Analysis of vicilin (7S)‐class globulin in cocoa cotyledons from various genetic origins

Cocoa cotyledons contain vicilin (7S)‐class globulin (VCG), a major storage protein. It is the native source of oligopeptides and free amino acids which have been identified as precursors of cocoa‐specific aroma and are formed through proteolysis during fermentation. High‐resolution electrophoresis of native proteins isolated from ripe, unfermented cocoa cotyledons harvested from different cultivars was used to determine genetic differences of the genotypes. Flavour differences have been reported to exist after standard fermentation in cocoa beans harvested from various genotypes. In this paper, SDS‐PAGE and 2D IEF/SDS‐PAGE polypeptide separation patterns are shown which were separately isolated from cotyledons of various genetic origins. Cotyledons from three cultivars belonging to genetically distant varieties and a hybrid, Criollo, Forastero and Trinitario, did not reveal any analytical identity differences of VCG subunit polypeptide bands. Additionally, proteins of cotyledons harvested from three of those clones which were reported to produce genotype‐specific flavour differences in raw cocoa after standard fermentation, SCA 12, UIT1 and PBC 140, when analysed in the same way, did not indicate differences. Thus the cotyledon storage proteins from various genetically different cocoa trees are, within methodological limits, the same. We conclude that aroma differences in raw cocoa harvested from various genotypes are the result of other genetic, physiological or curing‐related factors, but are not due to genetic differences of aroma precursors derived from storage proteins. © 2002 Society of Chemical Industry     

The Precursors of Chocolate Aroma: Application of Gas Chromatography in Following Formation during Fermentation of Cocoa Beans

SUMMARY— An imporved method of roasting milligram order quantities of chocolate aroma precursors in a flash heater attached to a gas chromatograph has permitted semi-quantitative measurements of the aroma volatiles produced from samples withdrawn at intervals from fermenting heaps of cocoa beans. The observed increase in aroma concentration with increased duration of fermentation, and the time at which maximal concentration occurs, are in line with previous experience and with recent investigations on the production of free amino acids and reducing sugars, these substances being strongly suspected as aroma precursors.     

Aroma precursors and methylpyrazines in underfermented cocoa beans induced by endogenous carboxypeptidase

Cocoa-specific aroma precursors and methylpyrazines in underfermented cocoa beans obtained from fermentation induced by indigenous carboxypeptidase have been investigated. Fermentation conditions and cocoa bean components were analyzed during 0 to 3 d of fermentation. Underfermented cocoa beans were characterized as having hydrophilic peptides and free hydrophobic amino acids much higher than unfermented ones. These 2 key components of cocoa aroma precursors may be produced from the breakdown of proteins and polypeptides by endogenous carboxypeptidase during the fermentation process. The enzyme was activated during fermentation. Polypeptides of 47, 31, and 19 kDa were observed in the samples throughout the 3-d fermentation period; however, only the first 2 polypeptides were remarkably reduced during fermentation. Since the 1st day of fermentation, underfermented cocoa beans contained methylpyrazines, a dominant group of cocoa-specific aroma. This might be due to microbial activities during fermentation, observed through a decrease of pH value and an increase of temperature of cocoa beans. The concentration of tetramethylpyrazines was significantly increased during the 3 d of fermentation. This may increase the cocoa-specific flavor to the beans.     

酶解联合高温处理对可可粉风味的影响

本文考察了酶解和高温联合处理对可可风味的作用。首先以游离氨基酸含量为评价指标,研究了5种蛋白酶对脱脂可可粉酶解液中游离氨基酸的影响,并进一步采用顶空-气相色谱-质谱联用（headspace gas chromatography mass spectrometry,HS-GC-MS）结合香气活力值（odor activity value,OAV）以及感官评价研究了高温（90~180 ℃）对可可挥发性物质的作用。结果表明,酶解处理提高了总氨基酸含量,为酶解前的1.4~5.0倍,且与未经酶处理的对照样（12种挥发物）相比,5种联合处理的样品分别检测出29（天冬氨酸酶）、31（酸性蛋白酶）、29（风味蛋白酶）、30（菠萝蛋白酶）、32（木瓜蛋白酶）种挥发物,挥发性物质种类明显增加。当反应温度达到150 ℃以上更有助于可可中具有坚果和奶油香气的杂环化合物的生成;由感官评价可得,经风味蛋白酶处理并在150 ℃反应2 h的可可风味最佳。基于OAV与感官评价的偏最小二乘（partial least squares,PLS）模型验证可得:酶解和高温处理使可可在保持原有巧克力香气的同时增加花果香和奶香风味,有助于提升可可整体香气品质。     

