Fermented Cocoa Beans Research Articles

Determination of the microbial community in Amazonian cocoa bean fermentation by Illumina-based metagenomic sequencing

High-throughput DNA sequencing (HTS) was used to investigate the microbial community of cocoa bean spontaneous fermentation in the Amazon biome. The characterization was described in three localities from Para state (Amazon biome) and one locality from Bahia state, the top Brazilian state for cocoa production, for comparison of the microbial community structure. The microbial community profile was determined through a metagenomics approach using the Illumina HiSeq platform. Our data showed that the microbial community is composed of a consortium consisting of yeast (i.e., Pichia and Candida), filamentous fungi (i.e., Aspergillus, Penicillium and Fusarium), lactic acid bacteria (i.e., Leuconostoc, Lactococcus, and Lactobacillus), acetic acid bacteria (i.e., Acetobacter and Gluconobacter), enterobacteria and bacilli. Interestingly, Acetobacter pasteurianus was found in higher relative abundance than expected, and three acetic acid bacteria species were identified for the first time in cocoa fermentation (i.e., Komagataeibacter hansenii, Roseomonas sp. and Acidisphaera sp.). Some microorganisms were only detected in Amazonian cocoa and not detected in Bahia cocoa, including six yeast species (i.e., Starmerella bacillaris, Candida temnochilae, Zygosaccharomyces bailii, Candida glabrata, Saccharomycopsis crataegensis and Saccharomycopsis amapae) and seven lactic acid bacteria (i.e., Lactococcus lactis, Fructobacillus pseudoficulneus, Weissella confusa, Pediococcus pentosaceus, Lactobacillus reuteri, Lactobacillus murinus and Lactobacillus pontis). This study provides the first description of the microbiome in cocoa fermentation from the Amazon region, and these data open new perspectives for exploring these microorganisms in biotechnology applications, as well as their use as a starter culture to obtain high-quality cocoa beans.

The Change of Characteristics and Antioxidant Activity of Cocoa Vinegar During Fermentation of Pulp Liquids by Adding <i>Tape</i> Yeast

The pulp liquids as a by-product of cocoa beans fermentation is potential to be used as a raw material for making cocoa vinegar, but unfortunately the content of acetic acid is relatively low and there have been no studies related to antioxidant activities. The objectives of the present study were to examine the effect of addition of tape yeast and fermentation time of pulp liquids to the changes of characteristics and antioxidant activities of cocoa vinegar and to define the best treatments of the addition of tape yeast and fermentation time for making cacao vinegar. The experiment in this study used factorial Group Randomized Design (GRD) with 2 factors. Factor I is the addition of tape yeast consisting of 5 levels, namely: without tape yeast (control), addition of 0.05; 0.10; 0.15 and 0.20% (w/v) and factor II is fermentation time which consists of 6 levels, namely: 5, 10, 15, 20, 25 and 30 days. The characteristics of cacao vinegar observed were as stated in SNI 01-437-1996, including: acetic acid, acidity (pH), total soluble solid (TSS), total sugar, and alcohol; and the antioxidant activity, namely: total phenolic and antioxidant capacity. The results showed that: (1) the changes in the characteristics of cocoa vinegar consisting of acetic acid content, acidity (pH), total soluble solids (TSS), total sugar content, and alcohol content; and the antioxidant activity based on the total phenolic content and antioxidant capacity occurred during fermentation with the addition of tape yeast, (2) the best treatment for making cocoa vinegar is the addition of 0.10% tape yeast and 25 days fermentation time, with its characteristics namely: 4.09±0.01% acetic acid content, acidity (pH) of 3.40±0.00, TSS of 5.05±0.07 (^oBrix), 0.49±0.02% total sugar content, and 0.00% alcohol content; and antioxidant activities, namely: total phenolic content of 78.94±6.54 (mg/100g GAE) and antioxidant capacity of 12.50±0.14 (mg/L GAEAC).

