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.     

固定化酵母粒子强化及其对甜高梁茎秆汁液乙醇发酵的影响

采用正交试验设计开展了三亚乙基四胺(TTA)和戊二醛(GLU)的浓度和处理时间对海藻酸钙固定化酵母粒子的化学强度影响的试验研究,并以甜高粱茎秆汁液为原料,在5 L的反应器中进行乙醇发酵试验,考察强化后的固定化酵母粒子对乙醇发酵的影响.结果表明,最优的固定酵母粒子强化处理的方案为TTA浓度为0.5%,处理时间为120 min;GLU浓度为0.5%,处理时间为8 min.连续8批次的甜高粱茎秆汁液乙醇发酵试验结果表明,最优组合强化后固定化酵母粒子用于乙醇发酵时,平均乙醇得率和变异系数(CV%)分别为84.78%和8.08%,而未强化的固定化酵母籽子为84.32%和9.68%,可见,最优组合强化后的固定化酵母粒子的发酵性能略优于未强化的固定化酵母籽子.该文为固定化酵母发酵甜高粱茎秆汁液制取生物乙醇技术的研究提供了参考.     

固定化酵母粒子强化及其对甜高粱茎秆汁液乙醇发酵的影响

采用正交试验设计开展了三亚乙基四胺(TTA)和戊二醛(GLU)的浓度和处理时间对海藻酸钙固定化酵母粒子的化学强度影响的试验研究，并以甜高粱茎秆汁液为原料，在5 L的反应器中进行乙醇发酵试验，考察强化后的固定化酵母粒子对乙醇发酵的影响。结果表明，最优的固定酵母粒子强化处理的方案为：TTA浓度为0.5%，处理时间为120 min；GLU浓度为0.5%，处理时间为8 min。连续8批次的甜高粱茎秆汁液乙醇发酵试验结果表明，最优组合强化后固定化酵母粒子用于乙醇发酵时，平均乙醇得率和变异系数(CV%)分别为84.78%和8.08%，而未强化的固定化酵母籽子为84.32%和9.68%，可见，最优组合强化后的固定化酵母粒子的发酵性能略优于未强化的固定化酵母籽子。该文为固定化酵母发酵甜高粱茎秆汁液制取生物乙醇技术的研究提供了参考。     

桂西北喀斯特峰丛洼地降水氢氧稳定同位素的季节变化特征

喀斯特地区特殊的二元水文地质结构增加了水文过程研究的难度。大气降水是流域水循环的重要输入因子,研究降水氢氧稳定同位素的季节变化规律及其影响因素,有利于探讨该地区水汽来源、径流划分、水分滞留时间等水文过程。该文以桂西北环江地区典型喀斯特峰丛洼地为例,基于2009年4月-2011年8月的降雨、温度资料以及月降水氢氧稳定同位素组成数据,分析了降水分布及其氢氧稳定同位素组成的季节变化规律,并探讨了各种环境因素的影响。结果表明,当地大气水线与全球大气水线存在一定的偏离,表明研究区降水云气形成过程中汽、液两相同位素分馏不平衡的程度偏大。过量氘年平均值为14.21‰,旱雨季不同,反映了水汽来源的差异。降水氢氧稳定同位素季节变化呈现正弦波动的特性,波动周期和振幅受纬度及季节因素的影响明显大于降水量及温度,表明大尺度环境因素的影响占主导地位。该研究为基于氢氧稳定同位素技术的喀斯特地区流域尺度水文过程研究奠定了基础,对深入分析该地区植被恢复与重建的生态水文效应提供参考。     

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.     

基于电导率酒精发酵过程在线监测的研究

本试验对酒精发酵过程中电导率与还原糖、酒精度、pH的变化规律以及氯化钙和硫酸铵等盐类对电导率的影响进行了研究,进而探讨电导率在线监测酒精发酵过程的适用性。结果表明：电导率与还原糖和酒精度之间存在着一定的逻辑关系,利用电导率的变化能间接地反映还原糖和酒精度的变化;pH值与电导率之间存在着一定的线性关系(发酵60 h前变化规律相同);氯化钙和硫酸铵两种盐类在一定范围内对电导率的变化不产生影响。电导率达到最低点时,还原糖和酒精度均符合酒精发酵终点的参数指标,因此,该研究结果可用于发酵过程的在线监测和发酵终点的判定。     

Interaction between fenhexamid and yeasts during the alcoholic fermentation of Saccharomyces cerevisiae

The behavior of the fungicide fenhexamid, N-(2,3-dichloro-4-hydroxyphenyl)-1-methyl-cyclohexanecarboxamide, has been studied at concentrations corresponding to the limits fixed for grapes (3 mg kg(-1)), or higher, during the alcoholic fermentation. The presence of the fungicide did not affect the amount of alcohol produced. The amount of fenhexamid in the liquid phase decreased by ca. 15%, but the missing fenhexamid was recovered unchanged from yeasts. This suggests that the fungicide is not degraded during the fermentation process, but adsorbed by yeasts. Two constituents of Saccharomyces cerevisiae cell wall, chitin and glucan, tested as potential adsorbents, exhibited affinity for fenhexamid.     

