Discrimination for sake brewing methods by compound specific isotope analysis and formation mechanism of organic acids in sake.

Organic acids in sake affect its aroma and color and help control the activity of microorganisms. This study used liquid chromatography coupled with isotope ratio mass spectrometry and solid-phase extraction to determine the stable carbon isotope ratios (δ13C) for malic acid, lactic acid, and succinic acid in 49 sake samples. The mean δ13C of lactic acid was -25.6 ± 2.1‰ in kimoto samples and -20.2 ± 2.5‰ in sokujo sample. According to linear discriminant analysis using δ13C of lactic acid, 87.8% of kimoto and sokujo samples were correctly identified. The proportion of brewers' lactic acid in sake could be calculated from the δ13C value of lactic acid for the first time. The productions of malic acid and succinic acid may be conducted by some kinds of fermentation and the mechanism of the tricarboxylic acid cycle by using δ13C of malic acid and succinic acid.

Carbon isotope ratio of organic acids in sake and wine by solid-phase extraction combined with LC/IRMS.

We developed an analytical procedure for determining the δ13C values of organic acids in sake and wine using solid-phase extraction combined with liquid chromatography/isotope ratio mass spectrometry (LC/IRMS). First, the solid-phase extraction (SPE) procedure was performed and various tests were conducted to extract organic acids from alcoholic beverages using the simulated sake sample. Under the optimal SPE procedure, high recovery rates (96-118%) and good accuracies (≤ 0.7‰) were thus achieved for the simulated sake and wine samples. Next, we determined the δ13C of organic acid (tartaric acid, malic acid, lactic acid, succinic acid) in 9 sake and 11 wine samples. Finally, the δ13C values of lactic acid in nine sake samples suggested that lactic acid had been added during the brewing process. The high correlation between the δ13C values of tartaric acid and malic acid in 11 wine samples was consistent with their common source, grapes. This analytical method may help to identify when organic acids have been added to sake and wine and to elucidate the process of organic acid production therein. Graphical abstract.

Analysis of malto-oligosaccharides and related metabolites in rice endosperm during development.

MAIN CONCLUSION: Linear glucans with degree of polymerization of up to 23 were detected in rice endosperm at the very early developmental stage of endosperm and considered to play an important role in the de novo synthesis of branched glucans. Little is known concerning the contribution of malto-oligosaccharides (MOS) and longer linear glucans to the starch biosynthesis in cereal endosperm. In the present study, the changes in the amount of major metabolic intermediates including MOS and linear glucans with a degree of polymerization (DP) of ≤ 9 and ≥ 10, respectively, in rice endosperm were measured during the development. Significant amounts of linear glucans of at least DP23 were present in the endosperm at 3 and 5 days after pollination (DAP), whereas most MOS of DP up to 8 were detected in the endosperm throughout the development up to 20 DAP. It was also found that a significant amount of simple sugars such as sucrose, glucose, and fructose, and organic acids such as malic acid, citric acid, and succinic acid were present in the developing endosperm. Although the levels of metabolites are not directly related to the extent of the metabolic flux, the present results suggest that MOS and linear glucans as well as these sugars and organic acids are involved in starch biosynthesis of rice endosperm. It is thought that linear glucans might play a role in starch biosynthesis in rice endosperm, presumably as the precursor for the subsequent synthesis of branched glucans involved in the initiation process that is possibly active in the endosperm at the very early developmental stage.

Compound Specific Carbon Isotope Analysis in Sake by LC/IRMS and Brewers' Alcohol Proportion.