Characteristics of Dried Cocoa Beans (Theobroma cacao L.) Color Using Response Surface Methodology

The research aimed to study the effects of storage time of cacao fruit before fermentation, aeration rate and temperature of fermenter during fermentation on the color value of dry cocoa beans, including the value of L *, a *, b *, chroma, hue, and RGB, and their relationship using response surface methodology.. The results showed that storage time of cacao fruits before fermentation, aeration rate and temperature of the fermenter had an influence on the color values of L *, a *, b *, chroma, hue, and RGB of dried cocoa beans produced. The highest brightness prediction value of chroma color was ± 26 obtained in the treatment of the storage time of 9-day, aeration rate of 0.4 liters/minute, and fermenter temperature of 50 ℃. Meanwhile, the highest hue prediction value was 51.8 obtained in the treatment of storage time of 9 days, aeration rate of 0.3 liters / minute, and fermenter temperature of 50 ℃. Furthermore, the RGB color of dried cocoa beans showed that the color range of R (red) and G (green) of cocoa beans after fermentation was higher when compared to cocoa beans without fermentation, while the value of color B (blue) had a different tendency showing that dried fermented cocoa beans had a lower blue range compared to unfermented cocoa beans.

Comparative study of different cocoa (Theobroma cacao L.) clones in terms of their phytoprostanes and phytofurans contents

Cocoa has been widely discussed as a bioactive food rich in sensory stimulation and health benefits. However, no information has been provided concerning phytoprostanes (PhytoPs) and phytofurans (PhytoFs) in cocoa. These compounds are of interest because they play a role in the regulation of immune function. The present study included 31 cocoa clones. The PhytoPs and PhytoFs were quantified by UHPLC-QqQ-MS/MS. The total PhytoPs and PhytoFs contents ranged from 221.46 to 1589.83 ng g−1 and from 1.18 to 13.13 ng g−1, respectively. The profiles of the PhytoPs and PhytoFs identified in the cocoa beans showed significant differences among the clones analysed. The results indicate that dry fermented cocoa beans are rich in PhytoPs and PhytoFs, which may represent an additional benefit of the consumption of foods derived from cocoa.

Investigation on Potential Starter of Bacillus spp. for Ivorian Cocoa Beans Fermentation Improvement

To investigate some interesting functional properties of Bacillus spp. in cocoa fermentation processing, 702 strains of Bacillus sp. strains were isolated from six main Ivorian cocoa producer regions. Three mains technological and useful properties for good cocoa beans fermentation were monitored. Among the 386 pectinolytic Bacillus spp. strains, 86.79% of them were confirmed for enzymes production in liquid medium. These 335 pectinolytic Bacillus strains displayed variety of pectinolytic activity with 50.45% of them producing both pectinase and polygalacturonase (PG) enzymes. Among these 169 strains, 70% displayed both the ability to acidify the medium and to degrade the citrate. In mimic conditions of pH and temperature variations as for fermentation heap, 11 strains were able to maintain their activity ranged within 2.12 ± 0.02 to 0.41 ± 0.03 µmol/min/mg of proteins for PG and 2.36 ± 0.03 to 0.25 ± 0.03 µmol/min/mg of proteins for pectinase. Two species (T10T2 and T6HS10) identified as Bacillus subtilus and Bacillus thuringiensis, were selected as the best based on pectinase production level. They were able to continuously produce both pectinolytic enzymes in different conditions of organic acids and ethanol contents. The highlighted properties in this study and the performance of these two strains may make them best candidate as starters for cocoa fermentation control.

Forcing fermentation: Profiling proteins, peptides and polyphenols in lab-scale cocoa bean fermentation.

This study encompassed the lab-scale fermentation of cocoa beans in 300-g heaps under controlled laboratory conditions, in order to replicate the microbial dynamics and metabolomic changes that usually occur in large-scale spontaneous fermentations. Growth profiles of yeast and acetic acid bacteria (AAB) with the native assortment of microbes as well as with the use of a starter culture were very similar to those observed in literature. Greater production of acetic acid by AAB not only led to more acidic-tasting liquor but also contributed to bitterness, due to polyphenol preservation. It also brought about a drastic drop in pH leading to greater proteolytic activity. Peptides generated through proteolysis also showed incredible similarity to those reported in literature, in particular, those speculated to be involved in cocoa-specific flavour. A closer look at the naturally occurring peptide repertoires of our fermentation trials, generated by the breakdown of cocoa storage protein, pointed to a potential peptide responsible for cocoa-specific aroma.

THE EFFECT OF FARMER CHARACTERISTICS ON PERCEPTIONS OF THE FERMENTED COCOA BEANS TECHNOLOGY IN BENGKAYANG REGENCY, WEST KALIMANTAN.

