Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation

The potential impact of aromatic and pectinolytic yeasts on cocoa flavour was investigated using two defined mixed starter cultures encompassing strains of Pichia kluyveri and Kluyveromyces marxianus for inoculating cocoa beans in small scale tray fermentations. Samples for microbial and metabolite analysis were collected at 12–24 hour intervals during 120 h of fermentation. Yeast isolates were grouped by (GTG)₅-based rep-PCR fingerprinting and identified by sequencing of the D1/D2 region of the 26S rRNA gene and the actin gene. Pulsed Field Gel Electrophoresis (PFGE) was conducted on isolates belonging to the species P. kluyveri and K. marxianus to verify strain level identity with the inoculated strains. Furthermore, Denaturing Gradient Gel Electrophoresis (DGGE) was performed to follow yeast and bacterial dynamics over time including the presence of the bacterial inoculum consisting of Lactobacillus fermentum and Acetobacter pasteurianus. Yeast cell counts peaked after 12 h of fermentation with the predominant species being identified as Hanseniaspora opuntiae and Hanseniaspora thailandica. P. kluyveri and K. marxianus were found to compose 9.3% and 13.5% of the yeast population, respectively, after 12 h of fermentation whilst PFGE showed that ~ 88% of all P. kluyveri isolates and 100% of all K. marxianus isolates were identical to the inoculated strains. Despite never being the dominant yeast species at any stage of fermentation, the un-conched chocolates produced from the two inoculated fermentations were judged by sensory analysis to differ in flavour profile compared to the spontaneously fermented control. This could indicate that yeasts have a greater impact on the sensory qualities of cocoa than previously assumed.     

Investigation of chocolate produced from four different Brazilian varieties of cocoa (Theobroma cacao L.) inoculated with Saccharomyces cerevisiae

Properly conducted cocoa fermentation is an important step for the production of high-quality chocolate. The aim of this work was to investigate the effect of four cocoa varieties (CCN51, PS1030, FA13, and CEPEC 2004) inoculated with Saccharomyces cerevisiae CA11 on microbial communities and the profile of volatile compounds and sensory characteristics of chocolate. The S. cerevisiae population increased significantly (p < 0.05) during the fermentations. The microbial communities varied according to cocoa variety fermentation as assessed by denaturing gradient gel electrophoresis (DGGE). The dominant yeasts were S. cerevisiae and Hanseniaspora guilliermondii, while Lactobacillus casei and Gluconobacter oxidans were the predominant bacteria in the four different fermentations analyzed. Sixty-one volatile compounds—including aldehydes (11), ketones (10), esters (14), acids (8), alcohols (8), pyrazines (5), furans (3), and others (caffeine and heptadecane)—were detected and quantified by GC–MS in the different chocolates. The sensory analysis showed that caramel was perceptible in the chocolate of PS1030, while CEPEC2004 was related to astringency, bitterness, and chocolate flavor attributes. The chocolates produced from FA13 and CCN51 were more similar in terms of sour and chocolate aroma. A “temporal dominance of sensation” (TDS) analysis showed that although the bitter attribute was dominant, the fruity, sweet, sour, astringent, and cocoa attributes were also perceptible, depending on the cocoa variety. These results suggest that the cocoa varieties had an influence on the chocolate's quality, which should be considered to obtain chocolate with different sensory characteristics or for better standardization of the process, even when using yeast as a starter culture.     

Fermentation of cocoa beans: influence of microbial activities and polyphenol concentrations on the flavour of chocolate

BACKGROUND: Spontaneous cocoa bean fermentation is characterised by a succession of microbial activities. Cocoa flavour precursors are developed during fermentation and drying of cocoa beans. Polyphenols and alkaloids contribute to astringency and bitterness of cocoa and chocolate. RESULTS: Population dynamics, metabolite target analyses, and chocolate production were performed for seven independent spontaneous cocoa bean heap fermentations in Ghana. Although the same micro‐organisms were involved in these heaps, carried out at different farms or in different seasons, heap temperatures and microbial metabolite concentrations were different. This could be due to heterogeneity and size of the heaps, but was mainly ascribed to microbial variability. Indeed, differences in microbial activity could be linked with the flavour of chocolates made from the corresponding dried, fermented cocoa beans. Whereas the polyphenol and alkaloid contents of cocoa beans were crop‐ and heap‐dependent, epicatechin and theobromine levels decreased during fermentation due to diffusion out of the bean cotyledons and polyphenol oxidation and condensation. Residual levels were responsible for the degree of bitterness of the final chocolates. CONCLUSION: Differences in microbial activities between different heap fermentations can result in dried fermented cocoa beans and chocolates with different flavour characteristics. Hence, fermentation control may direct the flavour of chocolate. Copyright © 2008 Society of Chemical Industry     

