Hydrogen isotopic profile in the characterization of sugars. Influence of the metabolic pathway

The site-specific natural hydrogen isotope ratios of plant metabolites determined by 2H nuclear magnetic resonance (SNIF-NMR method) can provide powerful criteria for inferring mechanistic and environmental effects on biosynthetic pathways. This work examines the potential of isotopic profiles for the main constituents of carbohydrates, glucose and fructose, to distinguish different photosynthetic pathways. An appropriate analytical strategy, involving three suitable isotopic probes, has been elaborated with a view to measuring simultaneously, in conditions devoid of isotopic perturbations, all (or nearly all) of the carbon-bound hydrogen isotope ratios. It is shown that the type of photosynthetic metabolism, either C3 (sugar beet, orange, and grape), C4 (maize and sugar cane), or CAM (pineapple), and the physiological status of the precursor plant exert strong influences on the deuterium distribution in the sugar molecules. Consequently, this isotopic fingerprint may be a rich source of information for the comparison of mechanisms in metabolic pathways. In addition, it can provide complementary criteria to ethanol as a probe for the origin of sugars.     

Improved detection of sugar addition to maple syrup using malic acid as internal standard and in 13C isotope ratio mass spectrometry (IRMS)

Stable carbon isotope ratio mass spectrometry (delta13C IRMS) was used to detect maple syrup adulteration by exogenous sugar addition (beet and cane sugar). Malic acid present in maple syrup is proposed as an isotopic internal standard to improve actual adulteration detection levels. A lead precipitation method has been modified to isolate quantitatively malic acid from maple syrup using preparative reversed-phase liquid chromatography. The stable carbon isotopic ratio of malic acid isolated from this procedure shows an excellent accuracy and repeatability of 0.01 and 0.1 per thousand respectively, confirming that the modified lead precipitation method is an isotopic fractionation-free process. A new approach is proposed to detect adulteration based on the correlation existing between the delta13Cmalic acid and the delta13Csugars-delta13Cmalic acid (r = 0.704). This technique has been tested on a set of 56 authentic maple syrup samples. Additionally, authentic samples were spiked with exogeneous sugars. The mean theoretical detection level was statistically lowered using this technique in comparison with the usual two-standard deviation approach, especially when maple syrup is adulterated with beet sugar : 24 +/- 12% of adulteration detection versus 48 +/- 20% (t-test, p = 7.3 x 10-15). The method was also applied to published data for pineapple juices and honey with the same improvement.     

Detection of sugar syrups in apple juice by delta(2)H per thousand and delta(13)C per thousand analysis of hexamethylenetetramine prepared from fructose

An improved procedure for determining (13)C and (2)H isotope ratios, using gas chromatography-isotope ratio mass spectrometry (GC-IRMS), has been developed for identifying the addition of low cost commercial sugar syrups to apple juices and related products. Isotopic techniques are commonly used to identify the addition of low cost sugars to fruit juices and are difficult to circumvent as it is not economically viable to change the isotopic ratios of the sugars. The procedure utilizes the derivative hexamethylenetetramine, which is produced through chemical transformation of a sugar degradation product and provides position-specific (13)C and (2)H ratios that relate to the parent sugar molecule. The new procedure has advantages over methods using nitro-sugar derivatives in terms of analysis time and sensitivity. The differences between the delta(2)H per thousand and delta(13)C per thousand values of the 100 authentic apple juices and beet and cane commercial sugar syrups permit their addition to be reliably detected.     

Liquid chromatography coupled to isotope ratio mass spectrometry: a new perspective on honey adulteration detection

A new procedure to determine individual sugar (sucrose, glucose, and fructose) 13C isotope ratios, using liquid chromatography-isotope ratio mass spectrometry (HPLC-IRMS), has been developed to improve isotopic methods devoted to the study of honey authenticity. For this purpose 79 commercial honey samples from various origins were analyzed. Values of delta13Choney ranged from -14.2 to -27.2", and delta13Cprotein ranged from -23.6 to -26.9". A very strong correlation is observed between the individual sugar 13C ratios, which are altered in the event of sugar addition, even at low levels. The use of Deltadelta13C [fruct-glu], Deltadelta13C [fruct-suc], and Deltadelta13C [gluc-suc] systematic differences as an authenticity criterion permits the sugar addition [C3, beet sugar; or C4, cane sugar, cane syrup, isoglucose syrup, and high-fructose corn syrup (HFCS)] to be reliably detected (DL = 1-10%). The new procedure has advantages over existing methods in terms of analysis time and sensitivity. In addition, it is the first isotopic method developed that allows beet sugar addition detection.     