Influence of carboxypeptidases on free amino acid,peptide and methylpyrazine contents of under‐fermented cocoa beans

A study of the action of two carboxypeptidases on free amino acids, peptides and methylpyrazines in under‐fermented cocoa beans was carried out. Carboxypeptidase B from porcine pancreas and carboxypeptidase Y from baker's yeast were used separately for digestion. Hydrophobic free amino acids (alanine, valine, isoleucine, leucine, phenylalanine and tyrosine) were predominantly produced in samples digested with both carboxypeptidase B and Y. The peptide patterns of samples digested with both carboxypeptidases were similar to that of the control. The concentration of 2,3,5,6‐tetramethylpyrazine in samples with carboxypeptidase B addition was significantly higher than those of 2,5‐dimethyl‐ and 2,3,5‐trimethylpyrazine; the concentration of 2,3,5‐trimethylpyrazine was highest (1727.86 µg per 100 g) in the sample with carboxypeptidase B addition that had been incubated for 24 h. These findings indicate that carboxypeptidase B from porcine pancreas was more prominent in the formation of cocoa‐specific aroma. © 2002 Society of Chemical Industry     

Extraction,identification and semi-quantification of oligopeptides in cocoa beans

Peptides and amino acids generated during cocoa bean fermentation are known to be the most important precursors for the development of cocoa aroma. Although cocoa fermentation and aroma development have been extensively studied, the cocoa oligopeptide fraction is under-investigated. In particular, the identification of specific peptide sequences and the quantification of cocoa peptides are scarce in the literature. The aim of this study was to investigate the presence of oligopeptides in fermented cocoa beans. Peptides were analysed by reversed phase LC/ESI-MS and LC/ESI-MS/MS, and the molecular masses of 44 different peptides were obtained by analysing the mass spectra associated with the most intense chromatographic peaks. Peptides were identified based on the exact molecular masses, mass fragmentation patterns and by comparison with vicilin and 21 kDa cocoa seed protein sequences. Semi-quantitative data on peptide presence in fermented cocoa samples of different geographic origin, different fermentation levels and on roasted products were also provided.     

Free amino acid amounts in raw cocoas from different origins

Raw cocoa is the processed and traded form of the cocoa seed. Fresh seeds undergo fermentation and a drying process before they are prepared for transport and shipping. Depending on the local situation in the producer region the seeds are collected from big estates and are fermented and dried in big lots or they originate from small farmer's crop trees. In the subsequent transport and trading chain raw cocoas may be mixed and stored before they finally are sold and brought to the consumer countries. Local or regional variations in cocoa plant material, fermentation procedures and drying processes finally result in a typical traded good with respect to the amino acids, which form an important part of the flavour precursors. These free amino acids and their composition result from the fermentation procedure of fresh seeds. In the course of the fermentation specific cleavage of cocoa storage proteins delivers the amino acid patterns. In this study the variation of free amino acid amount and distribution of 108 commercially fermented and traded cocoa samples and two Theobroma grandiflorum (Willd. ex Spreng.) K. Schum samples were determined. This examination showed clearly, that content and distribution of free amino acids in raw cocoa from different origins vary greatly (5–25 mg g⁻¹ fatfree dry matter), in some cases country and even region-specific differences were apparent. It is important to notice typical, region-specific variations in the amounts and compositions of free amino acids.     

Comparative study on the proteolytic activities and storage globulins in seeds of Theobroma grandiflorum (Willd ex Spreng) Schum and Theobroma bicolor Humb Bonpl,in relation to their potential to generate chocolate‐like aroma

The cocoa relatives T grandiflorum (cupuaçu) and T bicolor (macambo) are promising crop plants for sustainable agroforestry in the Amazon region of South America. The market for cupuaçu is expanding since the fruit flesh is utilised by the foodstuffs industry. Attempts to commercialise chocolate‐like wares from the seeds have failed so far because of unreliable product quality. It is not known whether this is due to an insufficient aroma potential of cupuaçu seeds. We therefore investigated the proteolytic enzymes and the seed storage globulins which are both decisive for the formation of aroma precursors in cocoa. We found that the activities of the aspartic endopeptidase and the carboxypeptidase in T bicolor and T grandiflorum differed slightly from those in cocoa. The specificity of the carboxypeptidase for hydrophobic amino acids was quite similar across the three species, while the optimal pH of the T grandiflorum enzyme was lower than that of the other species. The qualitative and quantitative differences between the globulins indicate a lower maximum yield of aroma precursors in T grandiflorum and a higher maximum yield of aroma precursors in T bicolor, compared to cocoa. We conclude that the quality of chocolate‐like products made from the studied cocoa relatives can be improved by adapting fermentation procedures to particular biochemical features of these seeds. Copyright © 2004 Society of Chemical Industry     