Cocoa Commodities, especially cocoa beans, have a great opportunity to be developed due to high market demand, this must be in line with the good quality of cocoa beans owned with the world market demand standards which means that at the farm level the cocoa farming must implement the Good Agricultural Practices (GAP) and Good Handling Practice (GHP). This study aims to determine the effect of the characteristics of cocoa farmers on farmers' perceptions of fermented cocoa beans. The method used in this study is the survey method. The research location is Sui Duri Village, Sui Raya District, Bengkayang Regency. Data analysis using logit regression. The results showed that the five characteristics of the farmers, namely age, education, land area, number of family dependents and farming experience had a positive influence on farmers' perceptions, this indicates that farmers' characteristics are very important on the farmers' decision to adopt cocoa bean fermentation technology.Keywords: Characteristics of farmers, cocoa, perceptions, Good Agricultural Practices (GAP), Good Handling Practices (GHP)

Metataxonomic comparison between internal transcribed spacer and 26S ribosomal large subunit (LSU) rDNA gene

Next-generation sequencing has been used to strengthen knowledge about taxonomic diversity and ecology of fungi within food ecosystems. However, primer amplification and identification bias could edge our understanding into the fungal ecology. The aim of this study is to compare the performance of two primer pairs over two nuclear ribosomal RNA (rRNA) regions of the fungal kingdom, namely the ITS2 and 26S regions. Fermented cocoa beans were employed as biological material and the fungal ecology during fermentation was studied using amplicon-based sequencing tools, making use of a manually curated 26S database constructed in this study, and validated with SILVA's database. To explore potential biases introduced by PCR amplification of fungal communities, a mock community of known composition was prepared and tested. The relative abundances observed for ITS2 suggest that species with longer amplification fragments are underestimated and concurrently species that render shorter amplification fragments are overestimated. However, this correlation between amplicon length and estimation is not valid for all the species analysed. Variability in the amplification lengths contributed to the preferential amplification phenomenon. DNA extracted from twenty fermented cocoa bean samples were used to assess the performance of the two target regions. Overall, the metataxonomic data set recovered similar taxonomic composition and provided consistent results in OTU richness among biological samples. However, 26S region provided higher alpha diversity index and greater fungal rRNA taxonomic depth and robustness results compared with ITS2. Based on the results of this study we suggest the use of the 26S region for targeting fungi. Furthermore, this study showed the efficacy of the manually curated reference database optimized for annotation of mycobiota by using the 26S as a gene target.

Screening of lactic acid bacteria and yeast strains to select adapted anti-fungal co-cultures for cocoa bean fermentation

Contamination with filamentous fungi during cocoa bean fermentation and drying reduces the quality of cocoa beans and poses a health risk for consumers due to the potential accumulation of mycotoxins. The aim of this study was to develop anti-fungal lactic acid bacteria (LAB)-yeast co-cultures by selecting anti-fungal strains best adapted to the cocoa bean fermentation process from 362 LAB and 384 yeast strains isolated from cocoa bean post-harvest processes. The applied multiphasic screening approach included anti-fungal activity tests in vitro and in vivo and assessment of the carbon metabolism and stress tolerance of the anti-fungal strains in a cocoa pulp simulation medium. The anti-fungal strains, Lactobacillus fermentum M017, Lb. fermentum 223, Hanseniaspora opuntiae H17, and Saccharomyces cerevisiae H290, were selected based on their high fungal growth inhibition capacity and their well-adapted metabolism. Up to seven filamentous fungal strains of the genera Aspergillus, Penicillium, and Gibberella were inhibited on average by 63 and 75% of the maximal inhibition zone by M017 and 223, respectively, and by 25 and 31% by the strains H17 and H290, respectively. Both Lb. fermentum strains converted the medium's glucose, fructose, and citric acid into 20.4–23.0 g/l of mannitol, 3.9–6.2 g/l acetic acid, and 8.6–10.3 g/l lactic acid, whereas the two yeast strains metabolized glucose and fructose to produce 7.4–18.4 g/l of ethanol. The Lb. fermentum strains were further characterized as particularly tolerant towards ethanol, acetic acid, and heat stress and both yeast strains tolerated high amounts of ethanol and lactic acid in the medium. Finally, the anti-fungal in vivo assays revealed that the two Lb. fermentum strains completely inhibited growth of the citrinin-producing strain, P. citrinum S005, and the potentially fumonisin-producing strain, G. moniliformis S003, on the surface of cocoa beans. Furthermore, growth of the aflatoxin-producer A. flavus S075 was inhibited after 10-14 days by all four selected anti-fungal strains, i.e. Lb. fermentum M017, Lb. fermentum 223, H. opuntiae H17, and Sacc. cerevisiae H290, at 51–95% when applied as single cultures and at 100% when the strains were combined into four co-cultures, each composed of a Lb. fermentum and one of the two yeast strains. As a conclusion, these four LAB-yeast co-cultures are recommended for future applications to limit the growth of filamentous fungi and the concomitant mycotoxin production during the fermentation of cocoa beans.

