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Dendrogram derived from a similarity matrix obtained from scoring DGGE fragment profiles for fragments' absence or presence from the population obtained from natural fermentation Nat or from inoculated with Kluyveromyces marxianus Km. The 21 kDa trypsin-inhibitor albumin protein was detected even at h in both treatments, while the main storage protein globulins vicilin disappeared after 72 h of fermentation. Degradation of the seed proteins occurred first for the treatment with K.
On the other hand, no major difference in protein profiles could be observed between treatments after the beans were dried Fig. Evaluation of protein degradation during fermentation and drying of cacao seeds under natural conditions a or when inoculated with Kluyveromyces marxianus b by total seed protein profiles evaluated by SDS-PAGE.
Lanes represent standard Std molecular weight; samples collected at 0, 24, 48, 72, 96 and h during fermentation and after drying. As a reference, a class of proteins with a molecular weight similar to one vicilin 47 kDa and albumin 21 kDa was used. The acidity of the fermented and dried cacao beans obtained from the conventional fermentation was In terms of color development, K.
Results from the bean-cut test from natural or inoculated fermentation, based on cotyledon color brown, brownish-purple and purple or cotyledon morphology fermented, partially fermented, less fermented. Sensorial analysis of the chocolate produced from both fermentation treatments revealed statistical differences in terms of the flavor attributes and global acceptability, with better results for the seeds fermented with K.
For aroma, there was no statistical significance Table 3. Sensorial analyses of the chocolates produced from cacao beans from natural or inoculated fermentation. A complete and efficient fermentation of cacao seeds is required to fully develop chocolate flavor, and proper aeration of the fermentation mass is a critical factor for a successful process. During fermentation, gravity causes mass flow drainage of seed-pulp liquids resulting from physical or biological rupture of cells from the pulp parenchymatous tissue, naturally increasing aeration.
Conventionally, aeration is promoted by the periodical turning of the fermenting seed mass, but it can be increased by partial removal of seed pulp before fermentation.source site
Removal of seed pulp mechanically or by treatment using pectinolytic enzymes can make fermentation more efficient Wood et al. The estimated pectinolytic activity of the MMIII hybrid described here was superior to yeast strains obtained from fermenting fruits da Silva et al. The effect of the introduction of this hybrid K. During the first hours of fermentation, liquids were drained from fermentation masses from both treatments at similar rates due to the effect of gravity at 1 h; Table 1.
The effect of yeast introduction on liquid drainage was noticed after 24 h, with an increase of one-third of drained volume in comparison with the traditional fermentation 1. The increase in drained volume might be associated with the pectinolytic activity from the introduced yeast, causing more degradation of cell walls from the pulp parenchymatous tissue Buamah et al. Introduction of the hybrid K. Beans fermented with the introduction of K. The relationship between increased drainage of liquids during fermentation and quality improvement by increased aeration corroborated previous results for small-scale fermentation experiments Sanchez et al.
The yeasts K. In our control conventional fermentations, these two species were detected based on DGGE profiles during the first 48 h Fig. According to the amplified fragment profiles from a single culture of C. Introduction of K. The anaerobic phase of the fermentation was represented by the major group established from profiles derived from samples from the beginning of the fermenta; Robnett et al.
However, a difference in the timing of degradation was noticeable between treatments Fig.
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Vicilins were first degraded in the fermentation inoculated with K. The 21 kDa trypsin inhibitor was strongly degraded only after 96 h for the conventional fermentation Fig. An increase in aeration and the subsequent incassie brilliant blue. In terms of quality attributes, inoculation with K. Another positive effect of K.
Previously, chocolate produced from beans derived from conventional fermentation was more acceptable by a taster panel when compared with those from artificial inoculation using a cocktail of microorganisms Schwan et al. Introduction of Saccharomyces in fermentation has produced good-quality cacao beans Samah et al. The beneficial effects of drainage caused by the yeast pectinolytic activity Sanchez et al.
