Fermented Cocoa Beans Research Articles

Application of yeasts isolated from fermented cocoa beans for biocontrol of pathogenic mold in chocolate fruit

Contamination by pathogenic mold in postharvest cocoa beans becomes a significant concern by most Indonesian farmers. Pathogenic mold can cause damage to cocoa beans by such as rotting diseases in fruit. One alternative that can be used to control pathogenic mold is using biological agents such as yeasts. Some group of yeasts can produce cellulase enzyme that can degrade cellulose, and it can possibly break the cell wall with of mold which composed of semi-crystalline chitin, p-need, and cellulose. This study aims to determine the yeast originated from fermented cocoa beans which can produce cellulase enzymes and their potential ability as a biocontrol for pathogenic molds in chocolate fruit. This study includes yeast isolation from fermented beans, screening of yeast isolates that produce cellulase enzymes, and in-vitro antagonistic testing against pathogenic molds on chocolate fruit. The results showed that there were 21 yeast isolates from fermented cocoa beans, and among all, there were five isolates which can produce cellulase enzymes, namely isolate C4.-3.3, C4.-3.13, C4.-4.9, C4.-4.10, and C4.-5.9. Yeast isolate C4.-4.10 can produce cellulase enzymes with an index of 0.32 U/mL. This research showed that the 5 yeast isolates have the low category of cellulase enzyme, and further study is needed to be done to confirm their ability to act as a biocontrol agent.

Cellulase-producing yeast isolated from fermented cocoa beans as biocontrol for pathogenic mold chocolate fruit collected from Sentul, Jawa Barat, Indonesia

Mold is one of the microorganisms that can cause damage in chocolate fruit by producing phytopathogenic toxins. Until now, pesticides have been widely used for controlling postharvest loss in fruit. Yeasts can produce secondary metabolites, which can inhibit the growth of pathogenic mold. In this study, we isolate, identify, and apply yeasts isolated from fermented cocoa beans to control the growth of pathogenic mold in chocolate fruit collected from Sentul, Indonesia. This research includes yeast isolation using the dilution method with YMA medium, mold isolation using direct planting method on PDA medium, screening ability to produce cellulase using diffusion method on CMC medium, and in-vitro antagonist testing using dual culture method on PDA medium. The results showed that 128 yeast isolates and 37 mold isolates were obtained in this study. The results of screening representative cellulase capability of 77 yeast obtained 6 positive yeast isolates produced cellulase with the highest cellulolytic index of 0.23-0.30. The 6-yeast antagonism test with the highest cellulolytic index showed that C3.3.1 isolate had the best ability to inhibit pathogenic molds with 37.36% inhibitory power.

The potential of dry fermented cocoa (Theobroma cacao L.) variety Lindak bean shell treated at different degrees of roasting as a functional food

Cocoa bean shell is a waste from the chocolate processing industry that has not been used optimally and still contains 5.78% polyphenols which has potential as a source of natural antioxidant compounds. The chocolate is processed in the industry with light, medium and high roasting, based on the desired product. The purpose of this study was to determine the polyphenol content and antioxidant activity of the dry fermented cocoa shells as a result of the best degree of roasting. The research method used was an experimental method using a Randomized Block Design (RBD) with 4 treatments roasting dried fermented cocoa beans of Lindak varieties by using different degrees of roasting (Without roasting; low roasting (110°C for 35 minutes), medium roasting (140°C for 30 minutes), and high roasting (190°C for 15 minutes)) and repeated 3 times. The results showed the dry fermented cocoa shells of Lindak varieties by high roasting treatment has the best phenolic total (5.24%) and antioxidant activity (37.92 ppm).

