1. Introduction
The cocoa tree, also called
Theobroma cacao L., has been known since ancient times. The harvest of cocoa comes from the American continent, although the area of provenance is not accurately known. Various investigations hold that its beginning was generated from the Toltec culture due to a Quetzalcóalt belief that credited the tree to have multiple healing virtues [
1]. From there, the knowledge and use of this tree expanded to other cultures, such as the Olmecas, Aztecs, and Incas [
2]. The productivity of the cocoa tree in convenient conditions starts at three years of age and can give cobs all year, depending on the area of production and altitude. From 5 years of age, the production is considered fulfilled, and its life expectancy is 100 years of age. However, the optimum time of productivity is around 40 years. The cocoa cob is a suitable ingredient to be used throughout the year. Nonetheless, during the stages of its development, farming can undergo various alterations that affect the production of cobs, such as phytosanitary problems, moniliasis, Black Pod, and Witch’s broom, which can cause up to 60% loss of production. All these changes are mainly produced by the effects of the climate [
3].
The cultivation of cocoa is predominantly practiced on a large scale in regions characterized by humid and hot climates in countries such as Ecuador, Colombia, Venezuela, Brazil, Peru, Bolivia, Trinidad and Tobago, and Mexico. It serves as a major source of income for agricultural families [
4].
Ecuadorian cocoa stands out for its global exportation to around 61 countries, making a significant economic impact on households [
5]. Since 2021, the total area of cocoa plantations in Ecuador was to an average of 626.962 hectares, representing 26% of the country’s total agricultural area [
6]. Within these territories, diverse varieties of cocoa are cultivated. Notably,
National Cocoa Fino de Aroma (NCFA) covers 25% of the planted area, while the remaining 75% belongs to
Colección Castro Naranjal 51 (CCN-51) [
7]. Regarding the data registered in the Plant and Animal Protection Control and Regulation Agency, cocoa is exported in different formats: vegetable product and as a by-product of vegetable origin, creating other products such as oil, blocks, bars, peel, husk and film, chocolate, and other prepared foods that contain cocoa [
8,
9]. NCFA is appraised for its fruity, floral, herbal, and woody flavors, standing out from other varieties and being used to create intense and balanced chocolates. Its origin and domestication are in the Amazon region of the south of Ecuador [
8]. On the other hand, the CCN-51 variety, a cloned cocoa of Ecuadorian origin, was declared to be of high productivity in 2005, being valued for its high tolerance to diseases, efficiency, and quality [
10].
In Ecuador, only 20–23% of the total cocoa production is used in the final product, indicating that the cocoa beans are exclusively utilized [
11]. Between 77 to 80% of the remaining by-product is discarded, including the mucilage/pulp. The by-products derived from these raw products mean a significant source of nutrients that could be used to elaborate healthy foods, thus contributing to diversifying the range of foods presented to the population [
12].
The average nutritional composition of cocoa consists of proteins (11.5%), starch (7.5%), tannins (6%), water (5%), salts and trace elements (2.6%), organic acids (2%), theobromine (1.2%), and caffeine (0.2%), among others [
13]. However, the nutritional composition of the by-products of cocoa processing is not well studied because they are considered for use.
At an industrial level, the peel, placenta, and mucilage are often discarded. However, utilizing these resources to create other products introduces a new perspective on sustainability, which helps reduce environmental problems and creates additional sources of income [
14].
In recent years, by-products derived from cocoa mucilage have begun to be commercialized, although their usage is still relatively limited. Their adoption has been facilitated by their fruity flavor and natural sweetness, requiring no additional sugars. On the other hand, mucilage has been pasteurized to preserve its freshness, and later used as a natural sweetener in bars or in chocolate covers, besides being transformed into lyophilized powder to be sold in its most natural form [
15]. In the same context (its use), artisanal chocolate-makers and now also more famous brands are experimenting with including mucilage in their chocolate bars, taking it always as a more natural and healthier sweetener. It is interesting to note that cocoa mucilage exhibits various flavor profiles depending on its variety and origin, similar to cocoa beans [
16].
