Dataset of cocoa aspartic protease cleavage sites.
ABSTRACT: The data provide information in support of the research article, "The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors" (Janek et al., 2016) . Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS) and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans.
Project description:Cocoa beans (Theobroma cacao L.) are the principal raw material for chocolate manufacture. Before cocoa beans are ready for the chocolate industry, farm-based fermentation and drying processes are key determinants of bean quality and hence the price. To improve its value, cocoa beans were dried in a modified greenhouse (MGHD), conventional greenhouse (CGHD), and open sun (OSD) dryers. The drying behavior, kinetics, and quality were evaluated. The MGHD was constructed by modifying a conventional greenhouse with a fleece of black polyester material. Evaluation of air properties of the dryers without and with cocoa beans showed that the MGHD had average temperatures of 2 and 8°C above, and relative humidity of 12.28% and 25.48% below the CGHD and OSD, respectively. The drying data were fitted to four thin layer mathematical models. The Page and Overhult models gave favorable ranges of R 2 (.976 to .987), chi-square (3.7 × 10-4 to 9.9 × 10-4), and root mean square (RMSE; 0.0188 to 0.0307) for the three dryers. The cocoa beans dried in the MGHD took a shorter time to reach the expected 5%-8% moisture content and were of grade one quality.
Project description:Chocolate production suffered a vast impact with the emergence of the "witches' broom" disease in cocoa plants. To recover cocoa production, many disease-resistant hybrid plants have been developed. However, some different cocoa hybrids produce cocoa beans that generate chocolate with variable quality. Fermentation of cocoa beans is a microbiological process that can be applied for the production of chocolate flavor precursors, leading to overcoming the problem of variable chocolate quality. The aim of this work was to use a cocktail of microorganisms as a starter culture on the fermentation of the ripe cocoa pods from PH15 cocoa hybrid, and evaluate its influence on the microbial communities present on the fermentative process on the compounds involved during the fermentation, and to perform the chocolate sensorial characterization. According to the results obtained, different volatile compounds were identified in fermented beans and in the chocolate produced. Bitterness was the dominant taste found in non-inoculated chocolate, while chocolate made with inoculated beans showed bitter, sweet, and cocoa tastes. 2,3-Butanediol and 2,3-dimethylpyrazine were considered as volatile compounds making the difference on the flavor of both chocolates. Saccharomyces cerevisiae UFLA CCMA 0200, Lactobacillus plantarum CCMA 0238, and Acetobacter pasteurianus CCMA 0241 are proposed as starter cultures for cocoa fermentation.
Project description:This survey reports for the first time the changed of quality of fermented cocoa (Theobroma cacao L.) beans. The quality evaluation and simultaneous detection of amino acids, flavor, procyanidin, color, fat, protein, antioxidant activity, and enthalpy were obtained for different fermentation stages of cocoa beans. The results showed that total essential amino acids contents ranged from 2.64 g/100 g to 3.68 g/100 g. A total of 88 compounds identified at the end of the fermentation belonged to alcohols, acids, esters, ketones, pyrazines, aldehydes, and terpenoids. One of the chemical groups that were present in highest abundance in the consummation treatments was acids, representing 56.04% of the total extracted area, followed by alcohols (22.95%) and ketones (9.40%). The colors of the beans in different fermentation stages were different, from deep purple to deep red-brown. Fermented cocoa beans were shown to be 53.45% and 13.51% bean butter and protein content, respectively. The value of denaturation enthalpy (?H) ranged from 30.4 (J/g) to 43.38 (J/g). The 3-day fermented sample had the highest ?H (43.38 J/g). When the fermentation process was complete, the procyanidin concentration of the beans decreased, with the final yield of procyanidin at 6.2%. During fermentation, the antioxidant capacity of beans gradually reduced. The fermenting of cocoa beans had a significant effect on the quality formation. The findings of this study constitute a basis for further investigations on the quality formation of cocoa during fermentation.
