Banana and apple extracts with efficient microorganisms and their effect on cadmium reduction in cocoa beans (Theobroma cacao L.)

Abstract

This research was aimed to assess the impact of efficient microorganisms and fruit extracts (Banana and Apple) on fermentation of cacao collected in Ecuador. The work was focused on compliance with European Union regulation 2021/1323, which sets cadmium (Cd) limits at 0.80 mg kg^–1 for cocoa products that are exported. The study conducted experiments on cacao of two Ecuadorian provinces, Manabí and Esmeraldas, employing two types of bioinoculants (Biol Albiobatch and EM-1 20%). Bioinoculants were analyzed for their potential in the reduction of Cd, using a Trifactorial statistical design (Completely randomized trifactorial design, CRTD) 2 × 2 × 2 added + 2. Ten treatments with three replicates were used, including two unamended controls. The Rohan Modified micro-fermentation method was employed, considering various parameters such as cacao fruit morphology, temperature, pH, and °Brix. After fermentation, the cocoa beans were sun-dried to reduce moisture content, and their fermentative state was assessed. Cd levels were determined using the USDAP 3050B method. The results showed that different treatments affected cacao fruit morphology, temperature, pH, and Brix differently. Notably, the Biol EM-1 + Banana treatment of cacao of Manabí exhibited the most favorable results in several aspects. The cutting test revealed significant improvements in fermentation quality for this treatment. Moreover, the use of microorganisms resulted in a substantial reduction in Cd levels, with cacao of Esmeraldas and Manabí provinces experiencing reductions of 63.25% and 58.20%, respectively. Overall, this research demonstrates the potential of efficient microorganisms and fruit extracts to enhance cocoa fermentation and reduce Cd content, contributing to the quality and compliance of Ecuador cocoa exports with EU regulations.

Introduction

Cocoa, prized for nutrition and flavor, drives Ecuador's income from the food and chocolate industry. Ivory Coast dominates with 33% global cacao production; however, Ecuador and Brazil are also key players in the global cacao production [1, 2]. Cocoa producing nations face economic risks due to elevated heavy metal levels in cocoa beans. These metals persist in chocolate, posing health risks. Cacao buying countries aim to safeguard human health and ensure safe chocolate consumption [3]. The European Union set maximum levels of cadmium (Cd) allowable in a cocoa paste product following the manufacture and industrialization of chocolate. There must be a total dry matter content of cocoa greater than or equal to 50%; and Cd must not exceed 0.80 mg kg⁻¹ [4].

According to previous research on the mitigation of Cd in the post-harvest of cocoa, Chang et al. carried out an application of microorganisms in the fermentation stage of cocoa [5]. They used Rhizobium japonicum, a Gram-negative nitrogen-fixing bacteria at a concentration of 300 and 600 mL for every 2 kg of fresh Trinitario cocoa mass, in cacao of two provinces of the Ecuador, i.e. Los Ríos and Guayas. Fermentation was carried out by Rohan boxes [6] and coolers. Trinitario, induced with the microorganism to reduce Cd levels. However, insights are lacking related to the research in hot areas where there is a high incidence and concentrations of Cd to see the conclusive effect of the rate of decrease of Cd by using microbial treatments during cacao fermentation.

Cd (Cd⁺²), a heavy metal, occurs at levels exceeding 1 mg kg^–1 in Ecuador’s cocoa crops, notably in provinces like El Oro, Esmeraldas, Manabí, and Guayas. In Ecuador, mining activities (particularly in the Amazon region) and use of agrochemicals are the principal cause of Cd⁺² into soils. In addition, emissions from metallurgical mines contain byproducts of metals like Zn, Cu, and Pb, which contaminate soils [7]. Metals present in the farmland soils subjected to phytoaccumulation (including cacao plants) through root uptake [8]. Subsequent translocation of metals in the plants causes harm to stems, leaves, flowers, and cocoa beans. Contamination affects beans via cotyledon penetration [9].