Stable carbon isotopic composition of the wine and CO2 bubbles of sparkling wines: detecting C4 sugar additions

Sparkling wines have become a popular beverage in recent years, and the production of these wines is subject to adulteration during fermentation. This study investigated the stable carbon isotopic composition (expressed as delta(13)C) of the wine and of the CO(2) bubbles produced during the second fermentation for a number of sparkling wines produced in different countries around the world. Carbon isotope ratio analyses were used to estimate the addition of sugar obtained from C(4) plants (sugar cane or corn). The average delta(13)C values of the Brazilian brut, demi-sec, and doux sparkling wines were -20.5 +/- 1.2 per thousand (n = 18), -18.1 +/- 1.3 per thousand (n = 9), and -15.8 per thousand (n = 1), respectively. These values were statistically heavier (more positive carbon isotope ratio values) than the average delta(13)C of sparkling wines produced in other parts of South America (Argentina and Chile, -26.1 +/- 1.6 per thousand, n = 5) and Europe (France, Germany, Italy, Portugal, and Spain, -25.5 +/- 1.2 per thousand, n = 12), but not statistically different from sparkling wines produced in the United States or Australia. The most likely explanation for differences in the carbon isotope ratios of wines from these different regions is the addition of C(4) sugar during the production of some sparkling wines from Australia, Brazil, and the United States. The isotopic composition of the CO(2) bubbles (delta(13)C-CO(2)) followed similar trends. The average delta(13)C-CO(2) of most of the Brazilian and Argentine sparkling wines was -10.8 +/- 1.2 per thousand (n = 23), indicating that the likely source of carbon for the second fermentation was sugar cane. Conversely, the average delta(13)C-CO(2) of most of the sparkling wines produced in Chile and Europe was -22.0 +/- 1.2 per thousand (n = 13), suggesting that a different sugar (most likely sugar beet) was most used in the second fermentation. It was concluded that in many cases, the carbon isotope ratios of sparkling wine and CO(2) bubbles can provide valuable information about the sugar sources.     

Generation of Maillard compounds from inulin during the thermal processing of Agave tequilana Weber Var. azul

During the cooking process of Agave tequilana Weber var. azul to produce tequila, besides the hydrolysis of inulin to generate fermentable sugars, many volatiles, mainly Maillard compounds, are produced, most of which may have a significant impact on the overall flavor of tequila. Exudates (agave juice) from a tequila company were collected periodically, and color, Brix, fructose concentration, and reducing sugars were determined as inulin breakdown took place. Maillard compounds were obtained by extraction with CH(2)Cl(2), and the extracts were analyzed by GC-MS. Increments in color, Brix, and reducing sugars were observed as a function of time, but a decrease in fructose concentration was found. Many Maillard compounds were identified in the exudates, including furans, pyrans, aldehydes, and nitrogen and sulfur compounds. The most abundant Maillard compounds were methyl-2-furoate, 2,3-dihydroxy-3,5-dihydro-6-methyl-4(H)-pyran-4-one, and 5-(hydroxymethyl)furfural. In addition, a series of short- and long-chain fatty acids was also found. A large number of the volatiles in A. tequilana Weber var. azul were also detected in tequila extracts, and most of these have been reported as a powerful odorants, responsible for the unique tequila flavor.     

