| The traditional Chinese Spirits authenticity identification, which is a powerful guarantee to establishing the credit enterprises system for liquor industry, is now an urgent technical problem to be conquered. Previous studies are mainly related to chromatographic fingerprint, spectroscopic fingerprint or Sensor array technology, which all focused on the micro-content compounds in Chinese Spirits. However, with the deep researches on physicochemical characteristics of Chinese Spirits and the development of blending techniques using computer, the models above can not distinguish the high imitation products. Stable isotopic technique is valuable in detecting cheap raw materials to foods, and now has been applied for authenticity assessment of honey, juices, wine, etc. It is illegal to add edible alcohol into traditional Chinese Spirits, however, there is no effective method to detect the edible alcohol in Chinese Spirit because of there similar chemical ingredients. In this study, the stable isotope ratio character of Chinese Spirits was studied, the influence factors were discussed, the changes of stable isotope ratio while edible alcohol added in were researched, and finally, one model for detecting the added edible alcohol in Chinese Spirit was formulated.Firstly, series of methods for stable isotope ratio measurement were developed according to the isotopic analysis requirement combined with the isotope fractionation principle and the chromatographic separation.(1) Method for the determination of ?18O of water based on the water/CO2 was developed. This method using a gas chromatography-isotope ratio mass spectrometry(GC-IRMS) for sample injection and measurement has the advantage of fast speed, low cost and more importantly it expands the application scope of GC-IRMS. In this part, influence factors such as memory effect, gas tight of reaction tube, robust of the aeration system were evaluated, and the reaction conditions were optimized. Results showed that the internal precision was better than 0.08‰, the standard deviation for water analysis was better than 0.2‰, but this method could not be used for Chinese Spirits analysis. This method has been adapted by two industry standards.(2) Carbon isotopic analysis of fermented sugar from grain was established on the base of elemental-isotope ratio mass spectrometry(EA-IRMS), the impact of sample preparation on carbon isotope ratio was evaluated, data showed this method could afford the accurate analysis.(3) We first developed the rapid and accurate method for hydrogen and oxygen isotope analysis of ethanol without extract ethanol from aqueous samples over the world. Samples with ethanol content from 1% to 100% can be determined using this method, the main ingredient that influence the analysis of ethanol, water, was eliminated by the equipment automatically after separation by GC column, the repeatability and reproducibility for oxygen and hydrogen isotope analysis were all better than 0.5‰ and 3‰, respectively. The accuracy for ethanol oxygen isotope analysis was validated through the FIT-PTS scheme(organized by the Joint Research Centre of the European Commission. Following this advantage, time, labor cost and sample consumption are all saved. Meanwhile, this sample preparation procedure can be used for ethanol carbon isotope analysis, which has been adapted by one Industry Standard in Chinese Spirits.(4) The oxygen isotope ratio of water in Chinese Spirit can be determined by GC-IRMS coupled with chromatography separation and temperature conversion. In this method, the memory effect and impacts of ethanol and air were not observed, the standard deviation was better than 0.5‰. Compared this method with the water/CO2 method, a strong correlation can be found between two values from the same water samples, which illustrated that this method can be used for accurate analysis for Chinese Spirits.(5) The carbon isotope ratio of isoamyl alcohol from fermented matrix and Chinese Spirits were determined based on the liquid-liquid extraction and GC-IRMS technique, the impact of other organic compounds can be eliminated with a better repeatability.Secondly, systematic researches were made to study the stable isotopic characteristic and variation associated with Chinese Spirit production, influence factors and the mechanism of stable isotope fractionation were detailed discussed. Some founds are as followings:(1) Water is one major ingredient of Chinese Spirit, and also the medium for production. It was observed that the heavy oxygen isotope in water will be enriched after the starch dextrinization of grain, but not varied during saccharification and alcoholic fermentation. However, data showed that the water component didn't derive from the fermented matrix, but the condensation of steam during distillation, and meanwhile the added water during blending process. Thus, the steam ?18O value of water in Chinese Spirit will not be influenced by fermentation conditions and the fermented materials, but the water used for steam and blending that varied between different geographical origin.(2) The hydrogen and oxygen isotope composition of ethanol will be simultaneously influenced by fermented sugar(from grain) and water, significant correlations were observed between ethanol/sugar and ethanol/water. The reason is that part hydrogen and oxygen atoms come from fermented sugar directly, however, there was oxygen isotope change reaction between metabolic intermediates and water during alcoholic fermentation that lead to the oxygen isotope ratio of ethanol was affected greatly by water used as fermentation medium; meanwhile, the alcoholic fermentation contains series of dehydrogenase reaction that introduced exogenous hydrogen atoms that altered the hydrogen isotope composition of metabolic intermediates. Data sowed the hydrogen isotope composition was affected greatly by fermented sugar. In addition, the distillation process will influence the hydrogen and oxygen isotope ratio of ethanol, but these two isotope ratios of crude spirits equaled to the fermented matrix which indicate these two character could be used for traceability of fermented sugars and water.(3) Strong correlation(R2=0.989) between carbon isotope ratio of ethanol and grain sugar was found, with the ?13C value of ethanol more depleted. Data showed that the difference of ?13C value between ethanol and sugar varied that related to the ?13C value of sugar. Monitoring the fermentation process, it was observed that the ?13C value of ethanol are not constant but four stage that caused by the metabolic pathway. It was pointed out that the difference between ethanol and fermented sugar were not caused by common isotope fractionation, but the non-statistical distribution of carbon isotope in glucose, and the fist constant value in ethanol appeared while the ethanol produced from glyceraldehyde-3-phosphate that degrading directly from the glucose, after this glyceraldehyde-3-phosphate was exhausted the dihydroxyacetone phosphate was used to produce ethanol with the ?13C value increased step by step. One hypothesis was proposed that the ?13C value of ethanol will not be impacted by fermentation conditions, and was verified by the orthogonal experiment. As to the distillation process, despite ?13C value was depleted with distillation time because of the inverse vapor pressure isotope effect, the ?13C value of ethanol in crude spirits equaled to the fermented matrix that can be used to identify the grain type for fermentation.(4) There are two pathways to produce isoamyl alcohol: Ehrlich metabolic pathway and Harris metabolic pathway. Both these metabolic pathway will impact the carbon isotope ratio of isoamyl alcohol and the type of nitrogen source will decide the major pathway for isoamyl alcohol production. Data showed that the carbon isotope ratio of isoamyl alcohol were correlated to the carbon isotope composition of sugar. Although ?13C of isoamyl alcohol will changed during distillation, the correlation between isoamyl alcohol and ethanol was found, thus the isoamyl alcohol can be used as the 13 C internal reference material to ethanol.Finally, the potentiality of stable isotopic technique in edible alcohol detection in Chinese Spirits was discussed and one discriminant model was established:(1) Results showed that the stable isotope character differed from different brewing manufactures because of their different fermented conditions and grain types used for production, but it was nearly a constant in the same manufacture. The edible alcohol can be chattered two types according their carbon and hydrogen isotope character, meanwhile, their was a strong correlation between the ?13C and ?D values.(2) The isotopic change of Chinese Spirit while edible alcohol added in was investigated, significant correlations between ?13C/?D value and the content of edible alcohol were observed, therefore one discriminant model was established on the base of carbon isotopic technique.(3) Combined with the ?13C value of isoamyl alcohol in samples, the database can be avoided which simplified the evaluation procedure and the cost. Results from blind samples from China Alcoholic Drinks Association and one Chinese Spirit manufacture demonstrated that samples mixed with edible alcohol can be separated, and the edible alcohol content can be calculated with discriminant error lower than 5%. |
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