Project description:Four Escherichia coli isolates were obtained from a batch of cocoa beans imported from Bolivia. The cocoa beans were rejected by a U.S. chocolate manufacturer due to poor microbiological quality. The four isolates were whole-genome sequenced and the sequences analyzed to identify genotypes, serotypes, and virulence and antimicrobial resistance genes.
Project description:Yeasts play an important role in the cocoa fermentation process. Although the most relevant function is the degradation of sugars and the production of ethanol, there is little understanding of the enzyme activities and attributes that allow them to survive even after drying. The present study explored the functional biodiversity of yeasts associated with Criollo Colombian cocoa fermented beans, able to survive after drying. Twelve species belonging to 10 genera of osmo-, acid-, thermo-, and desiccation-tolerant yeasts were isolated and identified from fermented and dry cocoa beans, with Pichia kudriavzevii and Saccharomyces cerevisiae standing out as the most frequent. For the first time, we reported the presence of Zygosaccharomyces bisporus in cocoa fermented beans. It was found that resistance to desiccation is related to the different degradation capacities of fermentation substrates, which suggests that associative relationships may exist between the different yeast species and their degradation products. Besides, the increased thermotolerance of some species was related to the presence of polyphenols in the medium, which might play a fundamental role in shaping the microbial community composition.
Project description:The growth of filamentous fungi during the spontaneous cocoa bean fermentation leads to inferior cocoa bean quality and poses a health risk for consumers due to the potential accumulation of mycotoxins. We recently developed anti-fungal cultures with the capacity to inhibit the growth of mycotoxigenic filamentous fungi on cocoa beans. However, it is not clear how these anti-fungal cultures affect the fermentation process and cocoa bean quality. For that, the anti-fungal co-cultures, Lactobacillus fermentum M017-Saccharomyces cerevisiae H290 (A) and Lb. fermentum 223-S. cerevisiae H290 (B), were applied to 180-kg box fermentations in Honduras in three time-independent replications each including a spontaneous control fermentation. The comparison of inoculated and spontaneous fermentation processes revealed that the co-cultures only marginally affected the fermentation process and cocoa bean quality. Microorganisms reached maximal levels of 6.2-7.6 log CFU/g of yeasts and acetic acid bacteria and 7.9-9.5 log CFU/g of lactic acid bacteria during all fermentations and led to maximal metabolite concentrations in bean cotyledons of 4-12 mg/g ethanol, 2-6 mg/g lactic acid and 6-14 mg/g acetic acid. The fermentation and drying processes resulted in 38-90 mg epicatechin equivalents/g in the cotyledons of dried beans. However, the co-cultures led to up to ten times higher mannitol levels in cotyledons of inoculated beans compared to beans during spontaneous fermentation, and caused a slower fermentation process, detectable as up to 8-12 °C lower temperatures in the centre of the fermenting pulp-bean mass and up to 22% lower proportions of well-fermented beans after drying. Co-culture B-with Lb. fermentum 223 -led to improved cocoa bean quality compared to co-culture A-with Lb. fermentum M017 -, i.e. cocoa beans with 0.5-1.9 mg/g less acetic acid, 4-17% higher shares of well-fermented beans and, on a scale from 0 to 10, to 0.2-0.6 units lower astringency, up to 1.1 units lower off-flavours, and 0.2-0.9 units higher cocoa notes. Therefore, the anti-fungal co-culture B is recommended for future applications and its capacity to limit fungal growth and mycotoxin production during industrial-scale cocoa bean fermentation should be investigated in further studies.
Project description:Classifying cocoa beans, considering some specific standards, is required and regulated around the world, if commercialization is intended, or it can be very useful, in order to select the best ones for a given proposal. This article presents an image dataset of cut test classified cocoa beans, provided by experts following the Brazilian regulative norm for that process. Fourteen classes, with 100 images for each, were registered in a device designed with standard constant artificial lighting and capturing settings and will be present in this paper. Images are hosted in the Núcleo de Biologia Computacional from Universidade Estadual de Santa Cruz, under the "tedais" name.