Ref. [
17] defines mucilage as a substance covering the seeds of cocoa, traditionally used as substrate in the fermentation process of the grains of cocoa, and is also an important element in the formation of precursor substances of scent and flavor. This by-product of cocoa production holds a high nutritional and functional value, due to its content of vitamins B, C, D, and E and minerals such as Ca, Fe, K, Mg, and Zn [
18]. Mucilage is capable of “metabolizing” during the fermentation of the bean of cocoa as it contains bacteria and yeasts that play a crucial role in the metabolism of sugars present in the mucilage. These microorganisms participate in the production of compounds that positively affect the organoleptic characteristics of cocoa, contributing to the unique flavor profile of the chocolate. Changes in the chemical composition of the mucilage layer can affect the production of acetic acid by yeasts and bacteria, and the consequences of these interactions are multiple.
Firstly, the organoleptic quality of the cocoa, including its flavor and scent, may be affected. In addition, changes in the production of acetic acid may influence the texture and acidity of the fermented cocoa, which has a direct impact on the final quality of the chocolate from these fermented beans of cocoa [
19].
The utilization of cocoa mucilage has gained significant importance in recent years, as evidenced by various studies. These studies have led to the development of different derived products, including non-alcoholic and alcoholic beverages, wines, vinegars, jellies, ice creams, gels, and marmalades [
15,
20]. Additionally, cocoa mucilage shows great potential in pectin production, making it an ideal substance for achieving consistency in marmalades and jellies [
21]. The emergence of cocoa mucilage as a valuable market product provides cocoa producers with a new source of income.
Previous investigations emphasize that mucilage should not be regarded as a mere by-product of cocoa, as it possesses chemical properties of significant value and a distinctive sensory profile [
22,
23]. Its tropical, fresh, and fruity characteristics resemble a blend of fruit juice. Some companies elaborate cocoa drinks exclusively from this mucilage (or white pulp), without the need for added sugars [
24,
25].
The aim of this study lies, firstly, in an evaluation of the pondered performance of the two varieties of cocoa,
Theobroma cacao L. (
National Cocoa Fino de Aroma (NCFA) and
Colección Castro Naranjal 51 (CCN-51)), harvested during the dry and rainy seasons. Such evaluation will be carried out regarding the different constituent parts of cocoa: cob, peel, grain, placenta, and mucilage, with special emphasis on the maturation stages (
Figure 1 and
Figure 2: underripe, ripe, and overripe). Secondly, a novel procedure will be implemented to extract overripe mucilage, with the aim of producing a fermented non-alcoholic beverage (kombucha) and conducting sensory evaluations for various formulations.
2. Materials and Methods
2.1. Research Design
This investigation took place in the Experimental Campus “La María” of the Technical State University of Quevedo (UTEQ), Los Rios Province, Ecuador. In
Figure 1, the different phases of the elaboration process are represented. The experimental period covered two consecutive years, where harvesting periods occurred in May, June, and July (dry months), as well as in November, December, and January (rainy months). The dry season in Quevedo generally spans from June to November. During this period, temperatures are warm during the day and can vary depending on the specific month, ranging between 22 °C and 32 °C. In contrast to the rainy season, Quevedo experiences more hours of sunlight during the dry season, averaging 7 to 9 h of sunlight per day. This increased sun exposure contributes to a drier and sunnier climate in the region. Additionally, humidity levels are lower during the dry season, fluctuating between 60% and 80%. Although humidity is lower than during the rainy season, it remains relatively high due to the tropical climate of the region. During the rainy season, which typically occurs between January and May, Quevedo experiences a significant increase in precipitation. The amount of rainfall tends to be higher, averaging between 1500 to 2000 mm per year. This can lead to temporary flooding and river overflow, especially in areas near bodies of water. Temperatures during the rainy season remained warm, with lows rarely dropping below 20 °C and highs that can exceed 30 °C. The combination of high temperatures and humidity during the rainy season can contribute to a feeling of mugginess in the region, with humidity levels typically ranging between 80% and 90%.