Towards a Starter Culture for Cocoa Fermentation by the Selection of Acetic Acid Bacteria

Acetic acid bacteria are involved in many food and beverage fermentation processes. They play an important role in cocoa bean fermentation through their acetic acid production. They initiate the development of some of the flavor precursors that are necessary for the organoleptic quality of cocoa, and for the beans’ color. The development of starter cultures with local strains would enable the preservation of the microbial biodiversity of each country in cocoa-producing areas, and would also control the fermentation. This approach could avoid the standardization of cocoa bean fermentation in the producing countries. One hundred and thirty acetic acid bacteria were isolated from three different cocoa-producing countries, and were identified based on their 16S rRNA gene sequence. The predominate strains were grown in a cocoa pulp simulation medium (CPSM-AAB) in order to compare their physiological traits regarding their specific growth rate, ethanol and lactic acid consumption, acetic acid production, and relative preferences of carbon sources. Finally, the intraspecific diversity of the strains was then assessed through the analysis of their genomic polymorphism by (GTG)5-PCR fingerprinting. Our results showed that Acetobacter pasteurianus was the most recovered species in all of the origins, with 86 isolates out of 130 cultures. A great similarity was observed between the strains according to their physiological characterization and genomic polymorphisms. However, the multi-parametric clustering results in the different groups highlighted some differences in their basic metabolism, such as their efficiency in converting carbon substrates to acetate, and their relative affinity to lactic acid and ethanol. The A. pasteurianus strains showed different behaviors regarding their ability to oxidize ethanol and lactic acid into acetic acid, and in their relative preference for each substrate. The impact of these behaviors on the cocoa quality should be investigated, and should be considered as a criterion for the selection of acetic acid bacteria starters.

Sensory evaluation of chocolate bar production materials of dry cocoa seeds in various fermentation treatments

The processing of chocolate bars is influenced by cocoa beans used. Chocolate bar is one of the downstream products with a simple processing process. The taste and aroma in candy comes from chocolate bars. Fermentation is an important activity in the formation of cocoa flavour. This study aims to determine the panellists’ acceptance of chocolate bars produced from various fermentation treatments of sun-dried cocoa beans. The materials used in this study were cocoa beans of varieties of Lindak. The fermentation variation consisted of two treatments, namely, the addition of the inoculum gradually (A1) and the addition of the inoculum simultaneously at the beginning of fermentation (A2). The inocula added were Sacharomyces cerevisiae (FNCC 3056), Lactococcus lactis (FNC 0086) and Acetobacter aceti (FNCC 0016). The control treatment was dry cocoa beans without fermentation (A0). The fermentation time was 120 h, then the fermented cocoa beans were processed into chocolate bars. The resulting chocolate bars were subjected to sensory analysis including evaluation of the taste of sepia, bitter, and sour taste and in the test the polyphenol content of chocolate bars was used. The results showed that there was a decrease in the septic, bitter and sour taste from treatment A0, A2 and A1, respectively. The highest polyphenol content was in A0, A2, and A1, respectively. Panellists stated that the preferred chocolate bar was made from cocoa beans with A1 treatment.

Influence of S. cerevisiae and P. kluyveri as starters on chocolate flavour.

Fermented cocoa beans (Theobroma cacao L.) are a pivotal raw material for chocolate production. A cocktail yeast applied in the cocoa fermentation process can promote the formation of pleasant metabolites. Saccharomyces, Pichia and Hanseniaspora have been widely used in fermentation to improve the final product organoleptic profile, highlighting that fermentation is a critical point for chocolate flavour precursor production. This study aims to evaluate the impact of Pichia kluyveri and Saccharomyces cerevisiae strains as starter cultures on the fermentation for two cocoa hybrids, FA13 and CEPEC2002. During fermentation processes, volatile organic compounds (VOCs) and protein profiles were assessed. Chocolates produced were also assessed regarding the presence of VOCs. Eighty VOCs were identified using gas chromatography coupled to mass spectrometry analysis. Mass spectrometry provided the protein profile evolution during fermentation and showed that the profiles changed with inoculation type (spontaneous versus inoculated fermentation). Chocolate obtained from FA13 inoculated with S. cerevisiae strain contained a greater amount of organics acids, being categorised as sourer than chocolate produced by spontaneous fermentation of FA13. CEPEC2002 inoculated with S. cerevisiae strain in co-culture with P. kluyveri strain generated less sour and sweeter chocolate than spontaneous fermentation only. Chocolates from inoculated assays with starter cultures were more accepted by evaluators, highlighting that P. kluyveri and S. cerevisiae influence the composition of VOCs. Besides, protein profiles also changed throughout fermentation. Further investigation should be conducted to clarify protein degradation dynamics during inoculated fermentations to define which of the microbial cultures positively affect the chocolate sensory characteristics. © 2021 Society of Chemical Industry.