Antifungal activity of yeasts and lactic acid bacteria isolated from cocoa bean fermentations

The aim of this study was to select yeast and LAB strains to be used as a mean of biological control against fungal growth during cocoa fermentation process. Their antifungal activity was assessed against six spoilage fungi isolated from both fermented and dried cocoa beans and belonging to Aspergillus and Penicillium genera. An initial screening was carried out by using the overlay method where the plates were examined for fungi inhibition zone around LAB and yeast streaks. Then, the most active strains were studied in inhibition test in 96-well microplates where mould growth was measured by microplate reader at 490 nm. The nature of their antifungal strenght (organic acid and/or proteins) was also evaluated. The most promising candidates as biological agents belonged to the species Lactobacillus fermentum, Lactobacillus plantarum, Saccharomyces cerevisiae and Candida ethanolica and their antifungal strength was attributed mainly to organic acid production (for LAB) and proteinaceous compounds (for yeasts) or to their synergic effect.

Improved Fermentation of Cocoa Beans with Enhanced Aroma Profiles

ABSTRACTThe 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.

A mathematical model of cocoa bean fermentation.

Cocoa bean fermentation relies on the sequential activation of several microbial populations, triggering a temporal pattern of biochemical transformations. Understanding this complex process is of tremendous importance as it is known to form the precursors of the resulting chocolate’s flavour and taste. At the same time, cocoa bean fermentation is one of the least controlled processes in the food industry. Here, a quantitative model of cocoa bean fermentation is constructed based on available microbiological and biochemical knowledge. The model is formulated as a system of coupled ordinary differential equations with two distinct types of state variables: (i) metabolite concentrations of glucose, fructose, ethanol, lactic acid and acetic acid and (ii) population sizes of yeast, lactic acid bacteria and acetic acid bacteria. We demonstrate that the model can quantitatively describe existing fermentation time series and that the estimated parameters, obtained by a Bayesian framework, can be used to extract and interpret differences in environmental conditions. The proposed model is a valuable tool towards a mechanistic understanding of this complex biochemical process, and can serve as a starting point for hypothesis testing of new systemic adjustments. In addition to providing the first quantitative mathematical model of cocoa bean fermentation, the purpose of our investigation is to show how differences in estimated parameter values for two experiments allow us to deduce differences in experimental conditions.

Dynamics and Biodiversity of Bacterial and Yeast Communities during Fermentation of Cocoa Beans.

Forastero hybrid cocoa bean fermentations have been carried out in a box (B) and in a heap (H), with or without the inoculation of Saccharomyces cerevisiae and Torulaspora delbrueckii as starter cultures. The bacteria, yeasts, and microbial metabolites (volatile and nonvolatile organic compounds) were monitored during fermentation to assess the connection between microbiota and the release of metabolites during this process. The presence of starter cultures was detected, by means of culture-dependent analysis, during the first 2 days of both fermentations. However, no statistical difference was observed in any of the physicochemical or microbiological analyses. Plate counts revealed the dominance of yeasts at the beginning of both fermentations, and these were followed by acetic acid bacteria (AAB) and lactic acid bacteria (LAB). Hanseniaspora opuntiae, S. cerevisiae, Pichia pijperi, Acetobacter pasteurianus, and Lactobacillus fermentum were the most abundant operational taxonomic units (OTUs) during both fermentation processes (B and H), although different relative abundances were observed. Only the diversity of the fungal species indicated a higher level of complexity in the B fermentations than in the H fermentations (P < 0.05), as well as a statistically significant difference between the initially inoculated starter cultures (P < 0.01). However, the microbial metabolite analysis indicated different distributions of the volatile and nonvolatile compounds between the two procedures, that is, B and H (P < 0.05), rather than between the inoculated and noninoculated fermentations. The box fermentations showed faster carbohydrate metabolism and greater production of organic acid compounds, which boosted the formation of alcohols and esters, than did the heap fermentations. Overall, the microbial dynamics and associations between the bacteria, yeasts, and metabolites were found to depend on the type of fermentation.IMPORTANCE In spite of the limited effectiveness of the considered inoculated starter strains, this study provides new information on the microbial development of box and heap cocoa fermentations, under inoculated and noninoculated conditions, as we coupled yeast/bacterial amplicon-based sequencing data with microbial metabolite detection. The information so far available suggests that microbial communities have played an important role in the evolution of aroma compounds. Understanding the pathways that microorganisms follow during the formation of aromas could be used to improve the fermentation processes and to enhance chocolate quality.