Cocoa fermentation is vital for high-quality beans, demanding effective harvest and post-harvest practices. Microorganisms are emerging solutions in the chocolate industry, enhancing bean quality and fermentation stages [10]. Applying efficient microorganisms and fruit extracts (Banana, Apple) to cocoa during fermentation aims to determine if Cd levels can be reduced in dried, fermented beans or roasted cocoa nibs. Therefore, the current research was aimed to use a combination of microbial inoculants + fruit extracts to improve the fermentative quality of fermented cacao by reducing the Cd in fermented cacao products. This research was carried out with the cacao of two provinces (i.e. Esmeraldas and Manabí) in Ecuador. The results of this investigation are certainly helpful in improving the economy of cacao farmers in the Pacific coast of Ecuador where farmers encounter a hot climate.

Materials and methods

Localization

The research was carried out in the city of Quevedo (Latitude: −1.02863 and Longitude: −79.46352) in Ecuador. The cocoa fruits were obtained from two farms belonging to two provinces of Ecuador. The Manabí is surrounded by cold waters enclosed between Calceta and Junín towns (0°55′45.5"N 80°08′42.3"W; Lat: –0.929304, Long: –80.145094). The another province is Esmeraldas, which located in the Tahigue enclosure in the Chincha parish of the Esmeraldas canton (0°45′31.6"N 79°35′16.8"W; Lat: 0.758777, Long: −79.588009). The median temperature in these regions is between 19 and 28 °C, with an average of 25 °C, with a precipitation of 1200 mm, in the north to 2650 mm. Physicochemical analysis variables were assessed in the bromatology lab at “La María” experimental farm, part of State Technical University of Quevedo, Ecuador. Plants and plant materials used in this study were complied with local or national guidelines.

Biol 1 and Biol 2

Biol 1 and Biol 2 were the two commercial inoculants used in this investigation. Biol manufactured by EMBIOECSA (Trade name), and hereafter, it is presented as EM-1 (efficient microorganism). The microbial composition of EM-1 is as follows: Saccharomyces cerevisiae (2.4 × 10⁵ colony forming units/mL) and Lactobacillus casei (1.3 × 10⁷ cfu/mL) (EMBIOECSA, 2021). Second microbial inoculant used in this research was Biol 2, commercially it is available as ‘Albiobach’ (Trade name). Biol 2 is a mixture of bacteria (1.3 × 10⁷ CFU/mL) and yeast cells (4.0 × 10⁶ CFU/mL). Bacteria that are commonly encountered in Biol 2 are Azotobacter chroococcum, Lactobacillus acidophilus, Saccharomyces cervissiae, Rhizobium japonicum [5, 11].

Experimental design

The study utilized an ANOVA (analysis of variance) in a Completely Randomized Design (CRD) with a 2 × 2 × 2 added + 2 trifactor model (trifactorial). There was total 10 treatments in triplicates (i.e. Two provinces (Manabí and Esmeraldas) × two EM (1 and 2) × two fruit extracts (banana and apple), with two controls. In this research, 20% EM induction (400 mL per 2 kg fermentative mass), and 3% fruit extract (Banana, Apple of 60 mL) were used (Table 1). Fermentation was carried out in a Rohan box whose dimension is 100 × 100 × 15 cm (length × width × height) and is made up of Laurel wood [6]. Cocoa mass capacity of a box is 45 kg; and a box has square compartments of 15 × 15 × 15 cm (length × width × height) with a flat base. There is a 0.5 cm slit which allows the runoff of the mucilage.

Table 1. Structure of ANDEVA

Source of variation		Degree of freedom
Treatments	t-1	7
Control 1		1
Control 2		1
Efficient microorganisms factor	(me-1)	1
Fruit extract factor	(Extract-1)	1
Provinces factor	(pro-1)	1
Int ME* Extract	(me-1) (Extract-1)	1
Int ME*Provinces	(Me-1)(p-1)	1
Int Extract* Province	(Extract-1)(p-1)	1
Int Mo* Extract*Provicnce	(Mo-1)( Extract-1)(Pro-1)	1
Experimental error	(me* Extract*pro) (r-1)	16
Total	me* Extract*pro*r-1	23

Analysis of variance was performed for the determination of data analysis of ANDEVA and the means were compared with Tukey's test (p ≤ 0.05) by using Infostat Statistical software (Updated version 29–09-2020 InfoStat; Versión: 2020 l). An ANOVA scheme and an experimental model were applied to compare the factors or variables studied in order to see the effect whether significant or not significant and to be able to reach concrete results and conclusions [12, 13] (Fig. 1a–h). The probability was calculated for different parameters, such as provinces (Manabí and Esmeraldas), fruit extracts (banana and apple extracts), microbial inoculants (Biol 1 and Biol 2) as, province × fruit extract, province × biol, extract × biol, province × extract × biol, control 1, 2 × all treatments, and control 1 × control 2; and all the results were presented in Supplementary Table 1–6.

