Study on the increase mechanism of the caffeine content during the fermentation of tea with microorganisms

The tea caffeine content had previously been shown to increase reasonably after being treated with mixed microorganisms for a period of time. In this study, single microorganisms were used in the fermentation of black and green teas in order to find which microorganism has the best effect on increasing the caffeine content. The results demonstrated that molds fermentation increased the caffeine content, but yeasts fermentation decreased the caffeine content. Among the three molds in this study, Aspergillus niger van Tieghem has the most remarkable effect, and the caffeine content in dry green tea increased from an initial 3.47% to 9.63%. The increase rate was 177.5% on the 16th day. Furthermore, the changes of caffeine and theophylline were of a similar trend. Possibly theophylline instead of theobromine is the precursor of caffeine in the living microorganisms. The new biosynthetic route is different from that in tea plants.     

冠突散囊菌与茶叶生物碱共培养液态发酵体系的构建及特性

选取茶叶中三种主要生物碱咖啡碱(Caffeine)、可可碱(Theobromine)、茶碱(Theophylline)作为构建单体-冠突散囊菌共培养液态发酵体系(10 d)的添加底物,考察了冠突散囊菌对茶叶中主要生物碱的发酵特性。结果表明,与对照组相比,咖啡碱(0.2mg/mL)的添加可刺激冠突散囊菌的生长与繁殖,发酵液中葡萄糖的含量下降更为迅速,pH值随着发酵时间的增加持续下降直至第9 d趋于稳定,可溶性蛋白含量呈现先增加后下降的趋势,但在发酵过程中,冠突散囊菌既不能将其作为碳源、氮源消耗来维持生长,也不能代谢分解咖啡碱;可可碱与茶碱(0.08 mg/mL)在发酵体系中的发酵情况与咖啡碱基本相似,两者均能刺激冠突散囊菌的生长与繁殖,但均不能将其作为碳、氮源消耗,且发酵结束后在各自发酵体系中分别检测到0.00091 mg/mL、0.00109 mg/mL的咖啡碱,这说明冠突散囊菌可能具有以可可碱、茶碱作为前体合成咖啡碱的能力。     

Theobromine and caffeine content of the cocoa bean during its growth

Changes in the concentrations of caffeine and theobromine in the cocoa pod during its growth were determined. Theobromine and caffeine concentrations increased markedly after about 12 weeks from flowering to the time of harvesting. During the stage when the bean was only a mucilaginous mass, the quantity of caffeine and theobromine was negligible. Caffeine and theobromine levels in the shell, once this is formed, increased rapidly until harvest.     

高效液相色谱法分析武夷岩茶中的生物碱含量

选取10种典型武夷岩茶(5种肉桂和5种水仙)作为研究对象,以绿茶、铁观音和红茶为对照,通过高效液相色谱法检测样品叶片及冲泡茶汤中的可可碱、茶碱和咖啡碱含量,并依据各样品的生物碱含量进行主成分分析。试验结果发现,武夷岩茶叶片中的咖啡碱、可可碱和茶碱含量分别约占茶叶干重的2.341%,0.065%,0.011%,低于绿茶、铁观音和红茶;茶叶经过60s沸水冲泡后,约46.56%,46.36%,50.15%的咖啡碱、可可碱和茶碱溶于茶汤中。此外,通过主成分分析发现,武夷岩茶的3种生物碱含量与绿茶、铁观音和红茶的生物碱含量有显著差异。     

液相色谱法测定饮料中咖啡因、可可碱和茶碱

采用高效液相色谱法同时测定饮料中的咖啡因、可可碱和茶碱.饮料样品经超声提取后,用高效液相色谱分离,二极管阵列检测器检测,建立标准工作曲线,以外标法定量.实验精密度为0.58%～1.85%,添加回收率范围为80.2%-96.4%.此方法快速,简便,准确,适用于饮料中咖啡因、可可碱和茶碱的测定.     