Effects of must concentration techniques on wine isotopic parameters

Despite the robustness of isotopic methods applied in the field of wine control, isotopic values can be slightly influenced by enological practices. For this reason, must concentration technique effects on wine isotopic parameters were studied. The two studied concentration techniques were reverse osmosis (RO) and high-vacuum evaporation (HVE). Samples (must and extracted water) have been collected in various French vineyards. Musts were microfermented at the laboratory, and isotope parameters were determined on the obtained wine. Deuterium and carbon-13 isotope ratios were studied on distilled ethanol by nuclear magnetic resonance (NMR) and isotope ratio mass spectrometry (IRMS), respectively. The oxygen-18 ratio was determined on extracted and wine water using IRMS apparatus. The study showed that the RO technique has a very low effect on isotopic parameters, indicating that this concentration technique does not create any isotopic fractionation, neither at sugar level nor at water level. The effect is notable for must submitted to HVE concentration: water evaporation leads to a modification of the oxygen-18 ratio of the must and, as a consequence, ethanol deuterium concentration is also modified.     

Tequila authenticity assessment by headspace SPME-HRGC-IRMS analysis of 13C/12C and 18O/16O ratios of ethanol

By use of headspace SPME sampling and a PLOT column, on-line capillary gas chromatography-isotope ratio mass spectrometry was employed in the combustion (C) and the pyrolysis (P) modes (HRGC-C/P-IRMS) to determine the delta(13)C(VPDB) and delta(18)O(VSMOW) values of ethanol in authentic (n = 14) and commercial tequila samples (n = 15) as well as a number of other spirits (n = 23). Whereas with delta(13)C(VPDB) values ranging from -12.1 to -13.2 per thousand and from -12.5 to -14.8 per thousand similar variations were found for 100% agave and mixed tequilas, respectively, the delta(18)O(VSMOW) data differed slightly within these categories: ranges from +22.1 to +22.8 per thousand and +20.8 to +21.7 per thousand were determined for both the authentic 100% agave and mixed products, respectively. The data recorded for commercial tequilas were less homogeneous; delta(13)C(VPDB) data from -10.6 to -13.9 per thousand and delta(18)O(VSMOW) values from +15.5 to +22.7 per thousand were determined in tequilas of both categories. Owing to overlapping data, attempts to differentiate between white, rested, and aged tequilas within each of the two categories failed. In addition, discrimination of tequila samples from other spirits by means of delta(13)C(VPDB) and delta(18)O(VSMOW) data of ethanol was restricted to the products originating from C(3) as well as C(4)/CAM raw materials.     

茶叶加工与样品制备对同位素分馏和测定的影响

为考察不同加工方式和样本制备目数对茶叶同位素分馏与测定的影响,建立茶叶中4种同位素δ¹³C、δ¹⁵N、δ²H、δ¹⁸O的元素分析-稳定同位素比率质谱(EA-IRMS)分析测试方法,研究红茶和绿茶加工过程中同位素分馏及不同目数样本制备的测试影响。结果表明,通过两点法或三点法构建的校准曲线具有很好的相关性,能够满足测试精度要求;相同原料所加工红茶和绿茶中4种同位素比率无显著差异,未发现同位素分馏效应;红茶和绿茶通过研磨后过80目、100目和120目筛与未过筛4种茶叶样品之间同位素比率无显著差异(P<0.05)。因此,相同原料的非发酵(绿茶)和发酵(红茶)加工以及不同目数样本制备对4种稳定同位素测定均无显著影响。本研究结果为茶叶稳定同位素数据库构建以及样品制备提供了方法基础。     

Detection of exogenous citric acid in fruit juices by stable isotope ratio analysis

A new method has been developed for measuring the D/H ratio of the nonexchangeable sites of citric acid by isotope ratio mass spectrometry (IRMS). Pure citric acid is transformed into its calcium salt and subsequently analyzed by pyrolysis-IRMS. The citric acid isolated from authentic fruit juices (citrus, pineapple, and red fruits) systematically shows higher D/H values than its nonfruit counterpart produced by fermentation of various sugar sources. The discrimination obtained with this simplified method is similar to that obtained previously by applying site specific isotopic fractionation-nuclear magnetic resonance (SNIF-NMR) to an ester derivative of citric acid. The combination of carbon 13 and deuterium measurements of extracted citric acid is proposed as a routine method for an optimum detection of exogenous citric acid in all kinds of fruit juices.     