On-farm implementation of a starter culture for improved cocoa bean fermentation and its influence on the flavour of chocolates produced thereof

Cocoa bean fermentations controlled by means of starter cultures were introduced on several farms in two different cocoa-producing regions (West Africa and Southeast Asia). Two starter culture mixtures were tested, namely one composed of Saccharomyces cerevisiae H5S5K23, Lactobacillus fermentum 222, and Acetobacter pasteurianus 386B (three heaps and one box), and another composed of L. fermentum 222 and A. pasteurianus 386B (seven heaps and one box). In all starter culture-added cocoa bean fermentation processes, the inoculated starter culture species were able to outgrow the natural contamination of the cocoa pulp-bean mass and they prevailed during cocoa bean fermentation. The application of both added starter cultures resulted in fermented dry cocoa beans that gave concomitant milk and dark chocolates with a reliable flavour, independent of cocoa-producing region or fermentation method. The addition of the lactic acid bacterium (LAB)/acetic acid bacterium (AAB) starter culture to the fermenting cocoa pulp-bean mass accelerated the cocoa bean fermentation process regarding citric acid conversion and lactic acid production through carbohydrate fermentation. For the production of a standard bulk chocolate, the addition of a yeast/LAB/AAB starter culture was necessary. This enabled an enhanced and consistent ethanol production by yeasts for a successful starter culture-added cocoa bean fermentation process. This study showed possibilities for the use of starter cultures in cocoa bean fermentation processing to achieve a reliably improved fermentation of cocoa pulp-bean mass that can consistently produce high-quality fermented dry cocoa beans and flavourful chocolates produced thereof.     

Effect of ingestion of dark chocolates with similar lipid composition and different cocoa content on antioxidant and lipid status in healthy humans

The association between in vitro antioxidant capacity of dark chocolates with different cocoa percentage and the in vivo response on antioxidant status was investigated. In a randomized crossover design, 15 healthy volunteer consumed 100 g of high antioxidants dark chocolate (HADC) or dark chocolate (DC). In vitro, HADC displayed a higher Total Antioxidant Capacity (TAC) than DC. In vivo, plasma TAC significantly peaked 2 h after ingestion of both chocolates. TAC levels went back to zero 5 h after DC ingestion whilst levels remained significantly higher for HADC. HADC induced a significantly higher urinary TAC in the 5-12 h interval time than DC. No change was detected in urinary excretion of F2-isoprostanes. Plasma thiols and triacylglycerol (TG) levels significantly increased for both chocolate with a peak at 2 h remaining significantly higher for DC after 5 h respect to HADC. Results provide evidence of a direct association between antioxidant content of chocolate and the extent of in vivo response on plasma antioxidant capacity.     

Fluidized bed roasting of cocoa nibs speeds up processing and favors the formation of pyrazines

Roasting is an important step in cocoa processing causing water loss and generating volatile compounds responsible for chocolate aroma like nitrogen-heterocycles. In this study, the comparison of two techniques, oven roasting, and fluidized bed roasting, in terms of effective water diffusivity (De) and activation energies of formation (Ea) of nitrogen-heterocycles was achieved with cocoa nibs. Fluidized bed roasting, recognized for its energy efficiency and low-footprint synthesis, was 16 times faster than oven roasting. The order of magnitude of De in fluidized-bed-roasted nibs was −8, while it was −9 in the oven-roasted nibs. Moreover, the a_w was 50% higher in fluidized-bed-roasted nibs than in the oven-roasted ones. The Ea of nitrogen-heterocycles ranged roughly between 40 and 80 kJ/mol. Those values were lower under fluidized bed roasting than under oven roasting. The more effortless water mobility within fluidized-bed-roasted cocoa demanded lower Ea, and favored the formation of nitrogen-heterocyclics.Industrial relevanceThis study can inspire cocoa manufacturers and equipment designers to pursue the formation of nitrogen-heterocycles during the roasting process of cocoa. It can be done either by adapting and scaling the current fluidized bed coffee roasters to cocoa beans or nibs; or by exploring other alternatives capable of leading enough water diffusivity and water activity in the cocoa nibs, as reported here. These physicochemical conditions undoubtedly boosted the formation of volatile compounds responsible for chocolate aroma, e.g., the pyrazines, without carrying the formation of typical-burn volatile compounds. This natural way of favoring the generation of pyrazines in cocoa nibs could contribute to clean labels by reducing or avoiding the subsequent use of flavorings. The implementation of efficient heat-transfer techniques during roasting, e.g., fluidized bed roasting, could reduce the processing cost and improve sustainability. Studies in the matter of sensory profile, and energy consumption/conversion are called for future research.     

