyanin-flavanol pigments in red wines by NMR and mass spectrometry

Three newly formed Port wine pigments were isolated by Toyopearl HW-40(s) gel chromatography and semipreparative HPLC. Furthermore, the pigments were identified by mass spectrometry (LC/MS) and NMR techniques (1D and 2D). These anthocyanin-derived pigments showed UV-visible spectra different from those of the original grape anthocyanins. These pigments correspond to malvidin 3-glucoside linked through a vinyl bond to either (+)-catechin, (-)-epicatechin, or procyanidin dimer B3 [(+)-catechin-(+)-catechin]. NMR data of these pigments are reported for the first time.     

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.     

Plant species affect the concentration of free sugars and free amino acids in different types of soil

Substantial amounts of low molecular weight organic compounds (LMWs) such as sugars and amino acids are transferred from plant roots into soil. These substances are released due to decomposition processes or leaching (exudation). Afterwards they can be metabolized by soil microorganisms into different compounds, or they can be partially re‐absorbed by the plants. The aim of this study was to clarify the influence of five wild plant species on the composition and pool sizes of LMWs extractable from three different soils. Four of the five species caused significant changes in soil LMW pools. In Chernozem, the sugar concentrations of soil with plants were up to 60 % higher than those of the bulk reference soil, and amino acids increased by as much as 207 %. The relative abundance of free amino acids in roots did not correlate with the relative abundance of amino acids in soil after six weeks of plant growth. The relative abundance of soil amino acids, that increased after plant growth, was strongly dependent on the type of soil and on the plant species present. We suggest that rather than rhizodeposition being dependent on soil type, it reflects differential microbial metabolization of amino acids in the respective soils.     

Reaction between hydroxycinnamic acids and anthocyanin-pyruvic acid adducts yielding new portisins

Three new anthocyanin-derived pigments were found to occur in a 2-year-old Port red wine. Their structures were elucidated through LC/DAD-MS and NMR analysis and were found to correspond to a pyranoanthocyanin moiety linked to substituted cinnamyl substituents. The structures of these compounds are very similar to the one already reported for portisins, with a phenolic moiety replacing the catechin moiety. The newly formed anthocyanin-derived compounds display a bathochromic shift of the lambdamax ( approximately 540 nm) when compared with their anthocyanin-pyruvic acid adduct precursor (lambdamax = 511 nm), which may be due to the extended conjugation of the pi electrons in the structures of those pigments. Studies performed in model solutions helped to clarify the formation mechanism of these pigments that can result from the nucleophilic attack of the olefinic double bond of a hydroxycinnamic acid to the eletrophilic C-10 position of the anthocyanin-pyruvic acid adduct, followed by the loss of a formic acid molecule and decarboxylation. The chromatic characterization of these malvidin-3-glucoside-derived compounds revealed a higher resistance to discoloration against the nucleophilic attack by water and bisulfite when compared to malvidin-3-glucoside that is almost converted into its colorless hemiacetal form. However, the resistance to discoloration of these new pigments is not as high as the one reported for catechin-derived portisins. This could be explained by the presence of a smaller group (hydroxycinnamyl group), which does not protect so efficiently the chromophore against nucleophilic attack at the C-2 position. The occurrence of these pigments in red wine highlights new chemical pathways involving anthocyanin-pyruvic acid derivatives as precursors for the formation of new pigments in subsequent stages of wine aging that may contribute to its color evolution.     

Differentiation of natural and synthetic phenylalanine and tyrosine through natural abundance 2H nuclear magnetic resonance

The natural abundance deuterium NMR characterization of samples of the amino acids tyrosine (1) and phenylalanine (2), examined as the acetylated methyl esters 4 and 6, has been performed with the aim to identify by these means the contribution in animals of the hydroxylation of the diet l-phenylalanine (2) to the formation of l-tyrosine (1), a feature previously revealed on the same samples through the determination of the phenolic delta(18)O values. The study, which includes also the NMR examination of benzoic acid (5) from 2 and of tyrosol (7) from 1, substantially fails in providing the required information because the mode of deuterium labeling of tyrosine samples of different origins is quite similar but indicates a dramatic difference in the deuterium labeling pattern of the two amino acids 1 and 2. The most relevant variation is with regard to the deuterium enrichments at the CH(2) and CH positions, which are inverted in the two amino acids of natural derivation. Moreover, whereas the diastereotopic benzylic hydrogen atoms of l-tyrosine (1) appear to be equally deuterium enriched, in l-phenylalanine (2) the (D/H)(3)(R)() > (D/H)(3)(S)(). Similarly, benzoic acid (5) shows separate signals for the aromatic deuterium nuclei, which are quite indicative of the natural or synthetic derivation. The mode of deuterium labeling of the side chain of 1 and 2 is tentatively correlated to the different origins of the two amino acids, natural from animal sources for l-tyrosine and biotechnological probably from genetically modified microorganisms for l-phenylalanine.     