Influence of the distillation step on the ratios of stable isotopes of ethanol in cherry brandies

Isotope ratio mass spectrometry and site-specific natural isotope fractionation-nuclear magnetic resonance were applied to determine the overall carbon isotope ratio (delta13C) and the hydrogen isotope ratios [(D/H)I and (D/H)II] of ethanol, respectively. Ethanol was obtained by distillation of fermented cherry mash from a pot still commonly used in fruit brandy production. Analyses of distillate fractions revealed that the distillation proceeds with a fractionation of ethanol isotopologues. The inverse vapor pressure isotope effect (VPIE) observed for the carbon isotopologues is in accordance with the data reported for distillation of ethanol in spinning band columns. In contrast, the inverse VPIE for hydrogen isotopologues of ethanol observed in spinning band columns could not be confirmed. To investigate whether the observed isotope fractionations might influence the applicability of stable isotope analysis for quality and authenticity assessment of fruit brandies, the collected distillate fractions were recombined to cuts, as is common practice in commercial fruit brandy production. Taking into consideration the limits of repeatability of the method, it could be demonstrated that the isotope fractionations observed do not impair the applicability of stable isotope analysis of the carbon and hydrogen isotopes of ethanol for the authenticity assessment of cherry brandies if the cuts are placed in accordance with common distillers' practice.     

Impact of mashing on sorghum proteins and its relationship to ethanol fermentation

Nine grain sorghum cultivars with a broad range of ethanol fermentation efficiencies were selected to characterize the changes in sorghum protein in digestibility, solubility, and microstructure during mashing and to relate those changes to ethanol fermentation quality of sorghum. Mashing reduced in vitro protein digestibility considerably, and a large amount of polymers cross-linked by disulfide bonds were developed during mashing. As a marker of cross-linking, protein digestibility of the original samples was highly related to conversion efficiency. gamma-Kafirin (%) neither correlated to ethanol yield nor conversion efficiency significantly. Solubility of proteins in an alkaline borate buffer in conjunction with SDS decreased substantially after mashing. Solubility and the SE-HPLC area of proteins extracted from mashed samples were highly correlated with ethanol fermentation. Ethanol yield increased and conversion efficiency improved notably with the increase of extracted proteins from mashed samples. SE-HPLC total area could be used as an indicator to predict ethanol fermentation. CFLSM images proved that sorghum proteins tended to form highly extended, strong web-like microstructures during mashing. The degree of protein cross-linking differed among samples, and more open microstructures were observed in samples with higher conversion efficiencies. The web-like protein matrix was found to hold not only starch granules but also some oligosaccharides or polysaccharides inside. The formation of web-like microstructures because of cross-linking reduced conversion efficiency.     

Stable isotope characterization of milk components and whey ethanol.

A multi-isotopic study of several components of milk has been carried out on commercial samples and on milk produced in feeding experiments involving different kinds of diets originating from C(3) or C(4) photosynthetic metabolisms and exhibiting a relatively wide range of isotope ratios. The dispersion of the carbon, hydrogen, and nitrogen isotope parameters of dried matter and of the lactose, protein, and lipid components has been estimated. In addition, the carbohydrates were represented by the site specific isotope ratios (SNIF-NMR) of ethanol resulting from standardized fermentation of lactose. The rates of response of the isotopic parameters to changes in the feeding materials is slower for the minor components, proteins, and lipids than for lactose and ethanol. For similar diets, the nonexchangeable sites of lactose and the methyl site of ethanol, in particular, are relatively enriched in deuterium in the case of polygastric animals, cow, goat, and ewe, as compared to the monogastric species, sow and mare, and woman. From an analytical point of view, the carbon and hydrogen parameters of ethanol provide efficient criteria for identifying a whey origin with respect to other agricultural and fossil sources.     

Behavior of a fenhexamid photoproduct during the alcoholic fermentation of Saccharomyces cerevisiae

The fungicide fenhexamid [N-(2,3-dichloro-4-hydroxyphenyl)-1-methylcyclohexanecarboxamide] degraded rapidly by UV or sunlight irradiation, yielding 7-chloro-6-hydroxy-2-(1-methylcyclohexyl)-1,3-benzoxazole (CHB) as a main photoproduct. CHB was isolated, and its effect on alcoholic fermentation of Saccharomyces cerevisiae was studied. The results indicate that the presence of CHB does not affect the extent of alcohol production. After 12 days, the amount of CHB in the fermentation medium decreased by ca. 65%. Only 25% of the missing CHB was recovered unchanged from yeasts, most likely because it was adsorbed on the yeast wall cell. The remaining part degraded during the fermentation process. Glucan and chitin, two potential adsorbents, which constitute yeast cell walls, exhibited affinity for CHB.     