New Hyperspectral Index for Determining the State of Fermentation in the Non-Destructive Analysis for Organic Cocoa Violet

This article covers a study of hyperspectral analysis in the process of fermentation of violet cocoa beans. The application of hyperspectral image processing techniques in cocoa is scarce. This article presents a study based on the calculation of spectral indices, finding a correlation with the biochemical parameters that indicate a correct evolution of the fermentation. Measurements of biochemical parameters were obtained experimentally: pH, temperature and humidity percentage; which were correlated with the hyperspectral indexes ARI2 (Anthocyanin Reflectance Index 2), RBE (Spectral Band Ratio), RBEAE (List of Spectral Bands and External Anthocyanin and Absorbances. They gave relevant information, the RBEAE index being the one with the highest contribution of Prediction on the evolution of fermentation. This study allows us to develop a strategy for the optimization of process and an estimation of parameters for the analysis of characteristics.

Characterization of Polyphenols from Various Cocoa (Theobroma cacao L.) Clones During Fermentation

Cocoa bean is a rich source of polyphenols, which are the largest group secondary metabolite with natural antioxidant property. Polyphenols from cocoabeans was reported to possess health benefits. Fermentation, one step in cocoa processing is needed to improve the quality of cocoa in which the concentration of cocoa bean polyphenols might decrease significantly through oxidation and exudation. Cocoa polyphenols content among different cocoa clones mightalso vary. The aims of this study were to determine total polyphenols, total flavanoid, epicatechin, and catechin content in several cocoa clones, those wereSulawesi 1, Sulawesi 2, ICCRI 03, and KW 617. Until now, characterization of polyphenols from those clones has not been reported. The effect of five daysfermentation to those parameters was also studied. The results of the study showed that fermentation and type of clones significantly affected total of polyphenols, total of flavanoids, epicatechin, and catechin content of the cocoa, there is also an interaction between fermentation and type of clones. Unfermented of Sulawesi 1 had the highest total polyphenols of 96.94±5.83 mg/g, total flavanoids of 90.92±1.89 mg/g, epicatechin of 52.50±0.46 mg/g, and catechin of 1.99±0.02 mg/g content compared to other clones. Among five days fermented cocoa beans, Sulawesi 2 showed the highest total polyphenols and total flavanoids content, while ICCRI 03 had the highest epicatechin and catechin content than other clones. Thus, in can be concluded that although fermentation is required to improve the flavor quality of cocoa, it significantly reduced the content of bioactive compounds. This effect varied amongdifferent cocoa clones.

Cocoa and chocolate are sources of vitamin D2

Cocoa beans are susceptible to fungal contamination and often contain substantial amounts of ergosterol, the precursor to vitamin D2. We hypothesized that sun-drying the fermented cocoa beans might lead to the conversion of ergosterol to vitamin D2. We quantified vitamin D in cocoa and cocoa-based foods by liquid chromatography-tandem mass spectrometry. Here, we show that cocoa beans from different growing regions contain vitamin D2. Particularly high vitamin D2 content was found in cocoa powder and butter. Among the chocolates, dark chocolate had the highest vitamin D2 content (ranging from 1.90 to 5.48 µg/100 g), white chocolate had the lowest vitamin D2 content (ranging from 0.19 to 1.91 µg/100 g), and chocolate nut spreads had a comparatively low vitamin D2 content, with an average of 0.15 µg/100 g. Thus, cocoa and chocolate are clearly a dietary source of vitamin D, therefore, it is necessary to update food composition databases.

Variation of triacylglycerol profiles in unfermented and dried fermented cocoa beans of different origins

Fermentation and drying are the two crucial processing steps required to produce cocoa beans with desired properties, especially taste and flavor. To understand their impact on the lipid profile of cocoa, the lipid composition of unfermented raw and fermented dried beans from six different origins was investigated using high-performance liquid chromatography-mass spectrometry methods. While the comparison of triacylglycerol profiles across the different origins showed only small variations in individual compound concentrations, the comparison along the fermentation status showed major differences regarding the occurrence of polar lipids. These compounds may serve as biomarkers for the fermentation status of the beans and a simple analytical method suitable for field trials is proposed. Finally, a hypothesis identifying key unsaturated triacylglycerols contributing to the hardness and softness of cocoa butter is presented.

