Fermentation of cocoa beans: influence of microbial activities and polyphenol concentrations on the flavour of chocolate

BACKGROUND: Spontaneous cocoa bean fermentation is characterised by a succession of microbial activities. Cocoa flavour precursors are developed during fermentation and drying of cocoa beans. Polyphenols and alkaloids contribute to astringency and bitterness of cocoa and chocolate. RESULTS: Population dynamics, metabolite target analyses, and chocolate production were performed for seven independent spontaneous cocoa bean heap fermentations in Ghana. Although the same micro‐organisms were involved in these heaps, carried out at different farms or in different seasons, heap temperatures and microbial metabolite concentrations were different. This could be due to heterogeneity and size of the heaps, but was mainly ascribed to microbial variability. Indeed, differences in microbial activity could be linked with the flavour of chocolates made from the corresponding dried, fermented cocoa beans. Whereas the polyphenol and alkaloid contents of cocoa beans were crop‐ and heap‐dependent, epicatechin and theobromine levels decreased during fermentation due to diffusion out of the bean cotyledons and polyphenol oxidation and condensation. Residual levels were responsible for the degree of bitterness of the final chocolates. CONCLUSION: Differences in microbial activities between different heap fermentations can result in dried fermented cocoa beans and chocolates with different flavour characteristics. Hence, fermentation control may direct the flavour of chocolate. Copyright © 2008 Society of Chemical Industry     

Investigating the fermentation of cocoa by correlating denaturing gradient gel electrophoresis profiles and near infrared spectra

Raw cocoa has an astringent, unpleasant taste and flavour, and has to be fermented, dried and roasted in order to obtain the characteristic cocoa flavour and taste. During the fermentation microbial activity outside the cocoa beans induces biochemical and physical changes inside the beans. The process is complex involving activity of several different groups of microorganisms which bring about numerous biochemical and physical changes inside the beans. Due to the complexity of these processes no thorough investigations of the interactions between the microbial activities on the outside of the beans and the chemical processes inside the beans have been carried out previously. Recently it has been shown that Denaturing Gradient Gel Electrophoresis (DGGE) offers an efficient tool for monitoring the microbiological changes taking place during the fermentation of cocoa. Near Infrared (NIR) spectroscopy has previously been used to determine various components in cocoa beans, offering a rapid alternative compared to traditional analytical methods for obtaining knowledge about changes in the chemical composition of the cocoa beans during fermentation. During a number of cocoa fermentations bean samples were taken with 24 h intervals to be dried and analysed by NIR. Cocoa pulp samples taken simultaneously during the same fermentations have previously been characterised using DGGE [Nielsen, D.S., Teniola, O.D., Ban-Koffi, L., Owusu, M., Andersson, T., Holzapfel, W.H. (2007). The microbiology of Ghanaian cocoa fermentations analysed using culture dependent and culture-independent methods. International Journal of Food Microbiology 114, 168-186.]. Here we report the first study where microbiological changes during the fermentation determined using DGGE are correlated to changes inside the beans determined by NIR using multivariate data analysis. Following data pre-processing (baseline correction followed by Co-shift correction or Correlation Optimised Warping) the DGGE spectra were analysed using Principal Component Analysis (PCA). A clear grouping according to fermentation time was seen demonstrating the microbial succession taking place during the fermentation. Subsequently the DGGE spectra were correlated to the NIR spectra using Partial Least Squares regression models (PLS2). Correlations of 0.87 (bacterial derived DGGE spectra) and 0.81 (yeast derived DGGE spectra) were obtained indicating the relationship between the microbial activities in the pulp and the (bio)chemical changes inside the beans. By comparing the X-block loadings of the PLS2 models and the DGGE spectra it was possible to directly link several microbial species with changes in the NIR spectra and consequently also with changes inside the beans.     

Effects of incubation and polyphenol oxidase enrichment on colour, fermentation index, procyanidins and astringency of unfermented and partly fermented cocoa beans

Incubation of unfermented and partly fermented cocoa beans in acetate buffer, pH 5.5, at 45 °C increased yellowness, total colour differences and fermentation index value of the cocoa bean powders and decreased cocoa procyanidins (monomers to pentamers), and their astringency. Fermentation index and (–)‐epicatechin content, equivalent to those of fully fermented beans, were reached by unfermented beans after 4–8‐h incubation, but not by partly fermented beans even after 16 h. During incubation of partly fermented cocoa beans enriched with polyphenol oxidase, yellowness and fermentation index value were increased, whilst (–)‐epicatechin was decreased. Tyrosinase had a less significant effect in yellow colour formation, but showed a significant reduction of (–)‐epicatechin and increase in fermentation index compared with crude cocoa polyphenol oxidase. However, both enzymes have similar effects on procyanidin degradation and astringent taste reduction. Incubation of cocoa beans for 16 h increased the cut test score of unfermented and partly fermented beans by 50 and 30%, respectively.     