Solid-substrate fermentation of corn fiber by Phanerochaete chrysosporium and subsequent fermentation of hydrolysate into ethanol

The goal of this study was to develop a fungal process for ethanol production from corn fiber. Laboratory-scale solid-substrate fermentation was performed using the white-rot fungus Phanerochaete chrysosporium in 1 L polypropylene bottles as reactors via incubation at 37 degrees C for up to 3 days. Extracellular enzymes produced in situ by P. chrysosporium degraded lignin and enhanced saccharification of polysaccharides in corn fiber. The percentage biomass weight loss and Klason lignin reduction were 34 and 41%, respectively. Anaerobic incubation at 37 degrees C following 2 day incubation reduced the fungal sugar consumption and enhanced the in situ cellulolytic enzyme activities. Two days of aerobic solid-substrate fermentation of corn fiber with P. chrysosporium, followed by anaerobic static submerged-culture fermentation resulted in 1.7 g of ethanol/100 g of corn fiber in 6 days, whereas yeast ( Saccharomyces cerevisiae) cocultured with P. chrysosporium demonstrated enhanced ethanol production of 3 g of ethanol/100 g of corn fiber. Specific enzyme activity assays suggested starch and hemi/cellulose contribution of fermentable sugar.     

Stereoselective formation of the varietal aroma compound rose oxide during alcoholic fermentation

The potent aroma compound rose oxide was quantified in several white wines by a headspace solid-phase microextration stable isotope dilution assay (HS-SPME-SIDA) and the enantiomeric ratios of the cis diastereomers were determined by enantioselective capillary GC. The most odor-active stereoisomer (23)-cis-rose oxide was detectable in all investigated white wines ranging from 0.2 to 12 microg/L. However, its contribution to the overall aroma in some white wine varieties can be neglected as indicated by a low odor activity value (OAV). The highest concentrations were found in Gewürztraminer wines, confirming the importance of rose oxide as a varietal aroma compound in this variety. Surprisingly, the enantiomeric ratio of cis-rose oxide in all investigated wines was substantially lower than in nonfermented musts and in some wines almost racemic cis-rose oxide was detected. Fermentation studies with a model must that contained deuterated water revealed that yeast is capable of reducing the precursor 3,7-dimethyl octa-2,5-dien-1,7-diol (geranyl diol I) yielding 3,7-dimethyl-5-octen-1,7-diol (citronellyl diol I) that gives rise to cis- and trans-rose oxide after acid catalyzed cylization. The deuterium labeling pattern of the resulting rose oxide stereoisomers and a clearly detectable kinetic isotope effect indicate that at least two different reductive pathways in yeast exist that yield cis-rose oxide with different enantiomeric ratios altering the genuine enantiomeric ratio in grape musts. The presence of (+)-cis-rose oxides in wines can therefore be attributed to the reductive yeast metabolism during fermentation. This observation corroborates recent findings that the modification of terpene derived varietal aroma is an integral part of yeast metabolism and not only a simple hydrolytical process.     

木质纤维素乙醇发酵研究中的关键点及解决方案

该文综述了近年来木质纤维素材料发酵生产乙醇的研究进展,提出了在木质纤维素原料发酵生产乙醇过程中的两大关键点,一是减少和消除原料预处理过程中抑制物及其有害影响;二是含木糖、葡萄糖、甘露糖、半乳糖等多种物质的混合物同时作为底物的乙醇发酵。在对已有研究结果比较和分析基础上,提出相应的解决方案。首先要采取综合的预处理过程,达到提高木质纤维素水解率的同时减少发酵抑制物;然后是提高发酵菌种对混合糖底物的利用能力和发酵生成乙醇的能力以及对抑制物的耐受性,以提高木质纤维素发酵生产乙醇的转化率。     

固定化酵母发酵甜高粱茎秆汁工艺参数对酒精得率的影响

以甜高粱茎秆汁为原料，在摇瓶和反应器上对固定化酵母发酵甜高粱汁制取酒精过程中温度、pH值及固定化酵母粒子填充率三个主要工艺参数对酒精得率的影响进行了试验研究。摇瓶单因素试验结果表明：固定化酵母发酵甜高粱茎秆汁液制取酒精过程中，较优的温度、pH值和粒子填充率分别为34℃、4.5和25%。摇瓶的验证试验结果表明，总发酵时间为6h，发酵结束时酒精得率和总糖利用率分别为95.15%和88.65%；反应器放大验证试验结果显示，总发酵时间10 h，发酵结束时，酒精得率和总糖利用率为96.72%和99.86%。该研究为甜高粱制取酒精技术提供了有益的参考。     