Physical characteristics of chocolate made from cocoa bean fermented at different duration: a preliminary study

Dark chocolate is one of the most popular chocolates due to its health benefit offered. One of the factors influencing its health effect is the fermentation process prior to the chocolate making. Fermentation change the cocoa bean chemical compound and affected the flavor and antioxidant content. Aside from that, changes in the inner structure of cocoa nib may also occur, resulting in different characteristics of cocoa solids. The aim of this study was to do a preliminary study on the impact of fermentation level (unfermented, half-fermented, and fermented cocoa bean) on the quality attributes of dark chocolate, namely color, glossiness, melting point and particle size. The results showed that fermentation duration influenced the characteristics of chocolate, to some extent. The color of chocolate made from fermented bean exhibited higher L*, C*, h° values than chocolate made from half-fermented and unfermented cocoa bean, consecutively. On the other hand, melting point of chocolate made from unfermented cocoa bean had higher propensity than that of chocolate made from half-fermented and fermented bean. Glossiness and particle size of the chocolates were comparable.

Effects of Processing Time and Temperature on Flavanol and Procyanidin, Proanthocyanidin and Antioxidant Activity of Cocoa Bean in Taiwan

Flavanol in cocoa is beneficial to cardiovascular health. This study investigated the effect of roasting conditions, temperature ranging from 110 to 150°C and time duration 15-35 min, on the level of flavanol and procyanidin (FP), total proanthocyanidin (PAC) and antioxidant activity of fermented cocoa beans in Southern Taiwan. The content of FP in unroasted and roasted cocoa bean was determined by the normal phase HPLC method and the antioxidant activity was assayed by DPPH and ABTS methods. High roasting temperature for long time decreased the content of FP and PAC in cocoa beans, where ranged from 55.30±1.95 mg (-)-epicatechin equivalents /g and 103.56±8.33mg procyanidin B1/g (at 110°C for 15 min) to 19.12±0.66 mg (-)-epicatechin equivalents /g and 51.58±0.86 mg procyanidin B1/g (at 150°C for 35 min), respectively. Meanwhile, as the roasting temperature rose, the DPPH and ABTS free radical scavenging ability of roasted cocoa bean extracts decreased gradually, varying from the maximum (IC50 = 104±2 µg/ml and 50.4±1.1mg TE/g) for unroasted bean to the minimum (IC50 = 144±4 µg/ml and 27.7±2.4 mg TE/g), and then slightly increased to IC50=134±4 µg/ml and 31.5±0.7 mg TE/g, respectively at most severe roasting treatment, showing the formation of melanoidins and polyphenols caused the synergistic effect, leading to the rise of the antioxidant activity. The first order kinetic reaction and the Arrhenius equation were applied to forecast the change of FP concentration during roasting. The activation energy of FP degradation is 11.2 kcal/mol. The obtained kinetic equation could be useful to predict the remaining amount of FP in cocoa beans under the designed experiment. The cocoa products with higher flavanol content and antioxidant activity could be possible by setting appropriate roasting conditions.

Effect of cocoa bean origin and conching time on the physicochemical and microstructural properties of Indonesian dark chocolate

Abstract Indonesian cocoa is cheaper and considered second grade compared with most other cocoa. However, the domestic chocolate industry is not well-developed due to significantly low consumption. To cope with these issues, product innovation through technical process improvement is required to stimulate the domestic chocolate industry. This study aimed to investigate the effect of cocoa bean origin and conching time on the physicochemical (water content, texture, color, crude fat content, and melting enthalpy) and microstructural properties of chocolate. The experiment was conducted under a completely randomized factorial design consisting of two factors: cocoa bean origin (100% fermented cocoa beans from Jember, 100% fermented cocoa beans from Southeast Sulawesi, 50% fermented cocoa beans from Jember + 50% non-fermented cocoa beans from Southeast Sulawesi, and 50% fermented cocoa beans from Southeast Sulawesi + 50% non-fermented cocoa beans from Southeast Sulawesi) and conching time (4, 6 and 8 h). The results showed that cocoa bean origin significantly affected the hardness, gumminess and color of chocolate, including the redness and yellowness level, whereas conching time affected water content, hardness, cohesiveness, elasticity, and crude fat content. Differential scanning calorimetry (DSC) analysis showed that the treatment with 100% fermented cocoa beans from Jember presented higher values of Tonset, ΔHmelt and area compared with those of the treatment with 100% fermented cocoa beans from Southeast Sulawesi, which presented higher values of Tpeak and Tend. Scanning Electron Microscopy (SEM) analysis showed a distribution of solid and dense particles with crystal interaction with chocolate structures.