Natural abundance hydrogen isotope affiliation between the reactants and the products in glucose fermentation with yeast

In glucose fermentation, the hydrogen source of products such as ethanol and glycerol is the medium and the sugar. The site-specific natural isotope ratios of the products, (D/H)(i), and that of the medium and sugar, (D/H)(k), may be related by a matrix, A, of redistribution coefficients, a(ik), that characterizes the specific genealogies of the hydrogen atoms. (D/H)(i) = [A](D/H)(k), where (D/H)(i) and (D/H)(k) are the column vectors of the isotope ratios of sites i and k that can be measured by (2)H NMR. The complete redistribution matrix was determined in a set of isotope labeling experiments. Thus, we obtained a mathematical model representing the hydrogen isotope affiliation during alcoholic fermentation. It not only provides information about the biochemical reaction mechanism but also can be used to estimate the isotopic data of the products, based on those of the substrate and the medium. The results prove, in a quantitative way, that the metabolites contain isotopic information about the precursor in a biotransformation and can be used to identify its origin. The method established for the study of the hydrogen-transfer mechanism can be applied to other chemical and biochemical reactions.     

Pulp and paper from blue agave waste from tequila production.

Pulping of blue agave waste, from the production of tequila, was evaluated by both chemical and biomechanical pulping processes. Two conventional and two organosolv systems were used to pulp the agave waste under a standard set of conditions. The soda-ethanol process was superior in terms of delignification and pulp properties in comparison to the soda and ethanol organosolv processes for pulping of agave waste; however, the kraft process gave the best strength properties. In general, the strength of the agave waste pulps was rather poor in comparison to wood and other agro-based pulps; however, the tear strength was relatively high. This result is typical of poorly bonded sheets and may be due to the coarseness of the agave fibers and/or loss of hemicelluloses in the steaming process for the tequila production. Fungal treatment of the agave waste with Ceriporiopsis subvermispora reduced the energy consumption for mechanical refining but gave biomechanical pulps with inferior strength properties. The blue agave chemical pulps should be suitable for blending with softwood kraft pulps for publication grade paper.     

Study of the influence of alcoholic fermentation and distillation on the oxygen-18/oxygen-16 isotope ratio of ethanol

A laboratory procedure for the analysis of the oxygen-18/oxygen-16 isotope ratios of ethanol derived from sugars and fruit juices by pyrolysis-isotope ratio mass spectrometry (IRMS) has been applied to the study of isotopic fractionation induced by the isotope effects of fermentation and distillation. For both processes, an experimental model has been established to describe and explain the observed fractionation phenomena. It is shown that reproducible results can be obtained when appropriate analytical conditions are used. Moreover, the ability of ethanol to act as a reliable indicator of the (18)O/(16)O ratio of sugars in orange juice (and therefore to be used as an internal reference for detecting water addition) is demonstrated both in theory and in practice.     

Multivariate analysis of FTIR and ion chromatographic data for the quality control of tequila

Principal component analysis (PCA) was applied to the chromatographic and spectroscopic data of authentic Mexican tequilas (n = 14) and commercially available samples purchased in Mexico and Germany (n = 24). The scores scatter plot of the first two principal components (PC) of the anions chloride, nitrate, sulfate, acetate, and oxalate accounting for 78% of the variability allowed a classification between tequilas bottled in Mexico and overseas; however, no discrimination between tequila categories was possible. Mexican products had a significantly (p = 0.0014) lower inorganic anion concentration (range = 1.5-5.1 mg/L; mean = 2.5 mg/L) than the products bottled in the importing countries (range = 3.3-62.6 mg/L; mean = 26.3 mg/L). FTIR allowed a rapid screening of density and ethanol as well as the volatile compounds methanol, ethyl acetate, propanol-1, isobutanol, and 2-/3-methyl-1-butanol using partial least-squares regression (precisions = 5.3-29.3%). Using PCA of the volatile compounds, a differentiation between tequila derived from "100% agave" (Agave tequilana Weber var. azul, Agavaceae) and tequila produced with other fermentable sugars ("mixed"tequila) was possible. The first two PCs describe 89% of the total variability of the data. Methanol and isobutanol influenced the variability in PC1, which led to discrimination. The concentrations of methanol and isobutanol were significantly higher (methanol, p = 0.004; isobutanol, p = 0.005) in the 100% agave (methanol, 297.9 +/- 49.5; isobutanol, 251.3 +/- 34.9) than in the mixed tequilas (methanol, 197.8 +/- 118.5; isobutanol, 151.4 +/- 52.8).     