以黄姜为原料发酵酒精的液化糖化条件的初步研究

利用黄姜根茎富含淀粉的特点,在温和的条件下研究其液化糖化性能,用来发酵酒精。黄姜粉加水调浆后,升至一定温度,加入适量α-淀粉酶进行液化处理,保温一段时间,然后降温至60℃,调pH值,加糖化酶糖化,保温处理使其中的淀粉转化完全。单因素试验结果表明：60目以上黄姜粉,料水质量比1∶5,α-淀粉酶10 U/g,在90℃时,30 min即可达到很好的液化;然后降温到60℃,调pH值4.0～4.5,加糖化酶60 U/g,90 min内基本完全糖化。利用酶解的方法转化黄姜中的淀粉是可行的,并且效果显著。     

Effect of organic acids and nitrogen source on alcoholic fermentation: study of their buffering capacity

The effect of tartaric acid and other organic acids on alcoholic fermentation was studied. Organic acids in media with high sugar concentrations and ammonium as the sole nitrogen source had an enormous impact on Saccharomyces cerevisiae metabolism during alcoholic fermentation. The main effect on yeast metabolism was the quick acidification of the media in the absence of organic acids. All of the organic acids used in this study (tartaric, malic, citric, and succinic acids) showed a buffering capacity, but not all of the acids had the same one. However, the results suggested that buffering should not be considered the only effect of organic acids on yeast metabolism. Nitrogen source also had a great influence on media pH. Ammonium consumption by yeasts produced a greater acidification of the media than when amino acids were used.     

Comparison of oil recovered from tea tree leaf by ethanol extraction and steam distillation

Two methods for the determination of oil and oil major components from tea tree (Melaleuca alternifolia) leaf are quantitatively compared. A microwave assisted ethanol extraction and a 2-h hydrodistillation technique were used on both dry and fresh leaf from a low and a high oil concentration tree. There was no significant difference between dry and fresh leaf. The distillation technique recovered 88% and 82% of the extractable oil for the low and high concentration material, respectively. For both samples this distilled oil was composed of lower absolute amounts of sesquiterpenoids and marginally lower amounts of monoterpenoids. Extending the distillation to 6 h increased the sesquiterpenoid recovery but this resulted in a reduction in both the absolute and relative amounts of the oxygenated monoterpenoids, terpinen-4-ol and 1,8-cineole.     

化学转化法测定水体中硝酸盐的氮氧同位素比值

采用化学转化法可同时测定各种水体样品中硝酸盐的氮、氧同位素比值。在弱碱性环境中,镀铜镉粒将硝酸盐还原为亚硝酸盐,然后在弱酸性缓冲体系中叠氮酸根将亚硝酸盐转化为氧化亚氮。通过带有预浓缩装置的同位素比值质谱计测定氧化亚氮的氮、氧同位素比值,并用公式将其换算为硝酸盐的氮、氧同位素比值。通过国际标准样品和实验室标准样品的多次检测证实化学转化法具有适用范围广、灵敏度高、操作简便、准确的特点,其转化过程仅需两步,且需样量仅为4.5μg NO3--N。δ15N和δ18O的测量精度分别为0.31‰和0.55‰,测定值与参考值的差异均小于1σ,测量的精确度和准确度均可满足样品分析的需求。     

Optimization of simultaneous saccharification and fermentation for the production of ethanol from lignocellulosic biomass

Simultaneous saccharification and fermentation (SSF) of alkaline hydrogen peroxide pretreated Antigonum leptopus (Linn) leaves to ethanol was optimized using cellulase from Trichoderma reesei QM-9414 (Celluclast from Novo) and Saccharomyces cerevisiae NRRL-Y-132 cells. Response surface methodology (RSM) and a three-level four-variable design were employed to evaluate the effects of SSF process variables such as cellulase concentration (20-100 FPU/g of substrate), substrate concentration (5-15% w/v), incubation time (24-72 h), and temperature (35-45 degrees C) on ethanol production efficiency. Cellulase and substrate concentrations were found to be the most significant variables. The optimum conditions arrived at are as follows: cellulase = 100 FPU/g of substrate, substrate = 15% (w/v), incubation time = 57.2 h, and temperature = 38.5 degrees C. At these conditions, the predicted ethanol yield was 3.02% (w/v) and the actual experimental value was 3.0% (w/v).