Determination of theobromine,theophylline and caffeine in cocoa samples by a high-performance liquid chromatographic method with on-line sample cleanup in a switching-column system

A simple reversed-phase high-performance liquid chromatographic method was developed for the determination of theobromine, theophylline and caffeine in cocoa samples. In the sample cleanup step, the procedure involves an on-line solid-phase extraction of analytes from cocoa samples into a home-made dry-packed pre-column with ODS-C₁₈ using a column-switching system. The separation was performed on a C₁₈ Nova-Pak column (150 mm × 3.9 mm, 4 μm) using a mobile phase consisting of a solution of 20% of methanol in water under isocratic conditions, at a flow-rate of 1.4 ml/min. The validation method revealed quantitative recoveries (>95.0%) with a coefficients of variation <3.2% and it also provided a good precision for data validation. The overlap of sample cleanup, analysis and recondition of the precolumn increases the sample throughput to 8 samples/h. Furthermore, the proposed method was successfully applied to analysis of cocoa samples “Trinitario”, “Forastero” and “Criollo” grown in different seasons of the year and fermented for 3 and 7 days, respectively. The results showed a slight reduction in the theobromine and caffeine content according to the fermentation times. In the same way, the theobromine/caffeine ratio was assessed, with the purpose of establishing a correlation with the genotype of the studied samples.     

可可碱、茶碱和咖啡碱的快速测定及其色谱保留行为

建立基于钛胶反相色谱固定相快速测定食品中可可碱、茶碱和咖啡碱的高效液相色谱（high performance liquid chromatography,HPLC）法。结果表明,可可碱、茶碱和咖啡碱的标准曲线线性良好（R2＞0.999 0）,线性范围分别为5～500、5～1 000 mg/mL和6～1 200 mg/mL,检出限分别为0.04、0.08 mg/mL和0.03 mg/mL,样品加标回收率分别为99.0%～102.0%、96.7%～106.7%和99.4%～102.3%,相对标准偏差（n=9）小于0.23%。3 种生物碱在HPLC上的保留行为由焓变ΔH0驱动,保留顺序与实验结果相符,依次为可可碱、茶碱和咖啡碱。建立方法操作简单、分析速度快,精密度和准确度均可满足测定要求。用于食品中可可碱、茶碱和咖啡碱的同时测定,结果满意。     

Evaluation of xanthine derivatives in chocolate – nutritional and chemical aspects

 Organic substances containing nitrogen are widespread throughout the whole of the natural world. Also included amongst these are the methylxanthine derivatives caffeine, theobromine and theophylline. These are very closely related and are to be found in extremely varying contents in different plants. Because of their pharmacological effect, which is fundamentally of a stimulative nature, methylxanthines have been significant since time immemorial as luxury, non-essential foodstuffs and as medication. These substances are often referred to as alkaloids. Theobromine is the major alkaloid in cacao (Theobroma cacao). Caffeine, on the other hand, is only to be found in cacao in very small quantities and theophylline only in trace amounts. Methylxanthines are said to contribute towards the typically bitter taste of cacao. In addition they represent important analytical parameters with regard to the evaluation of the quality of cacao-containing and chocolate products. The amount of methylxanthines in cacao depends on various influencing factors, the most essential ones being processing procedures, genotype, geographical origin and cacao bean weight. By determining the amount of theobromine and caffeine in cacao and/or cacao products, the amount of fat-free dry cacao can also be estimated. There are various methods available for the detection and determination of methylxanthines. UV-spectrophotometric determination and analysis using HPLC have attained major significance here. With regard to human nutrition, cacao and cacao products are the main natural sources of theobromine, but significantly less so with regard to caffeine. The presence of methylxanthines in cacao products leads to that they are sometimes the subject of consumer criticism, particularly because they are considered as luxury foodstuffs. The concluding evaluation of methylxanthines by dieticians and toxicologists reveals that plants that contain methylxanthines have been consumed for as long as can be remembered without having led to damage to health. Scientific research has shown that the consumption of these substances has no consequence whatsoever, as long as consumption is not excessive, which is the case for all foodstuffs. Methylxanthines in cacao products have no negative effects on the health of humans because their amounts in chocolates and other processed foodstuffs containing cacao are so low that they account for only a very small proportion of the whole of the human diet. Received: 15 November 1996 / Revised version: 22 January 1997     

Evaluation of xanthine derivatives in chocolate – nutritional and chemical aspects