The positive results indicated the robustness of the process and the endurance of the yeast, demonstrating a potential for large-scale application in controlled fermentation. Typically, beans produced in Brazil and Malaysia present incomplete fermentation, with low pH below the recommended range of 5—5. Introduction of a K. The potential of this yeast hybrid strain, which attacks the spongy parenchymatous cells of the seed pulp more efficiently, increasing liquid drainage and the consequent aeration of the seed mass for the first 24 h, was demonstrated.
This work also showed that changes in aeration at the beginning of the fermentation seemed to be more relevant to improve bean quality than the local microorganism population, and that the introduction of a yeast species that favors aeration might be sufficient to produce beans with superior quality in comparison with natural fermentation, without the need to inoculate with a complex mixture of microorganisms.
The authors wish to thank CNPq for their fellowships. We also wish to thank anonymous reviewers whose comments greatly improved the manuscript. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search. Article Navigation. Close mobile search navigation Article Navigation. Volume 8. Article Contents. Materials and methods. Fermentation of cacao Theobroma cacao L. Oxford Academic.
Google Scholar. Luiz Humberto Gomes. Priscilla Efraim. Flavio Cesar de Almeida Tavares. Antonio Figueira. Editor: Teun Boekhout. Cite Citation. Permissions Icon Permissions. Abstract Fermentation of Theobroma cacao cacao seeds is an absolute requirement for the full development of chocolate flavor precursors. Open in new tab Download slide. All values in the table did not differ significantly. Open in new tab. Search ADS. Google Preview. Vacuolar storage protein of cocoa seeds and their degradation during germination and fermentation.
Proceedings of the International Cocoa Conference: Challenges in the 90s. Parameters affecting the quality of processed cocoa powder: acidity fraction. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Pure yeast culture fermentation of cocoa Theobroma cacao L : effect on yield of sweatings and cocoa bean quality. Da Silva. The similarity of cocoa flavour of fermented seeds from fingerprinted genotypes of Theobroma cacao L.
The cocoa-pulp agroindustry and the uses of its residues in Bahia: Progress achieved in the last ten years. Occurrence and diversity of yeasts involved in fermentation of West African cocoa beans. Influence of organic acids on flavour perception of Malaysian and Ghanian cocoa beans. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit 26S ribosomal DNA partial sequences. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Evolution of cacao bean proteins during fermentation: a study by two-dimensional electrophoresis.
Designs to balance the effect of order of presentation and first-order carry-over effects in hall tests. Yeast involved in fermentation of Coffea arabica in East Africa determined by genotyping and by direct denaturating gradient gel electrophoresis. A photometric adaptation of the Somogyi method for determination of glucose. Yeast populations associated with Ghanaian cocoa fermentations analysed using denaturing gradient gel electrophoresis DGGE.
The microbiology of Ghanaian cocoa fermentations analysed using culture-dependent and culture-independent methods. Isolation and characterization of microorganisms involved in the fermentation of Trinidad's cacao beans. Increasing cocoa butter yield through genetic improvement of Theobroma cacao L: seed fat content variability, inheritance, and association with seed yield. The precursors of chocolate aroma: the distribution of free amino acids in different commercial varieties of cocoa beans.
Biochemical changes during fermentation of cocoa beans inoculated with Saccharomyces cerevisiae wild strain. A study of the yeast flora and the effect of pure culture seeding during the fermentation process of cocoa beans.
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SAS Institute Inc. Microbial fermentation of cocoa beans, with emphasis on enzymatic degradation of the pulp. Endopolygalacturonase secretion by Kluyveromyces marxianus and other cocoa pulp-degrading yeasts. An enrichment method for auxotrophic yeast mutants using the antibiotic Nystatin. Cloning and sequencing of the cDNA-encoding the major albumin of Theobroma cacao — identification of the protein as a member of the Kunitz protease inhibitor family.
Cloning and sequencing of a cDNA encoding the major storage proteins of Theobroma cacao. Cocoa-specific aroma precursors are generated by proteolytic digestion of the vicilinlike globulin of cocoa seeds. In vitro studies on the proteolytic formation of the characteristic aroma precursors of fermented cocoa seeds — the significance of endoprotease specificity. Published by Blackwell Publishing Ltd. Bean death occurs within two days allowing enzymes and substrates to interact freely inside the nibs.