Fig. 1 [Images not available. See PDF.]

Experimental location and material – Geographical map of South America (a) and Ecuador (b); samples of efficient microorganisms (c and d); weighing of cacao beans (e), cacao bean fermentation in Rohan boxes (f), dried cacao beans (g) and grounded cacao beans (h)

Collection of plant materials

When harvesting or collecting cocoa cobs, it should be taken into account that these fruits are in excellent condition and should be avoided by not collecting Moniliophthora contaminated cacao cobs. For this research, cocoa (CCN-51 variety of cacao) cobs were harvested in Esmeraldas and Manabí. From here onwards, the cacao of Esmeraldas and Manabí provinces were presented as ‘Cacao-ES’ and ‘Cacao-MN’, respectively. Approximately a total of 460 cobs were collected to reach the amount required for the executed study. After obtaining required pods, pulping was carried out, which consisted of removing the cocoa bean from the placenta of the fruit. In addition, a longitudinal or transverse cut was made to extract the cocoa beans. At the same time, once the cocoa beans were pulped, they were separated and the cocoa beans were stored in clean bins. These were placed in the cells of the micro-fermentation boxes that each cell supplies 2 kg of fresh cocoa mass.

Analysis of cocoa cobs

Weight of cocoa fruit The weight of the harvested cocoa cobs was taken, the weight divided by the number of healthy cobs [14].

Number of beans per cob -The number of almonds per cocoa fruit was counted directly and the average was determined [15].

Length of cocoa fruit The size of the length that corresponds in the interval from the part of the base at the junction of the peduncle of the cob to reach the apex of the cob was measured with the metric square instrument [16].

Diameter of the cocoa fruit Fruit was cut at its thickest part and the diameter measured [17].

Application dose of EM (efficient microorganisms)- Efficient microorganisms were used at 20% (v/w) (400 mL per 2 kg fermentative mass). Experimental layout followed 8 treatments, 3 repetitions, + 2 untreated controls, cocoa beas of two provinces (Supplementary Table 1). Induced doses aligned with field study objectives for Cd reduction and grain quality enhancement.

Preparation of fruit extracts A 3% (volume to weight 60 mL) solution of polyphenol oxidases was prepared from natural apple extract to assess its potential for enhancing grain quality and reducing Cd⁺² during fermentation. Six apples (100–150 g each) were mixed with 200 mL distilled water for fruit extraction at below 40 °C [18, 19].

Determination of pH For pH analysis, 10 g of cocoa beans were crushed and mixed with 10 mL of distilled water at 40 °C. This process was repeated for each treatment over 4 studied days by recording pH using a pH-meter (Thermo Orion, Waterproof, USA) [20]. The mixture was prepared in warm glass containers to measure pH changes [20].

Determination of total soluble solids (Units °Brix)—To measure this variable, a Refractometer (OPTi + 38-A1, Brix 95; Bellingham & Stanley, UK) was employed. Ten grams of cocoa beans were randomly selected and crushed in 10 mL of warm distilled water. Using an OPTi + Refractometer, the equipment was calibrated to 0.00 before applying 1–3 drops of the water, ensuring accurate treatment [21].

Temperature Determination—Maintaining optimal fermentative activity requires temperature control between 45 °C and 50 °C. Higher temperatures lead to over-fermentation, while lower temperatures hinder bean quality and fermentation. Temperature fluctuations were controlled through coolers. Temperature was monitored regularly using a thermometer [16].

Drying of cocoa beans

Post-fermentation cocoa drying is vital for flavor development. Sun-drying on wooden surfaces was used; and at this stage, mixing of beans was avoided due to farm variations [22]. Hygienic practices were maintained to prevent contamination. A humidity of 6–8% was achieved in 7–8 days [23].

Storage of cocoa almonds

Once the drying stage was finished, the cocoa beans were stored according to the alteration of their treatments and repetitions, these were placed in paper bags, this will help improve the quality of the cacao beans [24, 25].