Improved detection of added water in orange juice by simultaneous determination of the oxygen-18/oxygen-16 isotope ratios of water and ethanol derived from sugars

A procedure for the analysis of the oxygen-18/oxygen-16 isotope ratio of ethanol derived from the sugars of orange juice using the preparation steps of the SNIF-NMR method followed by pyrolysis-isotope ratio mass spectrometry is presented. The isotopic fractionation induced by the isotope effects of fermentation and distillation have been investigated, and it is shown that reproducible results can be obtained when appropriate analytical conditions are used. It is also shown that the oxygen isotope distribution in the water and organic matter pools of fruits remains quite stable during the harvest period and is not altered by the precipitation rate within the last few days before the fruits are picked. Due to the robustness of the method and the fact that most of the oxygen-18 enrichment from the initial sugars is still present in the end-product, ethanol appears as a convenient internal reference to circumvent the spatial and temporal variability observed for the oxygen-18/oxygen-16 isotope ratio of water. A very strong correlation is observed between the isotopic deviations of ethanol and water, which is altered in the event of a water addition, even at a low level. Combining the information brought by these two parameters leads to a more efficient authenticity testing tool, which avoids false positive cases and provides a lower detection limit for added water in juices not made from concentrate, whatever the origin of the sample tested.     

Preparation of ferulic acid from agricultural wastes: its improved extraction and purification

Ferulic acid (FA) is a phenolic antioxidant present in plants, which is widely used in the food and cosmetic industry. In the present study, various agricultural wastes such as maize bran, rice bran, wheat bran, wheat straw, sugar cane baggasse, pineapple peels, orange peels, and pomegranate peels were screened for the presence of esterified FA (EFA). Among the sources screened, maize bran was found to contain the highest amount of EFA. Pineapple peels, orange peels, and pomegranate peels were also found to contain traces of EFA. Alkaline extraction of EFA from maize bran was carried out using 2 M NaOH. Response surface methodology (RSM) was used for optimization of EFA extraction, which resulted in a 1.3-fold increase as compared to the unoptimized conventional extraction technique. FA was analyzed by means of high-performance liquid chromatography (HPLC). Purification was carried out by adsorption chromatography using Amberlite XAD-16 followed by preparative high-performance thin-layer chromatography (HPTLC). The recovery of Amberlite XAD-16 purified FA was up to 57.97% with HPLC purity 50.89%. The fold purity achieved was 1.35. After preparative HPTLC, the maximum HPLC purity obtained was 95.35% along with an increase in fold purity up to 2.53.     

Specific natural isotope profile studied by isotope ratio mass spectrometry (SNIP-IRMS): (13)C/(12)C ratios of fructose, glucose, and sucrose for improved detection of sugar addition to pineapple juices and concentrates

The delta(13)C values of fructose, glucose, and sucrose have been determined in authentic pineapple juices. The sugar fraction is separated from the organic acids by an anionic exchange process. Then the individual components (fructose, glucose, and sucrose) are isolated on a preparative HPLC device using a NH(2)-type column. It is demonstrated that no significant isotope fractionation occurs when close to 100% of material is recovered and when the hydrolysis of sucrose is avoided. The control of the recovery rates and of the sucrose hydrolysis rate after purification is recommended for a reliable interpretation of the results. Correlations between the delta(13)C values of fructose (delta(13)Cf), glucose (delta(13)Cg), and sucrose (delta(13)Csu) can be characterized by systematic differences between these values. For the set of measurements on authentic pineapple juices and concentrates, the mean and the standard deviation of the differences are delta(13)Cf - delta(13)Cg = -0.6 +/- 0.6 per thousand, delta(13)Cf - delta(13)Csu = -1.3 +/- 0. 6 per thousand, and delta(13)Cf - delta(13)Csu = -0.7 +/- 0.5 per thousand. The determinations of the (13)C content of fructose, glucose, and sucrose enable a refinement of the detection of added sugars in fruit juices, re-enforcing the SNIP-IRMS method.     