Sake is a traditional Japanese alcohol. Nowadays, the consumption for Sake is increasing in worldwide and its popularity is growing. However, there are act of fraudulence by additional brewers' alcohol and sugar. Therefore, a method is needed to find illegal fraud on label. In this work, we analyzed the δ13C values of the ethanol (δ13Ceth) and glucose (δ13Cglu) in Sake by liquid chromatography combined with isotope ratio mass spectrometry for the first time. Further, we developed the criteria using δ13Ceth and δ13Cglu to check brewers' alcohol and sugar. In addition, there are some sake categories (Ginjyo and Futsu-shu) allowed to additional brewers' alcohol up to legally determined percentage. The experimental additions of brewers' alcohol from a C4 plant were conducted to Junmai, as sake by C3 plants. There was a strong correlation (R = 0.98, P < 0.05) between the percentage of added brewers' alcohol and the δ13C values. We developed the method using the relationship for calculating percentage of brewers' alcohol for the first time and estimated the percentage for commercial sake. Further, the price of sake was found to be inversely related to the percentage of brewers' alcohol in the sake.

Heart-cutting two-dimensional liquid chromatography combined with isotope ratio mass spectrometry for the determination of stable carbon isotope ratios of gluconic acid in honey.

Liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) is used to analyze various types of samples, including foodstuffs, to determine their authenticity and trace their origin on the basis of their stable carbon isotope ratios (δ13C). However, multicomponent samples are difficult to analyze. For example, determining the δ13C values of the organic acids in honey is complicated by the presence of large amounts of carbohydrates. Herein, we present a heart-cutting two-dimensional LC/IRMS method for analysis of honey samples. In this method, the organic acids in the samples were first separated from the carbohydrates by a size-exclusion column, and then the organic acids were separated from each other by a reverse-phase column connected to the first column via a switching valve. By means of this method, the δ13C values for three organic acids in high-carbohydrate-content simulated honey samples could be determined with high accuracy and precision (≤0.3‰ and ≤0.1‰, respectively). In addition, the gluconic acid δ13C values for 25 honey samples were determined with high precision and found to range from -31.7 to -28.5‰ (mean: -30.0 ±â€¯0.7‰). These values shed some light on the mechanism of gluconic acid production. Taken together, our results suggest that this two-dimensional LC method has the potential to be more effective than one-dimensional LC for use in isotopic research.

Stable carbon isotope ratios for organic acids in commercial honey samples.

Stable carbon isotope ratios (δ13C) for glucose, fructose, disaccharides, trisaccharides, and organic acids in 116 commercial honey samples were measured by LC/IRMS. On the basis of EA/IRMS and LC/IRMS authenticity criteria, 39 of the samples were judged to have been adulterated. The δ13C values for organic acids from pure honey, reported here for the first time, ranged from -33.6 to -26.5‰. The mean Δδ13C (glucose-organic acids) value was +3.7 ±â€¯0.9‰. Glucose and organic acid δ13C values were strongly correlated (R = 0.71, P < 0.001). Gluconic acid, the predominant organic acid in honey, has been reported to be produced via decomposition of glucose by bee glucose-oxidase and certain Gluconobacter spp. This fact was confirmed by isotope analysis.

Determination of carbon isotope ratios for honey samples by means of a liquid chromatography/isotope ratio mass spectrometry system coupled with a post-column pump.

RATIONALE: Liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) has been used to authenticate and trace products such as honey, wine, and lemon juice, and compounds such as caffeine and pesticides. However, LC/IRMS has several disadvantages, including the high cost of the CO2 membrane and blocking by solidified sodium persulfate. Here, we developed an improved system for determining carbon isotope ratios using LC/IRMS. METHODS: The main improvement was the use of a post-column pump. Using the improved system, we determined δ13 C values for glucose with high accuracy and precision (0.1‰ and 0.1‰, respectively; n = 3). The glucose, fructose, disaccharide, trisaccharide, and organic acid constituents of honey samples were analyzed using LC/IRMS. RESULTS: The δ13 C values for glucose, fructose, disaccharides, trisaccharides, and organic acids ranged from -27.0 to -24.2‰, -26.8 to -24.0‰, -28.8 to -24.0‰, -27.8 to -22.8‰, and - 30.6 to -27.4‰, respectively. The analysis time was a third to a half of that required for analysis by previously reported methods. CONCLUSIONS: The column flow rate could be arbitrarily adjusted with the post-column pump. We applied the improved method to 26 commercial honey samples. Our results can be expected to be useful for other researchers who use LC/IRMS.