 Organic substances containing nitrogen are widespread throughout the whole of the natural world. Also included amongst these are the methylxanthine derivatives caffeine, theobromine and theophylline. These are very closely related and are to be found in extremely varying contents in different plants. Because of their pharmacological effect, which is fundamentally of a stimulative nature, methylxanthines have been significant since time immemorial as luxury, non-essential foodstuffs and as medication. These substances are often referred to as alkaloids. Theobromine is the major alkaloid in cacao (Theobroma cacao). Caffeine, on the other hand, is only to be found in cacao in very small quantities and theophylline only in trace amounts. Methylxanthines are said to contribute towards the typically bitter taste of cacao. In addition they represent important analytical parameters with regard to the evaluation of the quality of cacao-containing and chocolate products. The amount of methylxanthines in cacao depends on various influencing factors, the most essential ones being processing procedures, genotype, geographical origin and cacao bean weight. By determining the amount of theobromine and caffeine in cacao and/or cacao products, the amount of fat-free dry cacao can also be estimated. There are various methods available for the detection and determination of methylxanthines. UV-spectrophotometric determination and analysis using HPLC have attained major significance here. With regard to human nutrition, cacao and cacao products are the main natural sources of theobromine, but significantly less so with regard to caffeine. The presence of methylxanthines in cacao products leads to that they are sometimes the subject of consumer criticism, particularly because they are considered as luxury foodstuffs. The concluding evaluation of methylxanthines by dieticians and toxicologists reveals that plants that contain methylxanthines have been consumed for as long as can be remembered without having led to damage to health. Scientific research has shown that the consumption of these substances has no consequence whatsoever, as long as consumption is not excessive, which is the case for all foodstuffs. Methylxanthines in cacao products have no negative effects on the health of humans because their amounts in chocolates and other processed foodstuffs containing cacao are so low that they account for only a very small proportion of the whole of the human diet. Received: 15 November 1996 / Revised version: 22 January 1997     

HPLC determination of caffeine and theophylline inPaullinia cupana Kunth (Guarana) and Cola spp. samples

Summary A reversed-phase high-performance liquid-chromatographic method for the determination of caffeine and theophylline in commercial guarana samples (drug obtained from the seeds ofPaulinia cupana Kunth,Sapindaceae of the Amazon Region) and inCola spp. samples is described and discussed.The methodology developed is simple and rapid with a minimum of samples preparation required. A comparision of five different techniques for the extraction of caffeine and theophylline is discussed. Furthermore the quantitative determination of caffeine and theophylline in five samples of Brasilian guarana, in two samples of dietetic products containing guarana, in two samples ofCola extract and in three ofCola seed powder are reported.

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HPLC-Bestimmung von Coffein und Theophyllin inPaullinia cupana- und in Cola spp.-Proben

Zusammenfassung Die Anwendung einer RP-HPLC für die mengenmässige Bestimmung von Coffein und Theophyllin in Guarana-Proben (Droge aus den Samen desPaullinia cupana-Baumgewächses Kunth, Familie:Sapindaceae) des Amazonas-Gebiet und in Cola spp.-Proben wird beschrieben. Die analytische Methode ist einfach, nicht zeitaufwendig und erfordert nur ein Mindestmaß an analytischen Operationen für die Proben-Zubereitung. Der Vergleich von 5 verschiedenen Extraktionstechniken für Coffein wird ebenfalls erörtert. Außerdem wurde der Gehalt von Coffein und Theophyllin in fünf verschiedenen brasilianischen Guarana-Proben, zwei Proben diätetischer Erzeugnisse mit Guarana-Gehalt, zwei Cola-Extraktund drei Cola-Samen-Proben bestimmt.

     

A new method based on electrospray ionisation ion mobility spectrometry (ESI-IMS) for simultaneous determination of caffeine and theophylline

In this work for the first time, simultaneous analysis of caffeine and theophylline was done by ion mobility spectrometry (IMS) only, without a powerful separation technique (e.g., HPLC). Ion mobility spectrometry with low cost, inexpensive maintenance and very fast analysis makes an attractive technique for the simultaneous determination of the caffeine and theophylline in foodstuff samples and biological matrices. In this study, the extraction protocol using molecular imprinted polymer-solid phase extraction (MIP-SPE) was successfully used to directly extract caffeine and theophylline from real samples. The results obtained provided the detection limits of 0.2 and 0.3 μg mL⁻¹ for caffeine and theophylline, respectively. The linear dynamic range of about two orders of magnitude was obtained for these compounds. Also, the proposed method was used to analyse various real samples of green tea and spiked human plasma, and the obtained results confirmed the capability of ESI-IMS for simultaneous detection of caffeine and theophylline.     