Use of the <sup>13</sup>C/<sup>12</sup>C, <sup>15</sup>N/<sup>14</sup>N and <sup>18</sup>O/<sup>16</sup>O Isotopic Ratios of Theobromine and Caffeine in the Characterization of Geographic Origin

The aim of this work is to characterize the geographical origin of cocoa beans and coffee beans. This study aims to contribute to the traceability of raw materials in order to fight against falsification. For this purpose, we based our work on the measurement of isotope ratios in 13C, 15N and 18O. The multi-element isotope ratios have been evaluated as a means to distinguish fermented cocoa beans of different geographic and varietal origins. The isotopic ratios of 13C, 15N and 18O were measured in theobromine obtained from samples of fermented cocoa beans. Twenty-two (22) samples of different geographical origins covering the four mainland cocoa producing areas were analyzed on the one hand and on the other hand, 16 caffeine samples from various origins were also analyzed. The treatment of the values resulting from these isotopic analyzes by statistical methods, namely the principal component analysis (PCA) makes it possible to visualize the discriminations between the different origins. The most discriminating variables identified as responsible for the geographic and varietal differences were the δ15N, δ13C and δ18O values of cocoa beans and certain extracts and tissues. We have shown that the isotope ratios are correlated with the altitude and precipitation conditions encountered in the different cocoa growing regions.

Isolation and identification of microbial species found in cocoa fermentation as microbial starter culture candidates for cocoa bean fermentation in Colombia.

Microbial activity involved in the cocoa beans fermentation process is essential to maintain and improve the organoleptic and nutritional qualities of chocolate; therefore, the aim of this investigation was to search and select microbial isolates with the potential to improve the quality of cocoa beans. Fermentation experimentswere conducted on farms located in Maceo (Antioquia), San Vicente de Chucurí (Santander), and Rivera and Algeciras (Huila), Colombia. Yeast, lactic acid bacteria (LAB), acetic acid bacteria (AAB), and mesophilic aerobic microorganisms were obtained from different fermentation batches. The growth of these microorganismswas tested in six treatments as follows: 50% cocoa pulp agar (CPA), high concentrations of glucose (10%), ethanol (5%), and acetic acid (7%), an acidic pH of 3.0, and a high temperature of 50oC for 24 h. The isolates with the highest growth were identified by 18S and 16S rRNA gene analysis, revealing a high diversity ofspecies associated with cocoa fermentation, including eight species of yeasts (Debaryomyces hansenii, Meyerozyma guillermondii, Wickerhanomyces anomalus, Pichia guillermondii, Pichia kudriavzevii, Trichosporon asahii, Candida parapsilosis, and Pichia manshurica), six species of LAB (Pediococcus acidilactici, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus farraginis, Lactobacillus rhamnosus, and Leuconostoc mesenteroides), four species of AAB (Gluconobacter japonicus, Acetobacter tropicalis, Acetobacter pasteurianus, and Acetobacter malorum/tropicalis), and three species of Bacillus spp. (Bacillusaryabhattai /megaterium, Bacillus subtilis, and Bacillus coagulans). In general, microbial populations increased in cocoa batches after 12 h of fermentation and decreased after 84-96 h. All the yeast isolates grew in 10% glucose and CPA, 85.7% in 5% ethanol, and 95% at a pH of 3.0. All the yeast isolates were affectedby 7% acetic acid and incubation at 50oC for 24 h. Eighty-five percent of the LAB grew in 10% glucose, 100% in 5% ethanol, 42.8% in CPA, 64% at a pH of 3.0, and 35.7% grew after being exposed to 50oC for 24 h; all were affected by 7% acetic acid. As for the AAB, 100% grew in 10% glucose, 71% in 7% ethanol, 100% grew in CPA, in 7% acetic acid, and at a pH of 3.0, while 100% were affected by incubation at 50oC. Three yeast isolates, W. anomalus, D. hansenii and M. guillermondii, three LAB isolates, P. acidilactici, L. brevis, and L. plantarum, and three AAB isolates, A. tropicalis, A. pasteurianus and G. japonicus, were selected as promising strains to be used in a microbial starter culture for cocoa bean fermentation to improve the organoleptic quality of cocoa.