Gas chromatography–mass spectrometry associated global analysis of rice root exudates under aseptical conditions

Components of rice root exudates were surveyed by gas chromatography–mass spectrometry (GC-MS) and the effect of phosphate (P) starvation was investigated. Rice seedlings were aseptically grown in controlled environments under P-supplied (+P) or P-deficient (–P) conditions. Root exudates were collected from the culture solution 4 and 8 days after treatment (D4 and D8, respectively), which contain the first and second 4-day intervals. The collected solution was lyophilized and then eluted with methanol. In the present study, primary metabolites, such as sugars, amino acids, organic acids and fatty acids, were mainly targeted. After derivatization with methoxyamine hydrochloride and N-methyl-N-trifluoroacetamide, the samples were analyzed by GC-MS. Sugars and various amino acids (such as aspartic acid and l -isoleucine) were mainly detected and the components changed with the growth stage rather than the P content in the culture solution. The relative amount of sugars (such as glucose and fructose) and amino acids (such as aspartic acid and l -isoleucine) decreased in the D8 samples. Of the 78 detected compounds, orthogonal partial least-squares discrimination analysis (O-PLS-DA) showed that four compounds were related to the P content of the nutrient solution and 58 were related to the sampling date (days after treatment). The results suggest that the physiological change during seedling development was unexpectedly large and that the response to the environment was rather small.     

Microclimate influence on mineral and metabolic profiles of grape berries

The grape berry microclimate is known to influence berry quality. The effects of the light exposure of grape berry clusters on the composition of berry tissues were studied on the "Merlot" variety grown in a vineyard in Bordeaux, France. The light exposure of the fruiting zone was modified using different intensities of leaf removal, cluster position relative to azimuth, and berry position in the cluster. Light exposures were identified and classified by in situ measurements of berry temperatures. Berries were sampled at maturity (>19 Brix) for determination of skin and/or pulp chemical and metabolic profiles based on (1) chemical and physicochemical measurement of minerals (N, P, K, Ca, Mg), (2) untargeted 1H NMR metabolic fingerprints, and HPLC targeted analyses of (3) amino acids and (4) phenolics. Each profile defined by partial least-square discriminant analysis allowed us to discriminate berries from different light exposure. Discriminant compounds between shaded and light-exposed berries were quercetin-3-glucoside, kaempferol-3-glucoside, myricetin-3-glucoside, and isorhamnetin-3-glucoside for the phenolics, histidine, valine, GABA, alanine, and arginine for the amino acids, and malate for the organic acids. Capacities of the different profiling techniques to discriminate berries were compared. Although the proportion of explained variance from the 1H NMR fingerprint was lower compared to that of chemical measurements, NMR spectroscopy allowed us to identify lit and shaded berries. Light exposure of berries increased the skin and pulp flavonols, histidine and valine contents, and reduced the organic acids, GABA, and alanine contents. All the targeted and nontargeted analytical data sets used made it possible to discriminate sun-exposed and shaded berries. The skin phenolics pattern was the most discriminating and allowed us to sort sun from shade berries. These metabolite classes can be used to qualify berries collected in an undetermined environment. The physiological significance of light and temperature effects on berry composition is discussed.     

Nuclear magnetic resonance spectroscopic study of beta-lactoglobulin interactions with two flavor compounds,gamma-decalactone and beta-ionone

Interactions between a well-characterized protein, beta-lactoglobulin, and two flavor compounds, beta-ionone and gamma-decalactone, were studied by 2D NMR spectroscopy. NMR spectra were recorded in aqueous solution (pH 2.0, 12 mM NaCl, 10% D(2)O) under conditions such that beta-lactoglobulin is present in a monomeric state. TOCSY and NOESY spectra were recorded on the protein and the complexes between protein and ligands. The spectra of the NH-CH(alpha) region showed the cross-signals due to the coupling between N- and C-bonded protons in the polypeptide backbone. The observed chemical shift variations in the presence of ligands can be assigned to changes in the protein conformation. It appears that the side chains of several amino acids are affected by binding of gamma-decalactone point into the central cavity (Leu46, Ile56, Met107, and Gln120), whereas binding of beta-ionone affects amino acids located in a groove near the outer surface of the protein (Leu104, Tyr120, and Asp129), as illustrated by molecular visualization. This NMR study provides precise information of the location of binding and confirms the existence of two different binding sites for aroma compounds on beta-lactoglobulin, which was suggested in previous competition studies by fluorometry or affinity chromatography and by structural information obtained from infrared spectroscopy.     