English

Lactic acid bacteria (LAB) strains isolated from six main Ivorian cocoa producer regions were investigated based on their biochemical properties in order to select the best one as potential starter. Three main technological and useful properties for good cocoa beans fermentation were monitored among the 568 isolated LAB strains. Thus, between the 408 cocoa-specific acidifying LAB strains identified, 05.88% (24 isolates) of them were able to maintain this activity in pH and temperature conditions as same as fermentation heap with an acidifying rate ranged within 0.03&plusmn;0.00 to 1.19&plusmn;0.070%. All these 24 cocoa-specific acidifying LAB strains displayed the ability to degrade the citrate while six (25%) of them were able to produce acetoin. These six LAB strains namely T1Gb8, T6C5, T6C12, T7C8, T9C9 and T11C5 were identified as Lactobacillus plantarum. They were the best able to continuously produce lactic acid in the required standards. Firstly under varying condition of temperature (30 &agrave; 45&deg;C) and pH (3 &agrave; 6) with sharp acid (acetic, lactic and citric acids) influence. Secondly beyond 8% of ethanol content. The highlighted properties in this study and the performance of these six strains may make them best candidate as starters for cocoa fermentation control. Key words: Lactic acid bacteria, cocoa-specific acidifying, technological properties, starter, Ivorian cocoa, fermentation. &nbsp

Origin and varietal based proteomic and peptidomic fingerprinting of Theobroma cacao in non-fermented and fermented cocoa beans

It is well known that the development of chocolate flavor is initiated during cocoa bean fermentation. Storage proteins undergo the most intensive breakdown yielding peptides and free amino acids, which both serve as flavor precursors. A comprehensive analysis of cocoa proteins and oligopeptides of non-fermented and fermented beans from various geographic origins allows the assessment of systematic differences with respect to their origin as well as fermentation status. Protein quantities as well as their profiles derived from two-dimensional gel electrophoresis, showed striking differences for non-fermented beans depending on their geographical origin. From fermented beans, oligopeptides were relatively quantified by utilizing UHPLC-ESI-Q-q-TOF and annotated based on their characteristic fragmentation pattern in the positive-ion mode. With >800 unique oligopeptides, excluding di- and tri-peptides, across 25 different samples, we are herein reporting on the largest collection of cocoa oligopeptides ever observed and identified. The detected diversity of peptides could not be correlated to the geographical origin but rather to the degree of fermentation. Our findings suggest that the variability in peptide patterns depends on the fermentation method applied in the country of origin ultimately indicating diversified proteolytic activities. Furthermore, our results showed that well-fermented and fair-fermented beans can be differentiated from partially fermented and under-fermented ones by higher numbers and total amounts of oligopeptides.

Analysis of the cocobiota and metabolites of Moniliophthora perniciosa-resistant Theobroma cacao beans during spontaneous fermentation in southern Brazil.

Cocoa bean fermentation is a spontaneous process involving a succession of microbial activities, yeasts, lactic acid, and acetic acid bacteria. The spontaneous fermentation of cocoa beans by Theobroma cacao TSH565 clonal variety, a highly productive hybrid resistant to Moniliophthora perniciosa and Phytophthora spp., was investigated. The natural cocobiota involved in the spontaneous fermentation of this hybrid in southern Brazil, was investigated by using both a culture-dependent microbiological analysis and a molecular analysis. The changes in the physicochemical characteristics and the kinetics of substrate utilization and metabolite production during fermentation were also evaluated. Yeasts (178) and bacteria (244) isolated during fermentation were identified by partial sequencing of the ITS and 16S rDNAs, respectively. After 144 h of fermentation, the indigenous yeast community was composed of Hanseniaspora spp., Saccharomyces spp., and Pichia spp. The bacterial population comprised Lactococcus spp., Staphylococcus spp., Acetobacter spp. and Lactobacilli strains. The kinetics of substrate transformation reflected the dynamic composition of the cocobiota. Substrates such as glucose, fructose, sucrose, and citric acid, present at the beginning of fermentation, were metabolized to produce ethanol, acetic acid, and lactic acid. The results described here provide new insights into microbial diversity in cocoa bean-pulp mass fermentation and the kinetics of metabolites synthesis, and pave the way for the selection of starter cultures to increase efficiency and consistency to obtain homogeneous and best quality cocoa products. © 2018 Society of Chemical Industry.