HPLC determination of caffeine and theophylline in Paullinia cupana Kunth (guarana) and Cola spp. samples

A reversed-phase high-performance liquid-chromatographic method for the determination of caffeine and theophylline in commercial guarana samples (drug obtained from the seeds of Paulinia cupana Kunth, Sapindaceae of the Amazon Region) and in Cola spp. samples is described and discussed. The methodology developed is simple and rapid with a minimum of samples preparation required. A comparison of five different techniques for the extraction of caffeine and theophylline is discussed. Furthermore the quantitative determination of caffeine and theophylline in five samples of Brasilian guarana, in two samples of dietetic products containing guarana, in two samples of Cola extract and in three of Cola seed powder are reported.     

Caffeine Inhibition of Ochratoxin A Production

The effect of caffeine and theobromine on growth and ochratoxin A production by Aspergillus ochraceus was determined using microbiological medium. Caffeine produced a small decrease in growth, while reducing ochratoxin production as much as 98%. Theobromine had relatively little effect on growth or ochratoxin production. Screening of caffeine for its effect on the growth of a number of Aspergillus and Penicillium species indicated that caffeine may have biological activity against a variety of mycotoxigenic molds.     

Kinetics and equilibria of tea infusion: Rates of extraction of theaflavin,caffeine and theobromine from several whole teas and sieved fractions

The rates of infusion of theaflavin and caffeine into water at 80°C have been measured for Kapchorua Pekoe Fannings, Kapchorua Pekoe Dust, Betjan Flowery Broken Orange Pekoe and Rupai Flowery Orange Fannings whole teas and sieved fractions. For Kapchorua PF fractions the rates of theobromine infusion were also determined. Caffeine always infused faster than theobromine, and both infused faster than theaflavin. Rate constants were obtained from the slopes of first order kinetic plots which yielded straight lines with small intercepts. The rate constants for all three constituents increased by a factor of 2.2 as leaf size decreased from 850–1000μm to 500–600μm. The rates of extraction from whole teas varied more than fourfold. Kapchorua PD gave the most rapid rates and Betjan FBOP the slowest. Comparisons of similarly sized fractions of teas showed that leaf size distributions accounted for half these differences and the intrinsic properties of the leaf for the remaining factor of two. The Kenyan CTC teas gave higher rate constants than the Indian orthodox teas. From the rate constants, the effective diffusion coefficients for theaflavin and caffeine in Betjan FBOP leaf were calculated and found to be some 100 times smaller than in water. This shows the diffusion processes within the leaf to be complex ones.     

EFFECT OF COLD STORAGE ON THE QUALITY CHARACTERISTICS OF ‘AU-RUBRUM’ PLUMS1

The effect of cold storage (0°C) and maturity on the physical, chemical and sensory characteristics of plums (cv. ‘AU-Rubrum’) was determined. The size, weight, soluble solids and total sugar contents of the more mature fruit were significantly higher (P<0.05) and the “L” and hue angle were significantly lower (P<0.05) than plums in the less mature class. ‘AU-Rubrum’ fruit contained glucose, fructose and sorbitol but had no sucrose. Artificially ripening fruit at 20°C had a significant effect (P<0.01) on all color values for both maturity 1 and 2 fruit. The titratable acidity and sugar contents of the fruit ripened at 20°C were lower for ripened than for unripened fruit. Storage of the plums at 0°C for 5 weeks followed by subsequent ripening at 20°C had no significant effect on physical, chemical or sensory characteristics. Data indicate that ‘A U-Rubrum’ plums which have one-half red ground color (maturity 1) will develop full color and complete the normal ripening process. Data also indicate that the plums can be stored for at least 5 weeks at O°C without loss of quality and that the cultivar has potential for shipping to distant markets.     