Project description:The aspartic protease pepsin is less specific than other endoproteinases. Because aspartic proteases like pepsin are active at low pH, they are utilized in hydrogen deuterium exchange mass spectrometry (HDX MS) experiments for digestion under hydrogen exchange quench conditions. We investigated the reproducibility, both qualitatively and quantitatively, of online and offline pepsin digestion to understand the compliment of reproducible pepsin fragments that can be expected during a typical pepsin digestion. The collection of reproducible peptides was identified from >30 replicate digestions of the same protein and it was found that the number of reproducible peptides produced during pepsin digestion becomes constant above 5-6 replicate digestions. We also investigated a new aspartic protease from the stomach of the rice field eel (Monopterus albus Zuiew) and compared digestion efficiency and specificity to porcine pepsin and aspergillopepsin. Unique cleavage specificity was found for rice field eel pepsin at arginine, asparagine, and glycine. Different peptides produced by the various proteases can enhance protein sequence coverage and improve the spatial resolution of HDX MS data. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.
Project description:BACKGROUND:Cocoa butter (CB) extracted from cocoa beans (Theobroma cacao) is the main raw material for chocolate production, but CB supply is insufficient due to the increased chocolate demand and limited CB production. CB is mainly composed of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0-C18:1-C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol (POS, C16:0-C18:1-C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0-C18:1-C18:0). In general, Saccharomyces cerevisiae produces TAGs as storage lipids, which consist of C16 and C18 fatty acids. However, cocoa butter-like lipids (CBL, which are composed of POP, POS and SOS) are not among the major TAG forms in yeast. TAG biosynthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT), and it is essential to modulate the yeast TAG biosynthetic pathway for higher CBL production. RESULTS:We cloned seven GPAT genes and three LPAT genes from cocoa cDNA, in order to screen for CBL biosynthetic gene candidates. By expressing these cloned cocoa genes and two synthesized cocoa DGAT genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some of the strains. In the best producer, the potential CBL content was eightfold higher than the control strain, suggesting the cocoa genes expressed in this strain were functional and might be responsible for CBL biosynthesis. Moreover, the potential CBL content increased 134-fold over the control Y29-TcD1 (IMX581 sct1Δ ale1Δ lro1Δ dga1Δ with TcDGAT1 expression) in strain Y29-441 (IMX581 sct1Δ ale1Δ lro1Δ dga1Δ with TcGPAT4, TcLPAT4 and TcDGAT1 expression) further suggesting cocoa GPAT and LPAT genes functioned in yeast. CONCLUSIONS:We demonstrated that cocoa TAG biosynthetic genes functioned in S. cerevisiae and identified cocoa genes that may be involved in CBL production. Moreover, we found that expression of some cocoa CBL biosynthetic genes improved potential CBL production in S. cerevisiae, showing that metabolic engineering of yeast for cocoa butter production can be realized by manipulating the key enzymes GPAT, LPAT and DGAT in the TAG biosynthetic pathway.
Project description:Full analytical data of Ecuadorian cocoa wastes (raw shells) and beans (as benchmark), are herein reported. A detailed characterization of production residues may pave the road to a zero-waste strategy for the cocoa industry. Multiple analytical techniques have been exploited to define the composition of the matrices, among them: elemental analyses, FTIR, Py-GC/MS/FID and UHPLC-ESI-MS/MS. Quali-quantitative data of carbohydrates, lipids, lignin, polyphenols, alkaloids and proteins have been obtained by Py-GC/MS/FID and UHPLC-ESI-MS/MS. Assignations are fully supported by literature references. The FAMEs composition of lipophilic UAE extract is also reported for sake of comparison with cocoa butter. This data collection completes a wider valorization work, "Cocoa bean shell waste valorisation; extraction from lab to pilot-scale cavitational reactors" (Grillo et al., 2018).