The initial pH within the nibs is about 6. The pH of Malaysian beans is often still below 5 after 5 days fermentation causing the objectionable high acidity while prolonging the fermentation beyond 5 or 6 days contributes to a high level of off-flavours in the cocoa bean. Since the cocoa beans grown in most of Peninsular Malay-sia contain a higher amount of pulp than other cocoa beans, e.
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The pressed beans have a rough exterior with a significant amount of pulp remaining which is unevenly distributed. Pod storage involves storing harvested pods for periods from 5 to 11 days after which the pods are broken open and although the pulp to nib ratio is reduced substantially, this reduction is inconsistent, resulting in a cocoa with only occasionally improved flavour.
In addition, pod storage in the field would leave the pods susceptible to attack from disease and pests such as rats, as well as to theft whereas pod storage in a warehouse is space and time consuming as well as expensive and not feasible on an industrial scale. Sun spreading involves placing the cocoa beans, fresh or from stored pods, in thin layers cm thick on trays or on a concrete Eloor in the sun for several hours to evaporate additional moisture. We have now developed a method of pretreating fresh co-coa beans to reduce the amount of pulp, which enables the subsequent fermentation to proceed in highly aerobic conditions, favouring acetic acid bacteria over lactic acid bacterial activity, causing the pH to remain above 5 during the fermentation and after which the cocoa beans have low acidity, are substantially free from off-flavours and show an enhanced cocoa flavour.
The freshly harvested cocoa beans are conventionally transported to the fermentary late afternoon and are weighed prior to treatment or overnight storage.
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By a depulper we mean a fruit or vegetable depulper which may be operated mechanically or by other means e. An example of a mechanical depulper which may be used in this invention is a paddle finisher comprising a perforated cylindrical screen inside which is a paddle or scraper system adapted to rotate, a means for feeding the fresh cocoa beans at an inlet end and a means for removing the depulped cocoa beans at the other end of the cylindrical screen. The cylindrical screen is usually static and may be positioned with its longitudinal axis horizontal.
AlternatiYely, this effect could be achieved by the cylindrical screen sloping upwards from the inlet end. Conveniently there are two paddles in the cylindrical screen.
The perforations in the cylindrical screen may have a diameter from 1 mm to 12 mm and preferably from 2 mm to 7 mm. Advantageously the machine parts are made of stainless steel. Preferably, the fresh beans may be fed into the inlet end of the depulper by mechanical means. Advantageously, the cocoa beans may be passed through the depulper more than once, preferably together with water.
If desired, the use of a depulper may be preceded by a pressing technique e. When the depulped beans are passed through the depulper a further time together with water, the amount of water may be up to 0. The predying step may be carried out in any conventional hot air dryer, such as the circular drier available at most S estate fermentaries , or any alternative drying system e. The cocoa beans treated in accordance with the present invention may be fermented by conventional methods. If the beans are too dry an appropriate amount of water may be added to the beans for the fermentation to proceed suitably.
Advantageously, the cocoa beans are placed into a fer-mentation box to give an initial shallow bed depth of from 13 to 30 cm, preferably from 18 to 27 cm and are later transferred e. During fermentation, the cocoa beans treated in accordance with the present in-vention form a porous mass allowing the fermentation to be highly aerobic.
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A shallow bed depth of between 10 cm and 20 cm is espe-cially desirable when the depulped beans have not been subjected to a predrying treatment. In such an instance the cocoa beans will have been pretreated by either a combination of hydraulic pressing followed by depulping with addition of water or a multiple pass through the paddle finisher with addition of water. The fermented beans are afterwards dried.
One advantage of the present invention is that the drying requires much less energy as the cocoa beans are already much cleaner and drier than after the usual fermentation. Preferably the drying time is kept similar to the stan-dard conditions and the hot air temperature is decreased from 70C - 80C to 55C - 70C. The present invention also provides a method of treating fresh cocoa beans before fermentation which comprises a quick-press stage with a hydraulic press to reduce the pulp content and afterwards predrying at a temperature from ambient to 70C to remove further pulp moisture.