Identification of soil organic phosphorus by 31P nuclear magnetic resonance spectroscopy

Abstract

Soil samples from different land use systems were collected before cropping (in spring) and after harvest (in fall) for organic phosphorus (P) extractions by 0.4M sodium hydroxide (NaOH) and characterization by ³¹P nuclear magnetic resonance spectroscopy. To prevent hydrolysis of organic P compounds prior to sample concentration, NaOH was removed from the NaOH soil extracts using a G‐25 Sephadex column. The ³¹P NMR spectra in the NaOH soil extracts showed the presence of glucose‐6 phosphate (up to 64%), glycerophosphate (up to 45%), nucleoside monophosphates (up to 91%), and polynucleotides (up to 58%) as the major forms of organic P in soils. The relative concentration of nucleoside monophosphates and polynucleotides decreased in some of the soils after harvest. The ³¹P NMR spectra of the extracts also revealed the presence of phosphoenolpyruvates, a previously unreported form of soil organic P.     

Isolation and structural characterization of new acylated anthocyanin-vinyl-flavanol pigments occurring in aging red wines

Three newly formed pigments were detected and isolated from a 2-year-old Port wine through TSK Toyopearl HW-40(S) gel column chromatography and characterized by UV-visible spectrophotometry, NMR, and mass spectrometry (ESI/MS). (1)H NMR and (13)C NMR data for these pigments obtained using 1D and 2D NMR techniques (COSY, NOESY, gHSQC, and gHMBC) are reported for the first time. The structure of the pigments was found to correspond to the vinyl cycloadducts of malvidin 3-coumaroylglucoside bearing either a procyanidin dimer or a flavanol monomer ((+)-catechin or (-)-epicatechin). Additionally, conformational analysis was performed for one of these newly formed pigment using computer-assisted model building and molecular mechanics. A chemical nomenclature is proposed to unambiguously name this new family of anthocyanin-derived pigments.     

Isolation and structural characterization of new anthocyanin-derived yellow pigments in aged red wines

Two newly formed yellow pigments that revealed unique spectral features were detected and isolated from an aged Port red wine by TSK Toyopearl HW-40(s) gel chromatography and characterized by UV-visible spectrophotometry, 1H NMR and 13C NMR, and mass spectrometry (LC-ESI/MS). The UV-vis spectra of these pigments showed maximum absorption at 478 nm that is significantly hypsochromically shifted from those of original grape anthocyanins and other pyranoanthocyanins, exhibiting a more yellow hue in acidic solution. The structures of these pigments correspond to methyl-linked pyranomalvidin 3-glucoside and its respective coumaroyl glucoside derivative. They were shown to arise from the reaction between acetoacetic acid and genuine grape anthocyanins. Isolation and NMR identification using 1D and 2D NMR techniques are reported for the first time for this new family of anthocyanin-derived yellow pigments occurring in red wines.     

Chemical and biochemical transformations during the industrial process of sherry vinegar aging

The work described here concerns a study of the chemical and biochemical transformations in sherry vinegar during the different aging stages. The main factors that contribute to the nature and special characteristics of sherry vinegar are the raw sherry wine, the traditional process of acetic acid fermentation in butts (the solera system), and the physicochemical activity during the aging process in the solera system. A number of chemical and biochemical changes that occur during sherry vinegar aging are similar to those that take place in sherry wine during its biological activity process (where the wine types obtained are fino and manzanilla) or physicochemical activity process (to give oloroso wines). Significant increase in acetic acid levels was observed during the biological activity phase. In addition, the concentrations of tartaric, gluconic, succinic, and citric acids increased during the aging, as did levels of amino acids and acetoin. A color change was also produced during this stage. Glycerol was not consumed by acetic acid bacteria, and levels of higher alcohols decreased because of the synthesis of acetates. On the other hand, in the physicochemical phase the microbiological activity was lower. Concentrations of some cations increased because of evaporation of water through the wood. A color change was also produced in this stage. Concentrations of different amino acids decreased because of reaction with carbonyl compounds. A precipitation of potassium with tartaric acid was also observed.     

