From Cocoa to Chocolate: Effect of Processing on Flavanols and Methylxanthines and Their Mechanisms of Action.

Despite the health benefits associated with the ingestion of the bioactive compounds in cocoa, the high concentrations of polyphenols and methylxanthines in the raw cocoa beans negatively influence the taste, confer the astringency and bitterness, and affect the stability and digestibility of the cocoa products. It is, therefore, necessary to process cocoa beans to develop the characteristic color, taste, and flavor, and reduce the astringency and bitterness, which are desirable in cocoa products. Processing, however, affects the composition and quantities of the bioactive compounds, resulting in the modification of the health-promoting properties of cocoa beans and chocolate. In this advanced review, we sought to better understand the effect of cocoa's transformational process into chocolate on polyphenols and methylxanthine and the mechanism of action of the original flavanols and methylxanthines. More data on the cocoa processing effect on cocoa bioactives are still needed for better understanding the effect of each processing step on the final polyphenolic and methylxanthine composition of chocolate and other cocoa products. Regarding the mechanisms of action, theobromine acts through the modulation of the fatty acid metabolism, mitochondrial function, and energy metabolism pathways, while flavanols mainly act though the protein kinases and antioxidant pathways. Both flavanols and theobromine seem to be involved in the nitric oxide and neurotrophin regulation.

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.

Exploring the contribution of fructophilic lactic acid bacteria to cocoa beans fermentation: Isolation, selection and evaluation.

Fructophilic lactic acid bacteria (FLAB) are a recently discovered group whose main characteristic is to prefer D-fructose over D-glucose. In this study, laboratory cocoa beans fermentation was analyzed by Illumina-based amplicon sequencing, indicating the presence of potential FLAB of the genera Fructobacillus and Lactobacillus. Eighty efficient fructose-fermenting isolates, obtained from fermenting cocoa pulp beans mass, were identified by 16S rRNA gene sequencing as Pediococcus acidilactici (n = 52), Lactobacillus plantarum (n = 10), Pediococcus pentosaceus (n = 10), Bacillus subtilis (n = 4), and Leuconostoc pseudomesenteroides (n = 4). The growth characteristics of all the 10 L. plantarum strains classified them as "facultatively" fructophilic bacteria, i.e., they grew on glucose without an external electron acceptor but the growth on fructose was faster. Among them, L. plantarum LPBF 35 was characterized by producing a range of aroma-impacting compounds (acetaldehyde, ethyl acetate, nonanal, and octanoic acid), being introduced into a cocoa fermentation process. Although the process started with approximately equal amounts of glucose and fructose, a concomitant, but faster utilization of fructose, was observed in cocoa fermentation conducted with L. plantarum LPBF 35 (with no residual fructose observed) when compared to control fermentation using a glucophilic strain (8.77 mg/g residual fructose) and a spontaneous process (8.38 mg/g residual fructose). L. plantarum LPBF 35 also showed an ideal profile of organic acid metabolism (citric acid consumption and lactic acid production) associated with cocoa fermentation. These results proved new insights on cocoa microbial activity and brings new perspectives on the use of lactic acid bacteria as starter culture.

Ion Mobility Spectrometry as a Potential Tool for Flavor Control in Chocolate Manufacture.

Chocolate has a complex flavor profile composed of more than 600 volatile compounds that mainly arise from the thermo-mechanical treatment during roasting and conching. The aim of this study was to evaluate the applicability of ion mobility spectrometry (IMS), as a real-time method for process monitoring in chocolate manufacture. It is evident from the ion mobility (IM) fingerprint spectra that individual processing steps affect the signal intensities at particular drift time regions. The analysis of individual IM spectra by principal component analysis (PCA) revealed that it is possible to distinguish with respect to conching temperature and time. PCA also allowed identifying those parts of the IM spectra that were predominantly affected by the respective treatment. It was, on the basis of the IM flavor fingerprints and subsequent PCA, possible to distinguish between the different states of processing of bulk cocoa. The results of the study imply that, using appropriate post-data treatment, IMS could be used for process control in cocoa processing.

The fate of Bacillus cereus and Geobacillus stearothermophilus during alkalization of cocoa as affected by alkali concentration and use of pre-roasted nibs.

Alkalization is a step of cocoa processing and consists of the use of alkali and high temperature to improve the sensorial and technological qualities of cocoa. Intense food processing can select spores, which can compromise safety and quality of the final product. Thus, the aim of this study was to evaluate the fate of B. cereus and G. stearothermophilus spores during the alkalization of pre-roasted (Pr) nibs (held at 120 °C) and unroasted (Ur) nibs (held at 90 °C) using potassium carbonate (0, 2, 4 and 6% w/w). In all conditions, log-linear inactivation kinetics with a tail was observed. The inactivation rate (kmax) for B. cereus varied from 0.065 to 1.67 min-1, whereas the kmax for G. stearothermophilus varied from 0.012 to 0.063 min-1. For both microorganisms, the lowest kmax values were observed during Ur nibs alkalization. The carbonate concentration increase promoted kmax values reduction. The highest tail values were observed for G. stearothermophilus in Ur nibs alkalization, reaching 3.04 log spores/g. Tail formation and low kmax values indicated that cocoa alkalization does not cause significant reductions on bacterial spore population. Therefore, the microbiological control should be primarily ensured by the raw material quality and by avoiding recontamination in the cocoa chain.

Cocoa Bean Proteins-Characterization, Changes and Modifications due to Ripening and Post-Harvest Processing.

The protein fractions of cocoa have been implicated influencing both the bioactive potential and sensory properties of cocoa and cocoa products. The objective of the present review is to show the impact of different stages of cultivation and processing with regard to the changes induced in the protein fractions. Special focus has been laid on the major seed storage proteins throughout the different stages of processing. The study starts with classical introduction of the extraction and the characterization methods used, while addressing classification approaches of cocoa proteins evolved during the timeline. The changes in protein composition during ripening and maturation of cocoa seeds, together with the possible modifications during the post-harvest processing (fermentation, drying, and roasting), have been documented. Finally, the bioactive potential arising directly or indirectly from cocoa proteins has been elucidated. The "state of the art" suggests that exploration of other potentially bioactive components in cocoa needs to be undertaken, while considering the complexity of reaction products occurring during the roasting phase of the post-harvest processing. Finally, the utilization of partially processed cocoa beans (e.g., fermented, conciliatory thermal treatment) can be recommended, providing a large reservoir of bioactive potentials arising from the protein components that could be instrumented in functionalizing foods.

The content of polyphenolic compounds in cocoa beans (Theobroma cacao L.), depending on variety, growing region, and processing operations: a review.

Polyphenols form the largest group of compounds among natural antioxidants, which largely affect the overall antioxidant and anti-free radical activity of cocoa beans. The qualitative and quantitative composition of individual fractions of polyphenolic compounds, even within one species, is very diverse and depends on many factors, mainly on the area of cocoa trees cultivation, bean maturity, climatic conditions during growth, and the harvest season and storage time after harvest. Thermal processing of cocoa beans and cocoa derivative products at relatively high temperatures may in addition to favorable physicochemical, microbiological, and organoleptic changes result in a decrease of polyphenols concentration. Technological processing of cocoa beans negatively affects the content of polyphenolic compounds.