By a quick-press stage we mean pressing the beans in press boxes for a short period of time usually from about S to 60 minutes, more usually from 15 to 30 mi-nutes until substantially no more juices are pressed out. After the quick-press stage the cocoa beans are prefera-bly predried at a temperature from 45C to 60C for a period of from 30 to minutes, more usually from 50 to minutes depending on the amount of beans.
Ater predrying the cocoa beans are fermented in highly aerobic conditions over a period of from 3 to 5 days. The initial bed depth is preferably from 13 to 30 cm and the cocoa beans are later transferred e. The processes of this invention may be applied to cocoa beans grown in any parts of the world, especially Malay-sian cocoa beans such as those grown in Peninsular Malaysia.
The present invention also provides a method of prepa-ring chocolate and cocoa products comprising roasting the dried cocoa beans produced as hereinbefore described in accordance with the invention, cracking, winnowing to remove shells and produce the nibs which are ground to give cocoa liquor which may be used to prepare chocolate, or may be pressed to extract cocoa butter and the resi-dual cake pulverised, cooled and sifted to give cocoa powder.
The chocolate and cocoa products utilising the fresh cocoa beans treated as hereinbefore described in accor-dance with the invention are of high quality. The beans were fermented in containers with a bed depth of 30 cms for 3 days with mixing after 1 day and 2 days only. The pH of the beans remained above 5 throughout the fermentation and the beans were dried after 3 days in a hot air dryer where 8 trays of 0. A mild air flow was introduced between the trays and the tempera-ture setting was 55C on the first day and 60 C on the following days. The drying was complete after 3 days.
Chocolate prepared from these beans was found to have low acidity, low off-flavour and enhanced cocoa flavour. Example 2 Fresh cocoa beans were passed through a paddle finisher as in Example 1 and the depulped beans were then passed through a second time together with water in a ratio of 0.
The beans were pressed by means of a Bucher hydraulic press, the piston pressure of bars being transferred through the plastic cover where it corresponds to a pressure of 12 bars on the beans. The pressing operation took 5 minutes. After shuffling the beans, a second pressing was carried out under the same conditions to reduce the pulp content more evenly throughout the mass.
The pressed beans were then treated as in Example 2 by passing through a paddle finisher with water to remove further pulp to give an overall weight loss of Chocolate prepared from these dried beans has low acidi-ty, low off-flavour and enhanced cocoa flavour. Example 4 Fresh cocoa beans were loaded at the plantation into stainless steel pressboxes at approximately kg in each box. The fully loaded pressboxes were transported to the fermentary in the late afternoon and weighed prior to being left overnight. The next morning the con-tents of two pressboxes of cocoa beans were passed through the paddle finisher with addition of 60 kg water evenly distributed.
The depulped beans were weighed and had lost The air flow forced through the S cm layer of cocoa beans was set at between 50CC. Afterwards, the same beans were transferred to the normal size fermentation boxes 1. Chocolate prepared from these dried beans had low aci-dity, low off-flavour and enhanced cocoa flavour.
Example 5 Fresh cocoa beans were harvested, delivered and weighed as in Example 4. On the next morning they were subjected to a quick-press by the hydraulic press. The quick-pres-sed beans were weighed and a The quick-pressed beans were then placed into the circular drier for predrying as in ExampIe 4. After predrying they were again weighed and an overall weight loss of The cocoa beans then underwent a fermentation process and a drying process as described in Example 4.
Example 6 Fresh cocoa beans were harvested, delivered and weighed as in Example 4. The contents of these pressboxes were passed through the paddle finisher the next morning, with the addition of water as in Example 4. The depulped beans were weighed and a The depulped beans were thinly and evenly spread in the sun for minutes. When weighed after this predrying, the cumulative weight loss was The treated beans were fermented and dried as described in Example 4.
Chocolate prepared from these dried heans had low acidi-ty, low off-flavour and enhanced cocoa flavour. A method according to claim 1 wherein the beans are passed through the depulper more than once. A method according to claim 1 wherein the depulped beans are passed through the depulper together with wa-ter. A method according to claim 1 wherein the depulped beans are passed through the depulper more than once together with water. A method according to claim 1 wherein the depulper is a paddle finisher.