Effect of mass and turning time on free amino acid,peptide-N,sugar and pyrazine concentration during cocoa fermentation

A response surface methodology (RSM) was used to determine the optimum condition for mass and turning time during cocoa fermentation. Mass and turning time were used as independent variables; concentrations of free amino acids, peptide-N, sugars and pyrazines were the dependent variables. The R² values for peptide-N, tetramethylpyrazine and total pyrazines were greater than 0·9. Both lower (10 kg) and higher (100 kg) cocoa mass together with lower (0 min) and higher (10 min) turning time gave products containing low concentrations of hydrophobic, total and other free amino acids, peptide-N, fructose, glucose and total reducing sugars; in contrast, those of acidic free amino acids gave higher concentrations. Trimethyl-, tetramethyl- and total pyrazines increased significantly (P<0·05) at higher mass (100 kg) and higher turning time (10 min). From the highest concentration of the important flavour precursors ie hydrophobic free amino acids, total reducing sugars and peptide-N, the recommended mass of cocoa beans and turning time for an optimum cocoa fermentation condition was 60 kg and 5 min, respectively. © 1998 Society of Chemical Industry.     

烤烟烟叶中游离氨基酸与内在质量关系的研究

系统地分析了烤烟烟叶中游离氨基酸与内在质量的关系，参试材料31份，每个样品分析鉴定25项指标，对试验数据分别进行简单相关分析，多元回归和偏相关分析以及典型相关分析。简单相关分析结果表明，游离氨基酸组分与内在质量之间存在一定的相关关系，其中缬氨酸、酪氨酸和游离氨含量较高，则烟叶香气较好，亮氨酸含量较高，烟叶杂气较少，丝氨酸，甘氨酸和游离氨含量高，则烟叶评吸总分较高，烟叶品质相对较好，多元回归和偏相关分析系统地分析了游离氨基酸组分对内在质量的贡献大小。其中对香气贡献较大的有精氨酸，苯丙氨酸和游离氨；对吃味贡献较大的有甘氨酸，对减少杂气贡献较大的是游离氨和脯氨酸，对评吸总分贡献较大的有精氨酸。苯丙氨酸、丝氨酸，总游离氨基酸和酪氨酸。典型相关分析结果表明，游离氨基酸与内在质量间的典型相关主要是由酷氨酸，蛋氨酸，苯丙氨酸，精氨酸，丝氨酸，脯氨酸，亮氮酸，苏氨酸，氨与香气，吃味，杂气，刺激性相关显著引起的。     

The Precursors of Chocolate Aroma: Production of Free Amino Acids during Fermentation of Cocoa Beans

SUMMARY— Amino acids are produced during the fermentation of cocoa beans at a rate which is in agreement with the already established rapid rate of flavour and aroma development. The role of these compounds as aroma precursors receives further confirmation as a result of these studies, and an objective method of assessing the "degree of fermentation" of cocoa beans has been described and tested on a number of different commercial varieties of cocoa.     

Hypoglycemic effects of cocoa (Theobroma cacao L.) autolysates

Fat, alkaloid and polyphenol contents of two clones of cocoa (UIT1 and PBC 140) were removed and the remaining powder was autolyzed at pH 3.5 and 5.2. Based on the results, autolysates of UIT produced at pH 3.5 exhibited the highest ability to inhibit α-amylase activity. However, no α-glucosidase inhibition activity was observed under the conditions specified. Autolysates produced under pH 3.5 caused the highest amount of insulin secretion. In streptozotocin-diabetic rats, all cocoa autolysates significantly decreased blood glucose at 4 h. To assure that the results from the assays were not due to the polyphenols of cocoa autolysates qualitative and quantitative tests were applied. According to their results cocoa autolysates were found to be free from polyphenols. Analysis of amino acid composition revealed that cocoa autolysates were abundant in hydrophobic amino acids. It can be suggested that besides other compounds of cocoa, its peptides and amino acids could contribute to its health benefits.     