Assessment of antioxidant activity of cane brown sugars by ABTS and DPPH radical scavenging assays: determination of their polyphenolic and volatile constituents

Seven cane brown sugars (four from La Réunion, two from Mauritius, and one from France) were investigated for their polyphenol content and volatile composition in relation to their free radical scavenging capacity determined by ABTS and DPPH assays. The thin layer coated on the sugar crystal was extracted by Soxhlet extractor with dichloromethane. The volatile compounds of brown sugars were studied by GC-MS, and 43 compounds were identified. The total phenolic content of brown sugars was determined according to the Folin-Ciocalteu method. Phenolic compounds were quantified in the brown sugar extracts by LC-UV-ESI-MS. Brown sugar aqueous solutions exhibited weak free radical scavenging activity in the DPPH assay and higher antioxidant activity in the ABTS assay at relatively high concentration. The brown sugar extracts showed interesting free radical scavenging properties despite the low concentration of phenolic and volatile compounds. Sugar is a common foodstuff traditionally used for its sweetening properties, which might be accompanied by antioxidant properties arising from molecules (polyphenols, Maillard products) other than sucrose of the cane brown sugars.     

Isotopic criteria in the characterization of aromatic molecules. 1. Hydrogen affiliation in natural benzenoid/phenylpropanoid molecules

The site-specific isotope ratios of several families of aromatic molecules are analyzed in terms of hydrogen affiliation and discriminating potential. Among the aromatic molecules produced by plants, many are biosynthesized by the shikimate pathway, but the terpenic pathway also forms some compounds with a benzenic ring. In compounds of the phenylpropanoid family, specific hydrogen connections are determined with cinnamic acid, a key intermediate in the formation of a large number of aromatic molecules. Then affiliations through the phenylalanine precursor, back to the parent d-erythrose 4-phosphate and phosphoenolpyruvate molecules and finally to glucose, are considered. Typical isotopic profiles of the benzenic ring in natural, as compared to non-natural, molecules are defined. The dispersion observed in the (D/H)i ratios of the lateral chains is illustrative of diverse mechanistic responses and the role of exchange phenomena. The isotopic patterns of aromatic molecules pertaining to the terpenic family are drastically different from those of the shikimate descendants, and they exhibit much less variability. They enable the stereochemical affiliation of individual hydrogen atoms to be traced back first to the parent atoms in the common intermediate, geranyl diphosphate, then to the glyceraldehyde 3-phosphate and pyruvate couple involved in the DOXP pathway, and ultimately to the glucose precursor. The results illustrate the aptitude of the site-specific isotope ratios not only to authenticate natural with respect to chemical molecules but also to characterize different metabolic pathways and to reveal differences associated with the nature of the plant precursor.     

Intramolecular carbon isotope distribution of acetic acid in vinegar

Compound-specific carbon isotope analysis of acetic acid is useful for origin discrimination and quality control of vinegar. Intramolecular carbon isotope distributions, which are each carbon isotope ratios of the methyl and carboxyl carbons in the acetic acid molecule, may be required to obtain more detailed information to discriminate such origin. In this study, improved gas chromatography-pyrolysis-gas chromatography-combustion-isotope ratio mass spectrometry (GC-Py-GC-C-IRMS) combined with headspace solid-phase microextraction (HS-SPME) was used to measure the intramolecular carbon isotope distributions of acetic acid in 14 Japanese vinegars. The results demonstrated that the methyl carbons of acetic acid molecules in vinegars produced from plants were mostly isotopically depleted in (13)C relative to the carboxyl carbon. Moreover, isotopic differences (δ(13)C(carboxyl) - δ(13)C(methyl)) had a wide range from -0.3 to 18.2‰, and these values differed among botanical origins, C3, C4, and CAM plants.