Exploring the Capability of Yeasts Isolated from Colombian Fermented Cocoa Beans to Form and Degrade Biogenic Amines in a Lab-Scale Model System for Cocoa Fermentation

Yeast starters for cocoa fermentation are usually tested according to their enzymatic activities in terms of mucilage degradation and flavor improvement, disregarding their influence on the production or elimination of toxic compounds as biogenic amines (BAs), important for human health. In this work, we tested 145 strains belonging to 12 different yeast species and isolated from the Colombian fermented cocoa beans (CB) for their capability of producing BAs in vitro. Sixty-five strains were able to decarboxylate at least one of the amino acids tested. Pichia kudriavzevii ECA33 (Pk) and Saccharomyces cerevisiae 4 (Sc) were selected to evaluate their potential to modulate BAs, organic acids, and volatile organic compounds (VOCs) accumulation during a simulated cocoa fermentation. The growth of Sc or Pk in the presence of CB caused a significant reduction (p < 0.05) of 2-phenylethylamine (84% and 37%) and cadaverine (58% and 51%), and a significant increase of tryptamine and putrescine with a strong influence of temperature in BA formation and degradation. In addition, our findings pointed out that Pk induced a major production of fatty acid- and amino acid-derived VOCs, while Sc induced more VOCs derived from fatty acids metabolism. Our results suggest the importance of considering BA production in the choice of yeast starters for cocoa fermentation.

Classification of fermented cocoa beans (cut test) using computer vision

Fermentation of cocoa beans is a critical step for chocolate manufacturing, since fermentation influences the development of flavour, affecting components such as free amino acids, peptides and sugars. The degree of fermentation is determined by visual inspection of changes in the internal colour and texture of beans, through the cut-test. Although considered standard for evaluation of fermentation in cocoa beans, this method is time consuming and relies on specialized personnel. Therefore, this study aims to classify fermented cocoa beans using computer vision as a fast and accurate method. Imaging and image analysis provides hand-crafted features computed from the beans, that were used as predictors in random decision forests to classify the samples. A total of 1800 beans were classified into four grades of fermentation. Concerning all image features, 0.93 of accuracy was obtained for validation of unbalanced dataset, with precision of 0.85, recall of 0.81. Although the unbalanced dataset represents actual variation of fermentation, the method was tested for a balanced dataset, to investigate the influence of a smaller number of samples per class, obtaining 0.92, 0.92 and 0.90 for accuracy, precision and recall, respectively. The technique can evolve into an industrial application with a proper integration framework, substituting the traditional method to classify fermented cocoa beans.

Co-culturing fructophilic lactic acid bacteria and yeast enhanced sugar metabolism and aroma formation during cocoa beans fermentation