Activation of remaining key enzymes in dried under-fermented cocoa beans and its effect on aroma precursor formation

Incubation-activation of remaining key enzymes in dried under-fermented cocoa beans and its effect on aroma precursor formation has been studied using defatted unfermented and partly fermented cocoa bean powders. Results of the study showed that aspartic endoprotease, carboxypeptidase and invertase were significantly inactivated during fermentation and drying, and the effect of fermentation was significantly lower than that of drying. The enzyme activities remaining in these beans were still sufficient to carry out enzymatic reaction during incubation. Peptide patterns, resulting from incubation of unfermented and partly fermented beans powders, were quite similar to the well-fermented patterns. Meanwhile, free amino acid concentrations of the unfermented beans were significantly increased during the first 4 h of incubation and then remained constant; however, with partly fermented beans, the formation continued and the hydrophobic and total free amino acid concentrations reached the value of well-fermented beans after 24 h of incubation. Reducing sugar concentrations of both unfermented and partly fermented cocoa beans could reach the level of well-fermented beans by incubation.     

Impact of starter cultures and fermentation techniques on the volatile aroma and sensory profile of chocolate

The sensory quality of chocolate is widely determined by the qualitative and quantitative composition of volatile compounds resulting from microbial metabolism during fermentation, and Maillard reactions taking place during drying, roasting and conching. The influence of applying mixed starter cultures on the formation of flavour precursors, composition of volatile aroma compounds and sensory profile was investigated in cocoa inoculated with cultures encompassing a highly aromatic strain of Pichia kluyveri or a pectinolytic strain of Kluyveromyces marxianus, and compared to commercially fermented heap and tray cocoa. Although only minor differences in the concentration of free amino acids and reducing sugars was measured, identification and quantification by dynamic headspace gas chromatography–mass spectrometry (HS/GC–MS) revealed pronounced differences in the composition of volatiles in roasted cocoa liquors and finished chocolates. 19 of the 56 volatile compounds identified in the chocolates were found in significantly higher amounts in the tray fermented sample, whilst significantly higher amounts of 2-methoxyphenol was measured in the two inoculated chocolates. The P. kluyveri inoculated chocolate was characterized by a significantly higher concentration of phenylacetaldehyde and the K. marxianus inoculated chocolate by significantly higher amounts of benzyl alcohol, phenethyl alcohol, benzyl acetate and phenethyl acetate compared to a spontaneously fermented control. Sensory profiling described the heap and tray fermented chocolates as sweet with cocoa and caramel flavours, whilst the inoculated chocolates were characterized as fruity, acid and bitter with berry, yoghurt and balsamic flavours. The choice of fermentation technique had the greatest overall impact on the volatile aroma and sensory profile, but whilst the application of starter cultures did affect the volatile aroma profile, differences were too small to significantly change consumer perception of the chocolates as compared to a spontaneously fermented control.     

ASSESSMENT METHODOLOGY TO PREDICT QUALITY OF COCOA BEANS FOR EXPORT

The existing quality assessment methods for cocoa such as the cut-test and sensory test were evaluated. The results were compared with the fermentation index and color measurement as indirect quality assessment methods. Results of the cut-test score and sensory evaluation methods indicated variability in results and therefore were not adequate to assess bean quality. The fermentation index method was useful to distinguish different bean samples from 5 color categories. The fermentation index related well with other quality parameters, such as pH, reducing sugars, free amino acids, and cocoa color pigments. Direct color measurement had a good relationship with fermentation index, pH, reducing sugars, and free amino acids, as evidenced by high correlation values. Color measurement was appropriate to estimate cocoa bean quality.     

Effect of polyphenol and pH on cocoa Maillard-related flavour precursors in a lipidic model system

Polyphenol and pH influence the flavour quality of cocoa beans during roasting. Amino acids and reducing sugars are flavour precursors in cocoa beans, which develop into cocoa-specific aroma through Maillard reactions during roasting. A central composite design was applied to determine the combined effect of polyphenol and pH on the flavour precursors during cocoa roasting at 120 °C for 45 min using a lipidic model system. Polyphenol was added at 40, 80 and 120 g kg⁻¹ and pH was adjusted to 4.5, 6 and 7.5. The response surface methodology revealed that a lower concentration of amino acids and reducing sugars was obtained at higher polyphenol concentration (120 g kg⁻¹) and lower pH value (4.5). Based on the constraints set, the best polyphenol concentration of 43–58 g kg⁻¹ and pH of 7.0–7.5 was found to be optimum for the formation of flavour precursors in this lipidic model study.     