Sensory profile,acceptability, and their relationship for diabetic/reduced calorie chocolates

The purpose of this study was to determine the sensory properties and acceptability of lab developed prototypes of conventional, diabetic (with no sugar), and diabetic/reduced calorie milk chocolates (no sugar and 25% calorie reduction) with high-intensity sweeteners, sucralose and stevioside, and partial fat replacement with whey protein concentrate (WPC). PLS was performed in order to relate sensory properties and consumer acceptability and to determine drivers of liking and disliking. There was no difference between conventional, diabetic and diabetic/reduced calorie milk chocolates for brightness, cocoa aroma, cocoa butter aroma, and cocoa flavor (p > 0.05). Acceptability was higher for sucrose substitution by sucralose than by stevioside and partial fat replacement reduced acceptability of flavor even more (p ? 0.05). Crucial attributes which determine consumer acceptability in samples are sweet aroma, melting rate, and sweetness, whereas bitterness, bitter aftertaste, adherence, and sandiness were drivers of disliking.     

Characteristics of cocoa butter and chocolates obtained from cocoa varieties grown in Bahia, Brazil

Due to the attack of cacao (Theobroma cacao L.) plants by the fungus Moniliophthora perniciosa which caused witches’ broom disease in the state of Bahia, Brazil, resistant clonal material with elevated productivity and superior physical characteristics has been selected for use in the genetic improvement program and by the cocoa producers themselves. The present study aimed to characterize the cocoa butter and chocolates produced from nine cacao varieties and also from the Amelonado cacao produced in the same region (denominated as Common), with a view to quality improvement. The cocoa butter was characterized for its solid fat content and triacylglycerol composition, and the chocolates for their moisture and protein contents, maximum particle diameter, pH value, acidity, rheological properties and instrumental texture, as well as a sensory test for acceptability with 50 consumers. A relationship was found between the triacylglycerol composition and solid fat curves of the cocoa butters and the instrumental texture of the chocolates, the highest values for the latter parameter being observed for the varieties with greater amounts of symmetrical triacylglycerols and greater solid fat contents at the temperatures evaluated. From a sensory point of view, it was shown that overall acceptance and buying intention were mainly determined by the attributes of “astringency,” “bitterness,” “acidity” and “chocolate flavor.” It was also shown that chocolates produced from varieties descendent from the Trinitario group were differentiated from those descendent from the Forastero group, showing the preference of consumers for these materials and the possibility of a gain in quality in the genetic improvement of cacao by including varieties descended from the Trinitario and Criollo groups in the crossing programs, associating disease resistance with product quality.     

Evolution of aroma volatiles during storage of sourdough breads made by mixed cultures of Kluyveromyces marxianus and Lactobacillus delbrueckii ssp. bulgaricus or Lactobacillus helveticus

Two mixed starter cultures were used for sourdough bread making to evaluate their ability to improve quality and increase bread shelf-life: Lactobacillus delbrueckii ssp. bulgaricus or Lactobacillus helveticus mixed with the lactose fermenting yeast Kluyveromyces marxianus as alternative baker’s yeast. Control sourdough breads (K. marxianus) without the addition of bacteria, were also prepared. The changes on the headspace aroma volatiles during storage were assessed using solid-phase microextraction (SPME) GC–MS analysis. The effect of these changes on bread flavour was evaluated by consumer preference evaluations and the results were co-evaluated with those from the GC–MS analysis. The obtained results showed differences in the volatile composition of the different types of breads examined, as well as dramatic decreases of the number and the amount of volatiles after five days of storage. The sourdough breads made with K. marxianus and L. bulgaricus, had a more complex aroma profile, longer shelf-life and achieved the highest scores in the sensory tests.     