烘焙温度对可可豆苦涩味的影响

对不同烘焙温度下的可可豆进行致苦涩味物质分离分析,基于感官评价和相对香味活力值的概念筛选出参与可可豆苦涩味形成的主要组分,并结合热裂解-气质联用技术研究烘焙温度上升时可可豆挥发性物质的变化规律。结果表明:(1)可可豆提取物苦涩味流分共检测出55种挥发性成分,其中醇类7种,酸类5种,酯类9种,醛类8种,酮类6种,烯烃类2种;(2)致苦涩味的关键组分为2-甲基丙醛、2-甲氧基苯酚、2-庚醇、戊醛、2-戊酮、苯甲醛、2,5-二甲基-4-羟基-3(2H)-呋喃酮,修饰组分为3-甲基丁醇、2,3-二甲基-5-乙基吡嗪、2,5-二甲基吡嗪、2,3-二甲基吡嗪、2-甲基丙醇;(3) 130~135℃为降低可可豆苦涩味的最佳烘焙温度。     

Improved Fermentation of Cocoa Beans with Enhanced Aroma Profiles

The cocoa (Theobroma cacao) fermentation was carried out using starter consortia of Saccharomyces cerevisiae, Lactobacillus plantarum, and Acetobacter aceti with 10% inoculum. The fermented beans were soaked with sterile water (1:3 wt/v) for the duration of 1, 3, and 5 h. The treated beans were dried to the moisture content of 7 ± 1%. The samples were then roasted, ground and subjected to steam distillation. The volatiles were extracted and identified using GC-MS. The samples soaked for 1 h scored 100% cut score along with 0.85% fermentation index. The volatile and non-volatiles were associated with beans treated in water after fermentation. Volatile compounds were classified as esters, alcohols, acids, aldehydes, ketones, pyrazines, hydroperoxides, and hydrocarbons. Most of the oxygenated aliphatic compounds and aromatics were potentially derived via an acetic acid and shikimic acid pathways, respectively. Untreated cocoa beans contained 12 sesquiterpene hydrocarbons potentially derived through the mevalonic acid pathway and contributed to harsh notes. Also, these metabolites were completely absent in the treated samples indicating depletion while soaking. Thus, the treated samples (1 h) had enhanced quality with high flavor notes with 8.5 rating on sensory evaluation. Thus, these soaking treatments with fermentation lead to improvement of flavor profiles which confers improved cocoa quality.     

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.     

Improving Malaysian cocoa quality through the use of dehumidified air under mild drying conditions

BACKGROUND: Various studies have been conducted in the past to improve the quality of Malaysian cocoa beans. However, the processing methods still remain crude and lack technological advancement. In terms of drying, no previous study has attempted to apply advanced drying technology to improve bean quality. This paper presents the first attempt to improve the quality of cocoa beans through heat pump drying using constant air (28.6 and 40.4 °C) and stepwise (step‐up 30.7–43.6–56.9 °C and step‐down 54.9–43.9 °C) drying profiles. Comparison was made against hot air drying at 55.9 °C. RESULTS: Product quality assessment showed significant improvement in the quality of Malaysian cocoa beans. Quality was found to be better in terms of lower acidity (higher pH) and higher degree of browning (cut test) for cocoa beans dried using the step‐up profile. All heat pump‐dried samples showed flavour quality comparable to that of Ghanaian and better than that of Malaysian and Indonesian commercial samples. Step‐up‐dried samples showed the best flavour profile with high level of cocoa flavour, low in sourness and not excessive in bitterness and astringency. CONCLUSION: Dried cocoa samples from the step‐up drying profile showed the best overall quality as compared with commercial samples from Malaysia, Indonesia and Ghana. The improvement of Malaysian cocoa bean quality is thus achievable through heat pump drying. Copyright © 2010 Society of Chemical Industry     