Glucose and fructose are the main fermentable sugars in cocoa pulp. During fermentation, glucose is consumed within 48–72 h and fructose only after 120 h, mainly associated with the preferential use of glucose by microorganisms. In the first stage of this study, the complete genome sequence of a lactic acid bacterium with high fructose consumption capacity (Lactobacillus plantarum LPBF35) was reported. The notable genomic features of L. plantarum LPBF35 were the presence of alcohol/acetaldehyde dehydrogenase gene and improved PTS system, confirming its classification as a “facultatively” fructophilic bacterium. Subsequently, this bacterium was introduced into cocoa fermentation process in single and mixed cultures with Pediococcus acidilactici LPBF66 or Pichia fermentans YC5.2. Community composition by Illumina-based amplicon sequencing and viable counts indicated suppression of wild microflora in all treatments. At the beginning of the fermentation processes, cocoa pulp consisted of approximately 73.09 mg/g glucose and 73.64 mg/g fructose. The L. plantarum LPBF35 + P. fermentans YC5.2 process showed the lowest levels of residual sugars after 72 h of fermentation (7.89 and 4.23 mg/g, for fructose and glucose, respectively), followed by L. plantarum LPBF35 + Ped. acidilactici LPBF66 (8.85 and 6.42 mg/g, for fructose and glucose, respectively), single L. plantarum LPBF35 treatment (4.15 and 10.15 mg/g, for fructose and glucose, respectively), and spontaneous process (22.25 and 14.60 mg/g, for fructose and glucose, respectively). The positive interaction between L. plantarum LPBF35 and P. fermentans YC5.2 resulted in an improved formation of primary (ethanol, lactic acid, and acetic acid) and secondary (2-methyl-1-butanol, isoamyl acetate, and ethyl acetate) metabolites during fermentation. The primary metabolites accumulated significantly in cocoa beans fermented by P. fermentans YC5.2 + L. plantarum LPBF35, causing important reactions of color development and key flavor molecules formation. The results of this study suggest that fructophilic lactic acid bacteria and yeast is a microbial consortium that could improve sugar metabolism and aroma formation during cocoa beans fermentation.

Packaging and storage of cocoa beans fermented with Lactobacillus plantarum HL-15 in Agricultural Technology Park Nglanggeran, Yogyakarta

In Gunungkidul, there is Agricultural Techno Park (ATP) Nglanggeran which processes fermented cocoa beans from local farmers for making chocolate products. This research aimed to obtain a suitable packaging method for fermented cocoa beans during storage in ATP Nglanggeran. This research was carried out using a completely randomized factorial design. The first factor was the addition of starter (B), which consisted of B1 (with Lactobacillus plantarum HL 15) and B2 (without Lactobacillus plantarum HL 15). The second factor was the packaging methods (K), which consisted of K1 (vacuumed PP plastic 0.8 mm in thickness), K2 (non-vacuumed PP plastic 0.8 mm in thickness), K3 (plastic container), K4 (vacuumed PP plastic 0.8 mm in thickness stored within the plastic container), and K5 (nylon sack). The third factor was the storage time (P), which were P0 (0 month), P1 (1 month), and P2 (2 months). The result showed that the samples with the addition of the starter had a lower number of fungal colonies than those without the addition. Besides, the packaging using vacuumed PP plastic 0.8 mm in thickness stored within a plastic container gave the best result on minimizing the total fungal growth contamination and the peroxide value.

Pod storage time and spontaneous fermentation treatments and their impact on the generation of cocoa flavour precursor compounds

SummaryMost of volatile flavour compounds occurred during the cocoa postharvest operations including pod storage and fermentation whom effects on these metabolites were aimed by the present study. Three different spontaneous fermentation treatments were performed on the farm, and the main isolated yeasts were identified using PCR‐DGGE technique. Fermented cocoa beans were solar‐dried, and their flavour compounds were identified using solid phase microextraction technique–gas chromatography–mass spectrometry. The main results showed that Pichia kudriavzevii was common yeast involved in the cocoa fermentation. Thirty flavour precursor compounds grouped into six chemical families including alcohols and pyrazines were identified in raw cocoa samples. ACP showed that short durations of pod storage and fermentation promote the formation of flavour precursors of cocoa. No spontaneous cocoa fermentation treatment influenced the occurrence of aroma compounds. The generation of flavour compounds depends only on the pod storage and the fermentative microorganisms of cocoa.

Oxygen Uptake Rate and Carbon dioxide Evolution Rate on the Fermentation Process of Cocoa Beans (Theobroma Cacao L.)