Phase 1. Fruit harvesting: Cocoa cobs were recollected in two specific moments of years, 2019 and 2020. For this study, two types were considered: National Cocoa Fino de Aroma (NCFA) and Colección Castro Naranjal 51 (CCN-51). One thousand eight hundred cobs were gathered in total. At the same time, the harvesting periods were April, May, and June (dry months) and October, November, and December (rainy months) in both years of study. Harvesting was conducted in the morning, selecting cobs at various stages of maturity, with each cob weighing approximately 300 g. After harvesting, they were transported to the processing plant at room temperature in fruit collection baskets, where Phase 2 took place.
Phase 2. Cleaning, preparation, and process of extraction of mucilage and other components of the cocoa cob: Cobs were put under cleaning and sanitizing treatment with tap water and sorted out according to the variety, time of the year, and maturation state. Maturation state is identified according to the color of the external part of the cob. In NCFA, the unripe ones are green, whereas the ripe ones are yellow. In the case of CCN-51, they are red and orangey yellow, respectively. In the case of overripe cobs, both varieties show a brown color.
After being washed, cobs were transported to proceed with the extraction of the grains, placenta, and mucilage with stainless steel machetes. The extraction of the components was performed using two longitudinal and two transversal cuts, which allowed the opening of the cob. In this process, the weight of the full cob, the peel, the bean, and the mucilage were measured. After writing down the weight of all the components, they were kept at refrigeration (20 °C) to later be pasteurized and fermented. Two liters were poured into the traditional glass wine bottles, and each bottle was sealed with a cork stopper.
Phase 3. Mucilage extraction treatment: The treatment followed the artisanal process, which involves wrapping the cocoa bean in a thin cloth and applying manual pressure to extract the mucilaginous liquid surrounding the cocoa bean. It should be emphasized that the extraction process was not taken to the final stage, as the cocoa bean requires a certain amount of mucilage to complete the fermentation process and acquire the essential characteristics necessary for chocolate production. Using this method, the extraction efficiency of the mucilage increases up to 80%.
After being washed with tap water, the pods were transported to proceed with the extraction of the beans, placenta, and mucilage, respectively. Stainless steel machetes were used to make cuts in the pod, performing two longitudinal and two transverse cuts to fully open the pod. During this process, the weight of the complete pod and its components, including the husk, the kernel, and the mucilage, was recorded. Subsequently, they were kept refrigerated at a temperature of approximately 20 °C.
In Phase 3, corresponding to the mucilage extraction treatment, an artisanal method was employed. This involved wrapping the cocoa bean in a fine linen cloth and manually applying pressure to the bean (graphical abstract). This way, the liquid surrounding the cocoa bean, known as mucilage, was obtained and collected in aluminum trays for later use. It is important to note that the extraction process was not carried out in its entirety, as the cocoa bean requires a certain amount of mucilage to remain integrated into its shell to complete the fermentation process and acquire the essential characteristics necessary for chocolate production. By using this method, the mucilage extraction efficiency increases by up to 80%.
In Phase 4: Elaboration of the non-alcoholic fermented beverage (kombucha-like): The experiment began (
Figure 1 in Phase 3) with ripe stage mucilage and involved eight treatment combinations (see
Table 1). The ingredients used were: 2 L of cocoa mucilage, 10 g of green tea, market yeast (120 mL of starting ground), and sucrose as a source of added sugar (regarding the treatments in
Figure 1). Factorial design A × B with two levels in Factor A (NCFA and CCN-51) and four levels in Factor B (40, 60, 80, 100 g/L). For this, 8 treatments with 4 repetitions each and a total of 32 samples of fermented beverages were evaluated.