菌种对芽菜发酵影响的研究

将选育的芽菜工作菌种,接种于菜中进行发酵。通过同时蒸馏萃取(SDE)-气质联用(GC-MS),分析不同发酵阶段芽菜的风味物质,并结合感官评定结果来判定芽菜的发酵情况。结果表明:芽菜接种发酵后的发酵周期比自然发酵缩短了近一个月,而且复合菌AN1∶AE发酵的芽菜产生的风味物质组分增多,口感最好,其次是AN∶AE,最后是自然发酵。     

On-farm implementation of a starter culture for improved cocoa bean fermentation and its influence on the flavour of chocolates produced thereof

Cocoa bean fermentations controlled by means of starter cultures were introduced on several farms in two different cocoa-producing regions (West Africa and Southeast Asia). Two starter culture mixtures were tested, namely one composed of Saccharomyces cerevisiae H5S5K23, Lactobacillus fermentum 222, and Acetobacter pasteurianus 386B (three heaps and one box), and another composed of L. fermentum 222 and A. pasteurianus 386B (seven heaps and one box). In all starter culture-added cocoa bean fermentation processes, the inoculated starter culture species were able to outgrow the natural contamination of the cocoa pulp-bean mass and they prevailed during cocoa bean fermentation. The application of both added starter cultures resulted in fermented dry cocoa beans that gave concomitant milk and dark chocolates with a reliable flavour, independent of cocoa-producing region or fermentation method. The addition of the lactic acid bacterium (LAB)/acetic acid bacterium (AAB) starter culture to the fermenting cocoa pulp-bean mass accelerated the cocoa bean fermentation process regarding citric acid conversion and lactic acid production through carbohydrate fermentation. For the production of a standard bulk chocolate, the addition of a yeast/LAB/AAB starter culture was necessary. This enabled an enhanced and consistent ethanol production by yeasts for a successful starter culture-added cocoa bean fermentation process. This study showed possibilities for the use of starter cultures in cocoa bean fermentation processing to achieve a reliably improved fermentation of cocoa pulp-bean mass that can consistently produce high-quality fermented dry cocoa beans and flavourful chocolates produced thereof.     

The Precursors of Chocolate Aroma: Production of Reducing Sugars during Fermentation of Cocoa Beans

SUMMARY— The sucrose in fresh cocoa beans is hydrolyzed to glucose and fructose during fermentation and the rate of the reaction confirms the possible inclusion of reducing sugars among the precursors of chocolate aroma. The optimal concentration of reducing sugars in the bean is reached at about the same time as maximal flavor development, and coincides approximately with the peak in amino acid concentration. An objective method for assessing the "Degree of Fermentation" of cocoa beans has been proposed and tested.     

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.     

Physicochemical properties,antioxidant content,volatile organic compounds and sensory profile of cocoa beans fermented with yeast starter cultures

This study determined the influence of selected yeast starters on the antioxidant content, volatile organic compounds and sensory qualities of fermented cocoa beans. Cocoa beans fermented with Hanseniaspora thailandica (MH979675), Pichia kudriazevii (MH979681), or a mixture of both species in fermentation (20 kg) were highly reproducible based on the physicochemical parameters (temperature, pH of nibs, fermentation index and cut test). The usage of H. thailandica improved the total polyphenols content of cocoa beans with fermentation index around 1.24 and 1.33. The correlation analyses suggested that phenolics and flavonoids compounds may not be the main constituents that led to the antioxidant activities of the fermented samples. Principal Component Analysis showed distinct differences in VOC profiles between spontaneous and fermentation using yeast. Sweet and spicy notes were only found in sample fermented with yeast. The inoculation of yeast in fermentation influenced the sensorial and antioxidant properties of cocoa beans.     

Performance of different drying methods and their effects on the chemical quality attributes of raw cocoa material

Studies were carried out to investigate the impact of different drying processes on the chemical quality traits of raw cocoa beans. The pH of less fermented cocoa is higher than the well‐fermented cocoa’s. The sun‐dried beans pH ranged from 4.5 to 5.5, while the pH of both oven‐ and mixed‐dried beans was between 3.8 and 5.2. The sun‐dried beans contained lower volatile acidity than oven‐dried beans. Artificially dried beans resulted in higher free acidity content when compared to both sun‐ and mixed‐dried beans. Ammonium nitrogen content in raw cocoa beans is not influenced by the drying methods. Free fatty acid content increases slowly but remains below the critical value of 1.75% whatever the drying processes. While oven‐dried beans show the FFA content above 0.70% both of sun‐ and mixed‐dried beans are associated with FFA content below 0.70%.     