Fermentation is one of the most critical stages in the postharvest handling process of cocoa beans. Temperature changes during the fermentation process characterize the success of the cocoa bean fermentation process. In general, aeration conditions during the fermentation process effects changes in fermentation air temperature. This study aims to determine cumulative oxygen uptake and cumulative carbon dioxide evolution during cocoa bean fermentation. Fermenter equipped with a controlled system of air temperatures and aeration rates was used to determine changes in oxygen uptake rate and carbon dioxide evolution rate during the fermentation process. The variables observed were including: length of pod storage time before fermentation (X₁; days), aeration rate (Xâ‚‚; l.min-1), and fermenter temperature (X₃; ℃). Response surface methodology was used to determine the relationship between cumulative oxygen uptake as well as cumulative carbon dioxide evolution and the observed variables (X₁, Xâ‚‚, X₃) The results shows that cumulative oxygen uptake and cumulative carbon dioxide evolution obtained in various treatments during the fermentation process are consecutive: 6.66-34.29 g Oâ‚‚ kgvs-¹ and 12.15-50.97 g COâ‚‚ kgvs-¹. Based on the variance analysis of response surface methodology, R² value of the cumulative oxygen uptake is about 88.1%, while R² value of the cumulative carbon dioxide evolution value is about 87.3 %. The highest amount of oxygen consumption and carbon dioxide production was obtained at X₁ = 5 days, Xâ‚‚ = 0.25- 0.4 l.min-1, and X₃ ≤ 50℃.

Development of a model for the alcoholic fermentation of cocoa beans by a Saccharomyces cerevisiae strain

The aromatic quality of chocolate requires the use of cocoa with high aromatic potential, this being acquired during the fermentation of cocoa beans. Traditional fermentation is still often carried out on a small scale with wild strains of yeasts and acetic bacteria and under poorly controlled conditions leading to cocoa quality ranging from best to worst. This study is the first part of a project aiming to control quality of cocoa to produce high aromatic quality chocolate by using a mixed starter of selected strains of yeast and acetic bacteria and by controlling the conditions of fermentation. To achieve this objective, a mathematical model of the alcoholic fermentation of cocoa beans has been developed. The growth, glucose consumption and ethanol production of Saccharomyces cerevisiae LM strain in synthetic broth were modeled for the most important intrinsic (pH, glucose, ethanol, free nitrogen and oxygen levels) and extrinsic (temperature, oxygen level) fermentation parameters. The model was developed by combining the effects of individual conditions in a multiplicative way using the gamma concept. The model was validated in liquid synthetic medium at two different inoculation levels 104 and 106 CFU/mL with an increase in temperature that recorded during spontaneous fermentations. The model clearly shows that the level of inoculation and the speed of the increase in temperature clearly drive yeast growth, while other factors including pH and ethanol, free nitrogen and oxygen levels have no significant impact on yeast growth.

Influence of two anti-fungal Lactobacillus fermentum-Saccharomyces cerevisiae co-cultures on cocoa bean fermentation and final bean quality.

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.

Profile of bioactive compounds, antioxidant and aromatic component from several clones of cocoa beans during fermentation

Commonly cocoa beans containing various chemical components, nutrients, and bioactive compound. The main bioactive compounds in cocoa beans consisting flavonoids and methylxantines has an positive influence on the health of the human body. Besides the fermented cocoa beans also produce a distinctive of cocoa aromatic. One of the main factors influencing the formation of aroma, chemical content and bioactive compounds of cocoa beans is the fermentation process. The purpose of research is to determine the effect of fermentation duration of cocoa clones with different varieties on the profile of bioactive compounds, antioxidant activity and aromatic components (volatiles) that occur in cocoa beans during fermentation. The treatment was arranged in a Randomized Block Design of two factors, the first factor is cocoa clones (Sulawesi 2/S2 clones and local clones) and the second factor is fermentation time (0, 2, 4 and 6 days). The observed parameters were polyphenol content, antioxidant activity, flavonoid bioactive compounds (catechin and epicatechin) and methylxanthine (theobromin and caffeine), as well as aromatic components (volatile) of fermented cocoa beans. The results of the study was showed that cocoa beans from local clones providing polyphenols, antioxidant activity and bioactive compounds (catechin, epicatechin and theobromine) are higher than S2 clones. While the high caffeine content and distinctive aroma of the pyrazine compound were produced from S2 cocoa beans on 6 days fermentation. The average content of polyphenols, antioxidants and bioactive compounds in non-fermented cocoa beans is higher than fermented beans, but the resulting scent is not strong. The results of the research are expected to obtain information about the profile of bioactive compounds, antioxidants and aromatic components of cocoa beans especially derived from Pinrang district has the potential improving community health.