Cob or pod index

This variable indicates the number of cocoa fruits required (20 pods) to obtain 1 kg of dried cocoa; this was calculated according to the following equation [26]:

Equation 1. Pod index (PI)

1

$$PI=\frac{Numberofcobs}{Driedalmondsweiht\left(g\right)}\times 1000$$

Seed index

Seed index was determined before cutting text. To calculate the seed index, 100 cacao beans (fermented and dried) were weighted in an analytical balance and average was determined. Seed index was determined using the following equation [27].

Equation 2. Seed index (SI)

2

$$SI=\frac{Hundredfermentedanddriedcocoabeansweight\left(g\right)}{100}$$

Test of testa (husk) and cotyledon

It was obtained by means of the weight of 30 g of fermented and dried cocoa beans; weight was determined in a precision analytical balance (Balanza Analítica MX, Mettler Toledo, USA). The husk and cotyledon test was done using the following equation [26, 28].

Equation 3. Husk and Cotyledon test

3

$$Husk\%=\frac{\left(Huskweight\right)}{Weightof30gcacao}\times 100$$

Cutting test

Hundred cacao beans were randomly selected, and the weight was measured on a precision analytical balance. A stylus was used to create cross-sections in dried beans. Observation helped to classify fermentation status: Well Fermented, Moderately Fermented, Violets, Slates, or Moldy. Standard guidelines were followed to ensure accurate classification based on quality and effects [26, 29].

Weight of cocoa beans in 100 g

To determine the weight of the cacao beans, 100 g were randomly taken, which subsequently took notes of the number of almonds to complete the 100 g [26].

Determination of Cd

The analysis of Cd was done in the laboratory INIAP (National Agricultural Research Institute), Quevedo, Ecuador. Cd content in cocoa after fermentation with EM and fruit extracts (Banana, Apple) was analyzed. The analysis was in compliance with EU 2021/1323 limits sought [4]. To analyze Cd²⁺ levels, a sample of 1 g of previously fermented dry cocoa was taken. This sample was crushed and subjected to wet digestion using 7 ml of nitric acid (HNO₃) and 3 ml of hydrogen peroxide (H₂O₂). The digestion was carried out in a microwave until reaching a temperature of 150 °C. Subsequently, the sample material was filtered and Cd²⁺ was determined (the USDAP standard 3050B method was used) in an atomic absorption spectrophotometer with a graphite furnace [6, 30]. The rate of decrease (D) of heavy metal was obtained with the formula:$$\%D=\frac{\left(Control-Treatment\right)}{\mathit{Control}}\times 100$$

Results and discussion

Physical–chemical variables—Initial variables of the cobs

According to the analysis of variance of the province factor, there was no statistical difference; however, Tukey’s probability was significant ((p ≤ 0.05)) with respect to the physical variables of the pods (Fig. 2). Details of samples (1 to 8) and the respective quantitative data were provided in Supplementary Table 1. In general, Cacao pod physical parameters are influenced by several factors such as, cacao variety, geography, soil and climate [26, 27, 31–34]. Graziani et al. found discrepancies between three types of cacao varieties (criollo, cacao foratero, cacao trinitario) considering the ‘width’ variable [14].

Fig. 2 [Images not available. See PDF.]

Effects of the interaction of fruit extracts (banana and apple) on the initial physical variables (a and b) of the cocoa cobs subjected to different treatments. (Sample 1 = E, B, Al; Sample 2 = M, B, Al; Sample 3 = E, A, Al; Sample 4 = M, A, Al; Sample 5 = E, B, EM-1; Sample 6 = M, B, EM-1; Sample 7 = E, A, EM-1; and Sample 8 = M, A, EM-1. Where E = Cocoa of Esmeraldas; M = Cocoa of Manabí; B Banana extract; A = Apple extract; Al = Albiobatch microbial inoculant; and EM-1 = Efficient microbial inoculant)

pH

In the pH variables (changes in the pH during 0–4 days fermentation, represented as pH0, pH1, pH2, pH3 and pH4, respectively) (Fig. 3), and the fermentation's impact showed statistically significant differences for pH0, pH1, pH2 (highly significant), and pH3 based on province. However, pH4 lacked statistical disparity, exhibiting uniformity across provinces per Tukey's probability (P ≤ 0.05). Notably, pH0 displayed variable behavior, with Cacao-MN exhibiting slightly higher pH (average 3.23) compared to Cacao-ES (average 3.16 pH). Two control values of pH at respective incubation days (0–4 days) were presented in Supplementary Table 2.