Performance of different fermentation methods and the effect of their duration on the quality of raw cocoa beans

This study aims to compare the effect of three cocoa fermentation methods and their duration on raw cocoa quality. Results showed a decrease in percentage of physical quality defects on fermentation method. Cocoa fermented for 4 days presented higher percentage of purple beans reached 45% and about 10% of slaty beans than cocoa fermented for 6 days whatever the process. Fermentation duration did not influence the mouldy beans that were around 1%. Formation of brown beans increased from 16% to 50% depending on the fermentation duration and process. Using wooden boxes allowed higher percentage of 77%–90% brown beans than others materials. Acidity of cocoa decreased on fermentation duration but beans treated in boxes were significantly (P = 0.05) acidic from 1.40 and 3.07 meq of NaOH g^?1. Fungal population did not vary in number depending both on the duration and the fermentation method with rates that ranged from 3.32 × 10⁷ to 8.63 × 10⁷ CFU g^?1.     

乳酸菌发酵酸豆奶中不良风味物质研究进展

随着植物基蛋白饮料在国内外的迅速发展, 酸豆奶必将进入新时代。酸豆奶是指以大豆为主要原料经过乳酸菌发酵后所得的产品, 在植物基蛋白饮料中占有重要地位, 良好的气味特征和口感特征对酸豆奶产品来说至关重要, 而豆腥味、苦涩味等不良风味的存在限制了酸豆奶的发展。酸豆奶风味物质种类繁多, 挥发性风味物质、非挥发性风味物质的成分和含量极大程度上决定了酸豆奶的感官品质。本研究就酸豆奶的发酵菌种、不良风味物质的来源、风味物质的分类和检测方法进行综述, 从发酵菌种与酸豆奶中不良风味物质的角度出发, 为解决酸豆奶在工业生产中不良风味问题提供思路。     

克东腐乳发酵过程中挥发性风味物质分析

克东腐乳是以微球菌为主要菌系发酵而成的细菌型腐乳,具有独特的质地和风味。本文采用同时蒸馏萃取法(SDE)提取,并结合气相色谱-质谱联用(GC-MS)技术对克东腐乳挥发性风味物质进行跟踪萃取检测研究,样品包括豆腐白坯、发酵液及生产过程中腐乳块和汤料,综合分析各种风味成分的产生情况及含量变化,并且与传统毛霉腐乳的挥发性风味物质种类及含量进行比较。分析表明随着发酵时间的延长,检测出的挥发性风味物质种类不断增加,酸、醛、酮、醇等类物质不断产生,而且相应的百分含量逐渐增加;酯类始终是主要的的风味物质,而在整个过程中酯类物质所占的百分比呈下降趋势。以克东腐乳为研究对象,揭示细菌型腐乳生产过程中风味物质的变化规律,为中国传统发酵食品建立基础性数据资料。     

发酵和焙烤对可可豆多酚、黄酮和风味品质的影响

本实验采用高效液相色谱仪（high performance liquid chromatography,HPLC）、色差仪、电子鼻分别检测未发酵、发酵和焙烤海南可可豆的总酚含量、总黄酮含量、色度和可可豆风味差异。结果表明：未经焙烤的未发酵豆总酚（464.03 mg/10 g）和总黄酮（126.86 mg/10 g）含量明显高于发酵豆总酚（211.86 mg/10 g）和总黄酮（61.98 mg/10 g）含量。105～145 ℃焙烤30 min,未发酵豆总酚和总黄酮含量分别为419.5～129.8 mg/10 g和77.8～16.8 mg/10 g;发酵豆总酚和总黄酮含量分别为182.53～86.25 mg/10 g和34.7～7.0 mg/10 g。其中,125 ℃焙烤20～40 min,未发酵豆总酚和黄酮含量分别为353.74～289.45 mg/10 g和42.86～32.20 mg/10 g;发酵豆总酚和黄酮含量分别为152.08～123.55 mg/10 g和25.12～21.14 mg/10 g。在105～145 ℃的温度焙烤下没食子酸含量变化显著。可可豆色度值的范围：L*值集中在40.0～47.0之间,a*值集中在5.0～6.8之间,b*值集中在4.0～8.5之间。未发酵和发酵可可豆之间,以及不同温度焙烤可可豆之间的电子感官风味分析结果差异较大。     