普洱茶固态发酵中转化咖啡碱为茶碱菌株的鉴定与应用

目的:在普洱茶渥堆发酵中分离并鉴定能够转化咖啡碱为茶碱的有效菌株,研究其在液态发酵与固态发酵中转化咖啡碱为茶碱的能力,为高茶碱茶叶的开发提供菌种。方法:采用咖啡碱琼脂固态筛选培养基,从普洱茶渥堆发酵中筛选出2株潜在菌株,通过18SrDNA序列测定结合菌落特征、分生孢子结构对菌株进行鉴定。采用不同浓度的咖啡碱液态培养基、茶汤液态发酵和茶叶固态发酵探究潜在菌株咖啡碱转化为茶碱的能力。结果:潜在菌株分别鉴定为黑曲霉(Aspergillus niger NCBT110A)和聚多曲霉(Aspergillus sydowii NRRL250)。经富含咖啡碱的液态培养发现,黑曲霉对咖啡碱的利用有限;聚多曲霉能促使咖啡转化为茶碱。在一定咖啡碱浓度下,聚多曲霉对咖啡碱的降解率在90%以上,促使一半以上的咖啡碱转化为茶碱。聚多曲霉接种的茶汤液态发酵、茶叶固态发酵表明,聚多曲霉发酵过程中咖啡碱显著性下降(p<0.05),茶碱急剧增加;在发酵结束时,咖啡碱的降解率分别为85.43%和87.37%,茶碱含量分别为(501.2±13.55) mg/L和3.3293%±0.2463%(w/w)。在咖啡碱存在情况下,聚多曲霉能将咖啡碱大幅度转化为茶碱。结论:本研究揭示了聚多曲霉(Aspergillus sydowii NRRL250)具备将咖啡碱转化茶碱的特性,为高茶碱茶叶或茶饮料的开发提供了优异菌种。     

尤溪苦茶资源苦涩味物质测定与分析

采用分光光度法和高效液相色谱法,对33?份尤溪苦茶资源的花青素、总黄酮、生物碱组分、儿茶素组分等显苦涩味物质的生化成分进行测定。结果表明,其物质成分特征指标均具有一定差异,20?个指标变异系数在9.74%～73.08%之间,说明尤溪苦茶苦涩味生化成分存在丰富的多样性和变异性。与4?份对照茶树相比,发现尤溪苦茶在花青素、咖啡碱、可可碱、茶碱、表没食子儿茶素没食子酸酯、儿茶素总量和酯型儿茶素的均值均高于对照,表明尤溪苦茶苦涩味突出的原因为高含量的花青素、咖啡碱和儿茶素及其相互协同作用。据此筛选出花青素含量特异资源8?份、高咖啡碱资源1?份、可可碱特异资源1?份以及高表没食子儿茶素没食子酸酯资源21?份,为苦茶的杂交育种、创新加工、功能性成分开发与次生代谢机理的相关研究提供良好的材料基础。     

Simultaneous spectrophotometric determination of caffeine and theobromine in Iranian tea by artificial neural networks and its comparison with PLS

In order to determine the amount of caffeine and theobromine, spectrophotometry was used as a simple, rapid and economical method. Because of severe overlapping between these components, artificial neural network was used. The 230–300 nm spectral window with 1 nm interval was used for data acquisition. An artificial neural network (5-5-3) with linear transfer function between input-hidden and hidden-output layers was trained and applied for prediction of concentration of these methylxanthines in four Iranian tea samples. The model was compared with PLS modeling method. HPLC technique was used as a standard method.     

HPLC Determination of Caffeine and Theobromine Content of Various Natural and Red Dutched Cocoas

Eighteen randomly selected cocoa samples were analyzed in duplicate by HPLC for caffeine and theobromine. The relative standard deviation for the method was determined to be 1.55%. The mean ± S.D. of the theobromine plus caffeine content (moisture free basis) was 2.85 ± 0.08% with a 90% confidence interval of 2.72–2.98% for red Dutched cocoa. The total ± SD. for natural cocoas was 2.82 + 0.16% with a 90% confidence interval of 2.56–3.08%.