Fig. 3 [Images not available. See PDF.]

Effect of fruit extract application on different variables such as pH (a), Brix (b), temperature (c) during the fermentation subjected to different treatments; and respective experimental work (d). Panel ‘a’, and ‘b’ are scatter plot with bars; and panel ‘c’ is scatter plot [Each scatter is a mean of 8 treatments with SD; and there are eight dots at each scatter, and each dot indicates the one sample]

In the province versus Biol interaction, statistical differences were absent between pH0 and pH3 (p ≤ 0.05), while pH1, pH2, and pH4 exhibited significant disparity according to the Tukey test. Notably, Biol application (EM-1) was effective in Cacao-MN. For pH1, the Cacao-MN with Biol Albiobatch showed 3.82. For pH2, Cacao-ES exhibited 4.30 with Biol EM-1, while Cacao-MN reached 5.69 with Biol EM-1, emphasizing Cacao-MN’s superior response to Biol EM-1 application (Fig. 3a).

Solórzano et al. carried out an investigation with different varieties of cocoa beans fermenting in wooden boxes, mentioning that the pH rises constantly during fermentation [20]. For this reason, the best ranges of pH are between 5.5 and 5.8. Pinargote et al. found an average pH range between 5.1 and 6.0 at the end of harvest time of fermentation with National cacao varieties [35].

In the course of cacao fermentation, Krähmer et al. showed a linear increase of pH values ranging from 5.00 to 6.00 [36];. Noor and Nazamid suggested that pH should be in the range of 5.20 to 5.49 [37], as this pH range improves the sensory quality of cocoa, where it found high chocolate flavor while low pH value i.e. 4.5 to 5.00 has an unfavorable effect in terms of the organoleptic analysis used in the research of the aforementioned authors. At a pH of 4.5–5.0, an unfavorable effect on cacao bean quality was found as determined by organoleptic analysis [37]. Alvarado et al. mentioned that the use of effective microorganisms in the fermentative stage of cacao beans has a positive effect on the increase in pH from 3.49 to 5.44 [38]; which is favorable for the production of chocolates with aroma and flavor characteristic of fine aroma cocoa. Chang et al. found pH of 5.26 as an effective variable during the cacao fermentation with yeasts and enzymes [39].

Brix value

The values of Brix 3 and Brix 4 of Cacao-MN exhibited a decreasing trend, and values were found to be 6.70 and 5.48 respectively. However, Brix 0, Brix 1, Brix 2 values showed decreasing linear relationship (Fig. 3b; ST-3).

The application of banana extract showed an acceptable decreasing conclusive effect of soluble solids, and corresponding values of 5.48 for the application of banana extract; while for Brix 0 to Brix 3, showed inconclusive decreasing linear values upon banana and apple extracts application. With regard to the interaction of province by fruit extract, a statistical difference was denoted in Brix 4 with a respective value of 5.48 being Cacao-MN that best suited Biol EM-1, while the other days (Brix0, Brix1, Brix2, Brix3) had uniformity between the values found during the fermentation stage. According to Cardenas et al. in their research they found that during fermentation at 24 h the cocoa had 15 Brix [40], and after 84 h they found that it decreased by 6°Brix. Loureiro et al. indicated that the initial soluble solids fluctuate between 20 to 15°Brix, and their conditioning with pH is what favors the development of microorganisms [21]. As long as the pH is below 5, the most important effect during fermentation is in the first 3 fermentative days.

In the same sense, Navia and Pazmiño found initial values of 14°Brix [41], and Garcia et al. found 20°Brix as an initial value during anaerobic fermentation of cacao by microbial inoculants [42]. Nonetheless, the °Brix values of present investigation are in line with the results reported by Garcia et al. [42].

A positive effect of enzyme extract of banana on Brix values was found in one investigation [43], which is attributed to enhanced microbial activity by banana extract.