Flavor Chemistry of Cocoa and Cocoa Products—An Overview

Cocoa originates from beans of the cocoa tree (Theobroma cacao L.) and it is an important commodity in the world and the main ingredient in chocolate manufacture. Its value and quality are related to unique and complex flavors. Bulk cocoas (Forastero type) exhibit strong basic cocoa notes, whereas fine varieties (Criollo, Nacional) show aromatic, floral, or smoother flavor characteristics. About 600 various compounds (alcohols, carboxylic acids, aldehydes, ketones, esters, and pyrazines) have been identified as odor‐active components. The specific cocoa aroma arises from complex biochemical and chemical reactions during the postharvest processing of raw beans, and from many influences of the cocoa genotype, chemical make‐up of raw seeds, environmental conditions, farming practices, processing, and manufacturing stages. There has been much research on cocoa flavor components. However, the relationships between all chemical components that are likely to play a role in cocoa flavor, their sensory properties, and the sources and mechanisms of flavor formation are not fully understood. This paper provides an overview on cocoa flavor from a compositional and a sensory perspective. The nonvolatile and volatile chemical components of cocoa and chocolate flavor, and their sensory properties correlated to the main influences involved in flavor formation, are reviewed.     

Effects of fat replacement and fibre addition on the texture,sensory acceptance and structure of sucrose‐free chocolate

Dietary fibre has been employed as a sucrose and fat replacement in chocolates and can influence the physical and sensory characteristics of the resulting product. Formulations of sucrose‐free chocolates were developed with the addition of inulin and β‐glucan concentrate as partial substitutes for cocoa butter using a mixture design. The effects of the combinations of the three ingredients provided for the design on the texture, microstructure and sensory acceptance of the chocolates were investigated. The substitution of cocoa butter for inulin or β‐glucan concentrate decreased the hardness of the chocolates. It was possible to replace 10 g of cocoa butter in a 100‐g control formulation with inulin and still maintain good acceptance, while this same substitution with β‐glucan resulted in less acceptable chocolate, with a mean score of 6.4 on a scale from 0 to 10. Scanning electron microscopy (SEM) was used to investigate the effects of fibre addition by observing the developed microstructure.     

A rapid method to evaluate the chocolate smoothness based on the tribological measurement

Cocoa is the primary ingredient in chocolate, and cocoa concentration attributes to the smoothness of the chocolate. Supporting this statement, we assessed the smoothness and melting properties of five chocolates of different cocoa concentration from three brands using the professional sensor evaluation and weight method, respectively. Artificial saliva was added to obtain the mixed chocolate solutions, and their viscosity and coefficient of friction (CoF) were measured for three rubbing pairs. The correlation of chocolate smoothness with their viscosities and average CoF (aCoF) was also discussed. The results indicated that the cocoa concentration significantly affected the smoothness of the chocolates among the three brands. The mixed solution of 50% chocolate could be a representative of oral processing, but their viscosities were not proficient enough to effectively characterize their smoothness. The aCoF of 50% chocolate solution could be rapid and effective to evaluate their smoothness. In addition, the Two-GCTPE rubbing pair was more suitable way to evaluate the smoothness of similar foods in the oral environment. The study results will provide a better insight into characterizing certain attribution of similar foods.     

Application of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and L. helveticus for sourdough bread making

The application of Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) as starter cultures for sourdough bread making was examined. Production of lactic and acetic acids, bread rising, volatile composition, shelf-life and organoleptic quality of the sourdough breads were evaluated. The amount of starter culture added to the flour, the dough fermentation temperature and the amount of sourdough used were examined in order to optimise the bread making process. The use of mixed cultures led to higher total titratable acidities and lactic acid concentrations compared to traditionally made breads. Highest acidity (3.41 g lactic acid/kg of bread) and highest resistance to mould spoilage were observed when bread was made using 50% sourdough containing 1% K. marxianus and 4% L. delbrueckii ssp. bulgaricus. The use of these cultures also improved the aroma of sourdough breads, as shown by sensory evaluations and as revealed by GC–MS analysis.     