2.2. Activities Involved in the Process of Elaboration of the Fermented Beverage
It started with the pasteurization of fresh cocoa mucilage in a metallic container, subjected to a temperature of 65 °C for 15 min (
Figure 3). Subsequently, tea preparation ensues, during which 10 g of green tea is added to a volume of 2 L of pasteurized mucilage. This mixture was heated for the following 8 min until it acquired a slightly dark color, aiding in preventing light filtration that may interrupt the fermentation process, resulting in a highly concentrated solution. Following this, sugar was added according to the designated treatment combination (see
Table 1) in the various treatments. To obtain the fermented beverage, the mucilage was left for approximately one hour until both the tea and mucilage reached an ambient temperature of 28 °C, at which point the mixing process was undertaken. Careful integration of both solutions was conducted in the same container, followed by gentle agitation to ensure proper homogenization. Subsequently, the liquid was poured into a glass container containing the SCOBY (Symbiotic Culture Of Bacteria and Yeast), ensuring complete coverage, and then tightly sealed with filter paper or absorbent towels, allowing for slight ventilation to facilitate gas exchange necessary during the fermentation process. The container was stored in a dark, dry place for 15 days and housed in a previously sterilized glass jar at a constant temperature of 25 °C. After this period, the product was packaged through a filtration process and placed in sterilized glass bottles. Final storage occurs under refrigeration to slow down or halt fermentation, ensuring product stability. Finally, the resulting beverage underwent physicochemical and sensorial analyses.
2.3. Physicochemical Analysis
For the physicochemical parameters (such as acidity, °Brix, pH, moisture, ash, protein, fat, fiber, vitamins, sugars, and polyphenols), cocoa mucilage samples of the CNFA and CCN51 varieties were used in a ripe state (as a raw material for the fermented beverage). Finally, the acidity, °Brix, and pH values were determined in the 8 formulations studied in the production of the beverage. All methodologies followed the AOAC for Physicochemical Analysis in food.
2.4. Sensorial Analysis
For the sensory analysis, a panel of experts comprising 30 semi-trained panelists participated. The beverage was analyzed after being elaborated for 16 days. In the hedonic test, the following attributes were evaluated: color, flavor, texture, smell, sweetness, and aftertaste. Each tester evaluated 8 samples. The sensory analysis report utilized the following scale: strongly dislike (1), dislike (2), neutral (3), like (4), and love (5).
2.5. Statistical Analysis
To compare the average numbers of the treatments, the Tukey multiple range test was used (p < 0.05), and Kruskal–Wallis (p < 0.05) non-parametric analysis was used for the sensorial results; both with the statistics software (InfoStat, 2017).
4. Conclusions
The varieties considered for this study were: Cacao Nacional Fino de Aroma y Colección Castro Naranjal 51 CCN-51. In total, 1800 cobs were recollected and grouped by variety, season of the year, and state of maturation, with the best state to obtain mucilage being the overripe stage, with over double performance than in unripe.
The performance based on the weight of the cobs obtained better results for the ripe varieties in the rainy season compared to dry, although the variability of the samples was higher in this last season for all the stages of maturation of both varieties of cocoa, apart from the National Cocoa in the winter season, which shows a range of weights from 430 to 600 g per cob and SD of 89.92.
The non-alcoholic fermented beverage (kombucha) received an excellent score, with the N40 (national + 40 g/L sugar) being the best combination in terms of flavor, texture, and sweetness. Aftertaste was not relevant to any of the panelists (the higher score was “neutral”), contrary to flavor and texture, which were determined in order to choose the best treatment and, finally, the acceptance of the new non-alcoholic fermented beverage based on mucilage and green tea.
The use of mucilage, with its high performance and significant nutritional value, positions it as a promising strategy to address the SDOs in underdeveloped countries since it contributes to zero hunger, health, well-being, and climate action.