Temperature

It was observed that for the province factor in all the days of study in terms of the temperature variable, statistical difference was observed in terms of the probability of Tukey at (p ≤ 0.05. Concurrently persists that Cacao-ES initially started with a Temp0 (Temperature 0) value of 32 °C, which was raised to 37.93 °C (Temp1), and the increasing trend was continued at the further days, i.e., 43.33 °C (Temp2), 45.67 °C (Temp3) and 48.67 °C (Temp4) (Fig. 3c and d, ST−4). The value found for the factor of apple extract in the Temp1 was 37.93 °C, which disagrees with the other days of temperature where they showed no statistical differences. At the same time when the biol factor was observed (Albiobacth and EM-1), statistical difference was denoted in the Temp1 and Temp2, where the biol that exerted effect was the EM-1 with a value of 37.93 °C for the Temp1 and 43.33 °C for Temp2. These values clearly evidencing the efficiency Biol EM-1 application, with respect to the other variables of temperature consideration, they did not register conclusive differences.

Application of microorganisms such as S. cerevisiae in the fermentation stage of cocoa increased in temperature (initial value was 30 °C), resulting in a cocoa liquor with adequate sensory qualities [44]. They mentioned that the temperature of 45–50 °C can cause death of the cacao embryo, penetrating the seed coat of the cocoa reaching the cotyledon and allowing the internal form to reach the required fermentation. To avoid temperature loss it is advisable to use tender banana leaves to cover the cocoa mass, in turn agrees that the use of microbial strains favors the fermentation stage [45], to increase the internal temperature of cocoa beans. Interestingly, the addition of microorganisms shortens the fermentation period in cocoa beans.

Physical variables (cut test)

A lowest humidity (i.e. 6.27%) was found in the Cacao-ES treated with banana fruit extract and EM-1 (P ≤ 0.05). However, the other physical variables of the cut test did not register the same conclusive behavior (Fig. 4a–d, ST-5).

Fig. 4 [Images not available. See PDF.]

Impact of fruit pulp treatment on cacao dry weight (a), Cotyledon % (b), quantity of fermented grains (c) and other physical variables (d). (Sample 1 = E, B, Al; Sample 2 = M, B, Al; Sample 3 = E, A, Al; Sample 4 = M, A, Al; Sample 5 = E, B, EM-1; Sample 6 = M, B, EM-1; Sample 7 = E, A, EM-1; and Sample 8 = M, A, EM-1. Where E = Cocoa of Esmeraldas; M Cocoa of Manabí, B Banana extract, A Apple extract, Al Albiobatch microbial inoculant, and EM-1 = Efficient microbial inoculant)

In a different investigation, it has been found that the fermentation induced with yeasts and polyphenols has reached up to 80% of the humidity of fermentation while beans not fermented with microorganisms or yeasts have reached a percentage of 20%. Increased humidity not only results in the acceleration of fermentation, but also improvement of yield quality [46]. Pangan, has carried out a research to understand the efficiency in types of fermenters, and the type of wood that reached more fermented beans 13% husk, and 79% cotyledon [47]. This evaluation was carried out to know the level of performance, in the same sense the authors [48] found referential data to which they fit with the present research. According to Medina and Velasquez, the application of yeasts and mixture of microorganisms, found about 70% of well-fermented beans, 20% represents violet grains and 10% represents slate and moldy grains, which allows improvement because the controls were below 50%, and the index of violet beans was higher [49]. On the other hand, in different investigations [50, 51], researchers have used 4 different fermentation methods (such as sack, heap, wooden boxes and tubs) for a period of 5 days, where it was evident that there was better fermentation in the wooden crates.

Cd analysis

Cd analysis in cocoa cotyledon for the province factor displayed a highly significant trend with Tukey's probability (p ≤ 0.05). Cacao-MN exhibited the lowest value (0.47 mg kg^–1), reflecting its adaptability and effective fermentation conditions. The apple extract exhibited superior efficiency in reducing Cd, supported by significant results through Tukey's test (p ≤ 0.05). Fruit extract by Biol yielded inconclusive statistical trends, as both fruit extracts combined with Biols effectively reduced Cd⁺² levels in cotyledons, as observed in the present study (Fig. 5a, b, ST-6).

Fig. 5 [Images not available. See PDF.]