7株米曲霉与沪酿3.042发酵制备郫县豆瓣用甜瓣子的对比研究

为了筛选高效的具有潜在应用价值的郫县豆瓣生产专用米曲霉菌株,研究了7株高产蛋白酶、α-淀粉酶的米曲霉试验菌株(24M-1、21M-2、25M-1、19M-2、BM-2、18M-1、DM1)与沪酿3.042米曲霉在制备甜瓣子中的发酵性能差异,考察了不同菌株发酵的种曲中中性蛋白酶、酸性蛋白酶、α-淀粉酶的酶活力差别;进一步评价了由试验菌株制作的种曲发酵的甜瓣子的氨基酸态氮、总酸、挥发性风味物质、感官评价等发酵特性指标。结果表明,菌株24M-1、DM1发酵甜瓣子的各项发酵特性指标表现突出,甜瓣子的综合品质较佳,明显优于沪酿3.042米曲霉发酵的甜瓣子,可进一步优化菌株培养条件及发酵工艺参数,为其在郫县豆瓣加工中的应用奠定了基础。     

Acidic Characteristics of Fermented and Dried Cocoa Beans from Different Countries of Origin

Thirty-nine fermented and dried cocoa bean samples from 13 countries were evaluated for pH, titratable acidity and concentrations of volatile and nonvolatile acids. The correlation coefficient between pH and log₁₀ titratable acidity was -0.94. Cocoa beans from Brazil and Far Eastern countries were highly acidic while those from Central American and South American countries were low in acidity. Samples from West African countries were intermediate with titratable acidity values from 0.12 to 0.15 meq NaOH/g sample and pH values from 5.20 to 5.49. Highly acidic beans were characterized by high concentrations of acetic and lactic acids. The high correlation between acetic acid and both pH (r=0.86) and titratable acidity (r = 0.91) indicated that this acid could be primarily responsible for high acidity in cocoa beans.     

The microbiology of Ghanaian cocoa fermentations analysed using culture-dependent and culture-independent methods

Export of cocoa beans is of great economic importance in Ghana and several other tropical countries. Raw cocoa has an astringent unpleasant taste and a spontaneous fermentation is the first step in a process leading to cocoa beans with the characteristic cocoa flavour and taste. The microbiology of Ghanaian cocoa fermentations was investigated using culture-dependent and culture-independent methods. Samples were collected at 12 hour intervals during 96-144 hour tray and traditional heap fermentations. Yeast, Lactic Acid Bacteria (LAB), Acetic Acid Bacteria (AAB) and Bacillus spp. were enumerated on suitable substrates and identified using phenotypic and molecular methods. The yeast and bacterial micro-populations involved in the cocoa fermentation were further investigated using the culture-independent method Denaturing Gradient Gel Electrophopresis (DGGE). A microbiological succession was observed during the fermentations. At the onset of fermentation yeasts were the dominating microorganisms. Lactic Acid Bacteria became dominant after 12-24 h of fermentation and remained predominant throughout the fermentations with AAB reaching high counts in the mid phase of fermentation. Bacillus spp. were only detected during heap fermentations where they reached high numbers during the later stages of fermentation. Hanseniaspora guilliermondii was the predominant yeast during the initial phase and Pichia membranifaciens during the later phases of fermentation. A number of other yeast species including three putatively undescribed species were isolated during the fermentations. Lactobacillus fermentum was the dominant LAB in most samples. Several other LAB including Lactobacillus plantarum, Leuconostoc pseudomesenteroides, Leuconostoc pseudoficulneum, Pediocococcus acidilactici and a putatively undescribed LAB species were detected during the fermentations. Acetobacter syzygii, Acetobacter pasteurianus and Acetobacter tropicalis were the predominant AAB in all investigated fermentations. During the later stages of heap fermentation Bacillus licheniformis and occasionally other Bacillus spp. were detected in high numbers. In general the culture-based findings were confirmed using DGGE. However, DGGE indicated that Lc. pseudoficulneum plays a more important role during the fermentation of cocoa than expected from the culture-based findings as it yielded a strong band in most DGGE fingerprints. Cluster analysis of the DGGE fingerprints revealed that the DGGE fingerprints clustered according to fermentation site. Within each fermentation site the profiles clustered according to fermentation time. The DGGE method seems to offer a relatively fast and reliable tool for studying yeast and bacterial dynamics during cocoa fermentations.