L-阿拉伯糖与氨基酸美拉德反应挥发性风味物质分析

L-阿拉伯糖反应型甜味香精香气柔和、丰富,使用此香精的终端产品有更自然和饱满的香气。用L-阿拉伯糖分别与丙氨酸、甘氨酸、苏氨酸、丝氨酸发生美拉德反应生成反应型甜味香精。采用固相微萃取-气相色谱-质谱联用技术,研究其挥发性风味化合物。结果表明：L-阿拉伯糖分别与4 种氨基酸反应均产生具有烤香和可可香的风味化合物2,5-二甲基吡嗪、2,3,5-三甲基吡嗪和2-乙基-3,5-二甲基吡嗪等。L-阿拉伯糖与苏氨酸发生美拉德反应产生带有青香风味的化合物薄荷酮。L-阿拉伯糖与丙氨酸发生美拉德反应产生带有花果香的风味物质5-茚醇。     

Effect of drying time,bean depth and temperature on free amino acid,peptide‐N,sugar and pyrazine concentrations of Malaysian cocoa beans

The effect of drying time on free amino acid, peptide‐N, sugar and pyrazine concentrations as well as the influence of bean depth and temperature on these compounds during cocoa drying was studied. Drying time, bean depth and temperature significantly decreased the concentration of free amino acids, peptide‐N, total reducing sugars and sucrose and increased the concentration of trimethyl‐, tetramethylpyrazine and total pyrazines in cocoa beans. The best drying treatment was obtained at the combination of bean depth/drying temperature of 8.3 cm/40 °C. This was based on the fact that it produced significantly high concentrations of hydrophobic free amino acids, peptide‐N and total reducing sugars and significantly low concentration of trimethyl‐, tetramethylpyrazine and total pyrazines. Drying treatment of 1.5 cm/60 °C significantly produced the lowest concentration of free amino acids, peptide‐N and total reducing sugars and the highest concentration of pyrazines. © 1999 Society of Chemical Industry     

不同氨基酸对普洱熟茶呈味物质和香气成分的影响

氨基酸是普洱茶发酵过程中重要的中间物质,对普洱茶内含物质的转化和品质都有着重要的影响。本文探讨了在发酵时添加不同氨基酸对普洱熟茶产品的主要呈味物质和挥发性香气成分的影响。结果表明,添加外源氨基酸组的水浸出物含量在34.82～39.46%之间,茶褐素含量在6.31～7.17%之间,游离氨基酸的含量在6.31～7.17%之间,这三项指标均高于对照组;茶多酚含量在7.76～8.73%之间,水溶性糖在3.07～3.89%之间,均低于对照组,咖啡碱含量与对照组相差不大。挥发性香气成分分析表明,醇类占香气成分的21.17～25.47%、醛类占15.64～18.71%、杂氧化合物类占13.75～17.92%、酯类占7.17～10.25%,这几类物质是主要的香气成分;实验组香气成分中的酯类和杂氧化合物类含量大于对照组,而它们又是陈年普洱茶的主要的特征香气成分。实验表明外源氨基酸对普洱茶汤中的呈味物质以及香气成分的形成有显著影响,能加快普洱茶特殊品质的形成。     

Factors influencing quality variation in cocoa (Theobroma cacao) bean flavour profile — A review

This review examined the factors that influence flavour volatiles of cocoa beans and the volume of work that needs to be done on these factors and their impact on the flavour volatiles of commercial cocoa beans. Cocoa bean flavour is one of the most important quality attributes as flavour is central to acceptability of cocoa beans and cocoa products such as chocolate. The complex composition of cocoa bean flavour depends on bean genotype, postharvest treatments such as pulp pre-conditioning, fermentation and drying, industrial processes such as roasting as well as the type of soil and age of cocoa tree. The bean genotype determines the chemical composition of the bean, specifically the contents of bean storage proteins, polysaccharides, and polyphenols. This determines the quantities and type of precursors formed during fermentation and drying processes leading to flavour formation, hence, influencing both flavour type and intensity. Cocoa bean fermentation and drying result in the breakdown of the storage proteins by endogenous proteases into amino acids and short chain oligopeptides while the polysaccharides are also degraded by invertase to glucose and fructose. The amino acids, oligopeptides, glucose and fructose react with each other during the roasting process to produce the typical cocoa flavour volatiles. Polyphenols are also oxidized by polyphenol oxidase during fermentation and drying which reduce the astringency and bitterness of the beans, thus, enhancing the flavour of cocoa beans. However, the extent to which other factors such as age of the cocoa tree and soil chemical compositions influence the formation of flavour precursors and their relationships with final flavour quality remains unclear. With increasing demand for sustainable production of high quality cocoa beans, greater understanding of factors contributing to the variations in flavour character would have significant commercial implications.