Nondestructive quantification of organic compounds in whole milk without pretreatment by two-dimensional NMR spectroscopy

In this study, various organic compounds in commercial whole milk were quantified simultaneously by 1H 1D and 1H - 13C HSQC 2D NMR spectra without any pretreatment. 2D NMR spectroscopy was applied to quantification of milk compounds for the first time. Milk fat content was easily determined to be 3.6 +/- 0.1%, and the lactose content was 47.8 +/- 1.0 mg/mL by 1H NMR spectra. From 1H-13C HSQC spectra, the concentrations of citrate, N-acetylcarbohydrates, and trimethylamine were determined to be 3.2 +/- 0.2, 2.9 +/- 0.1, and 4.0 +/- 0.6 mM, respectively. The latter two compounds were quantified in milk for the first time. Butyric acid, total monounsaturated fatty acids, and total polyunsaturated fatty acids of triacylglycerols were 6.2 +/- 0.5, 9.1 +/- 0.9, and 2.9 +/- 0.3 mM, respectively. The fatty acid compositions (mol %) of triacylglycerols were then calculated and were observed to be in good agreement with reference values. The results indicated that 1H 1D and 1H-13C HSQC 2D NMR spectroscopy is useful for the rapid and nondestructive determination of various compounds in milk.     

红曲中非酿酒酵母的分离鉴定及其对黄酒风味成分的影响

为探究红曲中非酿酒酵母对黄酒风味成分的影响,首先对红曲中的酵母菌株进行分离纯化,并采用分子生物学方法进行鉴定,然后将分离的非酿酒酵母菌株分别与红曲霉相结合,以纯种发酵的方式酿造红曲黄酒,分析各酒样中有机酸、氨基酸、挥发性物质等风味成分的组成及含量。结果表明,分离获得的4株非酿酒酵母CKS-2为威克汉姆酵母(Wickerhamomyces sp.)、CKS-3为季也蒙毕赤酵母(Meyerozyma guilliermondii)、CKS-4为胶红酵母(Rhodotorula mucilaginosa)、CKS-7为德尔布有孢酵母(Torulaspora delbrueckii),其中,CKS-7具有较好的产乙醇和苹果酸能力,发酵酒样中丝氨酸、组氨酸、色氨酸和亮氨酸的含量也较高,并且具有良好的合成2-苯乙醇、异戊醇、棕榈酸乙酯等挥发性风味成分的能力。本研究对红曲黄酒品质提升,以及筛选潜在可应用于红曲黄酒酿造的优良非酿酒酵母菌株具有一定作用。     

Studies on amino acids generated from rape seed cake in the soil

Among the several kinds of seed cakes, the cake made from rape seeds has been most widely employed for tobacco cultivation. Numerous investigations have been conducted by many workers, on the mineralization of organic nitrogen in rape seed cake applied to the soil and on the absorption of inorganic nitrogen degraded from the seed cake in soil by tobacco plants (8,9). However, little has been known about the organic nitrogen compounds which are generated through the process of decomposition of the seed cake. Recently, it has been believed that the plant roots are able to absorb the organic nitrogen compounds such as amino acids (10,11, 12). Therefore, it is considered that these organic nitrogen compounds produced by mineralization of the seed cake applied to the soil would have some effects upon the yield and quality of some crops such as tobacco plant with their physiological effects on plants. This paper deals with the changes in amino acids produced by the mineralization of rape seed cake in the soil.     

New qualitative approach in the characterization of antioxidants in white wines by antioxidant free radical scavenging and NMR techniques

The aim of this study was to obtain new information on antioxidant compounds in white wines. For this purpose, white wine degradation was promoted by a forced aged protocol, and six normally aged white wines from different vintages were analyzed. Both normal and forced aged wines were sequentially extracted using hexane and ethyl acetate. Apolar antioxidants were removed using hexane, and polar antioxidants were extracted with ethyl acetate. This last residue was subject to partial re-extraction with hexane and acetone. The antioxidant capacity of the wines and of each fraction was evaluated by two free radical methods, ABTS and DPPH. Normal aging provides a decrease in the total antioxidant capacity of wines. The antioxidant activity of ethyl acetate/acetone extracts was approximately 95% higher than that found for the hexane extracts. Concerning the forced aged wines, results showed that the wine submitted to a temperature of 60 degrees C for 21 days had higher antioxidant activity than that submitted to a temperature of 20 degrees C. With regard to the ethyl acetate/acetone extracts, oxygen and temperature treatment leads to a decrease in their antioxidant activity. NMR analysis was performed in the highest antioxidant capacity organic fractions (ethyl acetate/acetone extracts) and in the aqueous fraction of the control wine (T = 20 degrees C), in order to attempt the characterization of species involved in oxygen protection. Possible structures of antioxidant compounds in white wines were proposed. Two of these are tyrosol-like structures. This molecule is a well-known phenolic compound in wine, and it is reported to have antioxidative effects.