Assessing the shapes and speech sounds that people associate with chocolate samples varying in cocoa content

We report a series of three experiments designed to highlight the reliable crossmodal correspondences that exist between the cocoa content of various commercially-available chocolate products and both visually-presented shapes and nonsense words. The chocolates tested in this study included three kinds of Lindt chocolate and a milk chocolate truffle (‘Koko’ brand from Cadbury). Participants were given paper-based line scales, anchored at either end with either a nonsense word or simple outline shape. They tasted the chocolates and indicated whether their perception of the flavor better matched one or other of the items anchoring the scales by marking the appropriate point along the scale. The results demonstrate that certain chocolates were more strongly associated with angular shapes and ‘sharp’ inflected, high-pitched meaningless words, such as ‘tuki’ and ‘takete’. Specifically, Lindt extra creamy milk chocolate (30% cocoa) and Cadbury’s Koko milk chocolate truffles were both more strongly associated with rounded shapes and softer sounding, lower-pitched pseudo-words, such as ‘maluma’. By contrast, Lindt 70% and 90% cocoa chocolates were more strongly associated with sharper (angular) shapes and sounds, such as ‘takete’. These results demonstrate that the phenomenon of sound symbolism extends beyond the visual modality into the domain of flavor perception where, in particular, speech sounds carry meaning in terms of the taste/flavor of chocolates. These results have implications for the development of novel brand names for new products (such as, in this case, chocolate) that best connote the product’s likely sensory attributes.     

An investigation on the appropriateness of chocolate to match tea and coffee

The aim of this study was to provide some recommendations for selecting a befitting tea and coffee to match chocolate with different cocoa contents. Three coffee samples (chocolate flavored, vanilla flavored and unflavored coffee), four tea samples (black tea, green tea, vanilla flavored tea and citrus flavored tea) and three chocolates (30%, 70%, and 99% cocoa) were hedonically rated by eighty regular chocolate consumers. The beverages were then paired with each chocolate, and the consumers were asked to indicate the hedonic liking of the resulting pairings, and to indicate whether the chocolate or beverage flavor dominated the pairing flavor. This study showed that liking of chocolate and coffee pairings and chocolate and tea pairings significantly varied (p < 0.001) across samples. Consumers preferred pairings with 30% cocoa and 70% cocoa chocolate to pairings with 99% cocoa chocolate. Overall, coffee is significantly (p < 0.001) preferred to tea as a chocolate partner.Chocolate and beverage pairing liking was biased by the liking of the beverage tasted alone, the liking of chocolate tasted alone, beverage type, chocolate type and the level of flavor match between chocolate and tea or coffee in a given pairing. When chocolate and beverage flavor balanced out in a given pairing, chocolate and coffee/tea pairings were significantly preferred by the consumers. A significant decrease in acceptance was observed when beverage or chocolate flavor dominated the flavor of the pairing, much more so when the chocolate flavor dominated the pairing flavor. However consumers don't enjoy any preferred chocolate with any preferred tea or coffee because some flavors match better than do others. Indeed, consumers formulate their hedonic responses taking into consideration what flavors go well together more than they rely exclusively on their hedonic judgments of the chocolates, the teas, and the coffees tasted alone.     

Cocoa substitute: Evaluation of sensory qualities and flavour stability

In the past few years, the price of cocoa has climbed very rapidly, so there is increasing incentive in food industry to find a real cocoa substitute. A mixture of milled chicory roots and carob pods (1:2, w/w) was used for preparation of real cocoa substitute via Maillard technology. Comparative studies concerning sensory characteristics and headspace volatiles were carried out between the real cocoa and prepared cocoa substitute. The sensory evaluation revealed high quality of the cocoa substitute sample in comparison with the real cocoa sample. The comparative odour profile analysis showed that the cocoa-like and sweetish/caramel attributes scored higher in cocoa sample than in cocoa substitute sample, whereas the roasty/earthy attributes showed an opposite trend. The chocolate attribute exhibited the same quality in both samples. The storage of the cocoa substitute sample for 6 months gave rise to an increase in the chocolate attribute. The other attributes showed opposite behaviour. The headspace volatiles of both real cocoa and cocoa substitute were isolated and subjected to gas chromatography (GC) and gas chromatography–mass spectrometric (GC–MS) analysis. Most of the volatile compounds identified in real cocoa especially the key odorants were represented in the cocoa substitute sample. The two compounds, 2-phenyl-2-butenal and 5-methyl-2-phenyl-2-hex-2-enals, that are important contributors to chocolate aroma, showed noticeable increase after storage of cocoa substitute sample for 6 months.