Effect of fruit extract on the concentration of Cd in cacao cotyledon (10 × 10 dot plot) (a) percent of decrement range (D values showed in Violin plot) (b). The Cd values of control 1 and 2 were 1.22 and 1.35 mg kg^–1, respectively. (Sample 1 = E, B, Al; Sample 2 = M, B, Al; Sample 3 = E, A, Al; Sample 4 = M, A, Al; Sample 5 = E, B, EM-1; Sample 6 = M, B, EM-1; Sample 7 = E, A, EM-1; and Sample 8 = M, A, EM-1. Where E = Cocoa of Esmeraldas; M Cocoa of Manabí, B Banana extract, A Apple extract, Al Albiobatch microbial inoculant; and EM-1 Efficient microbial inoculant)

According to one investigation [52], the bacteria Ralstonia eutropha was found to be effective in lowering Cd⁺² in cacao beans. Some aerobic bacteria give way to Cd remediation, these microorganisms are allowed to be expelled by means of cocoa mucilage [53]. Application of microbial inoculants have significant importance in the reduction of Cd in cacao during the fermentation stage. Principal mechanisms behind Cd lowering by microorganisms are ‘Cd immobilization and sequestration’ [54]. Bacteria can interact with Cd by several mechanisms, such as biosorption, bioleaching, biotransformation, biodegradation, bioweathering, chemisorption, bioaccumulation etc. [54]. It is noteworthy that fermentation has a direct impact on the distribution and mobility of Cd in cacao. Cd exhibits outward migration from nib to mucilage and testa during fermentation [55]. The concentration of extractable Cd in cacao nibs is inversely proportional to the pH; Cd concentration increases with decreases in pH. Importantly, decreasing order of Cd in different parts of cacao fruit during fermentation is as follows: Testa > nib / placenta / pod husk > mucilage [56]. Thus, during the fermentation, the outward migration of Cd from nib to testa is against the concentration gradient. Nib Cd content is totally dependent on the nib pH acidification; nib Cd is decreased if there is a sufficient pH acidification during fermentation. At a pH 4.84 and day 6 fermentation, Pb and Ni were decreased by 60% and 50%, respectively in cacao beans [57]. However, the challenge is, too much acidification adversely affects the cacao flavor. Thus, the mitigation of Cd during cacao fermentation is an intractable problem. On the other hand, there are no concrete results on the lowering of Cd during or after cacao fermentation. Because, in some investigations, there was an enrichment of Cd after cacao fermentation was reported [58, 59]. Nevertheless, “Cacao fermentation and Cd mitigation” requires more future attention for the better and concrete understanding of fermentation’s role in Cd management [60].

Figure 5 shows the corresponding data of mg kg^–1 of Cd in the studied treatments and the rate of decrease of heavy metal i.e. ‘D’ values, in order to know the percentage of decrease between the cacaos of two provinces, resulting in the Cacao-ES having a higher rate of decrease compared to the Cacao-MN.

Conclusions

Physicochemical variables during the cacao fermentation were significantly affected by treatments with microbial inoculants and fruit extracts. There was an increase in pH (from 3.22 to 5.69) upon the application of microbial Biol EM-1 with apple extract. The maximum percent decrement (i.e. 71%) of variable °Brix was found in the application of Biol EM-1 with banana extract. In the variable dry weight of cocoa, difference was found in the application of Biol EM-1 and apple extract reaching a value of 906.33 g of bean weight on average. Likewise, the application of Biol EM-1 affects the moisture content, temperature and the cut test variables have a high valuation regarding the application of both Bioles.

The final Cd concentrations upon Albiobacth and EM-1 treatments were found to be 0.52–0.75 and 0.47–0.8 ppm respectively against 1.22–1.35 ppm of control. Geographically, Cd concentration in cacao beans is as follows: 1.4 ppm (South America), 0.5 ppm (East Africa and Central America), 0.3 ppm (Asia) and 0.09 ppm (West Africa) [22]. In the present investigation, treatment with EM-1 and apple extract, resulted in the decrease of 61–65% and 43–48% of Cd against control in the Cacao-MN and Cacao-ES, respectively. These results are significantly important in the mitigation of Cd during cacao fermentation. However, future studies are greatly warranted in the field of “Cacao fermentation–Cd mitigation” due to two main reasons – (i) Literature scarcity; (ii) Discrepancy in the results between the studies. Extent of trends in the Cd changes during fermentation and true effects of fermentation processing steps need to be more attention in the future studies.

Acknowledgements

LHVC, KEAV, FGIF are thankful to the Faculty of Postgraduate (Universidad Técnica de Manabí, Portoviejo, Ecuador) for encouragement throughout this study. Authors are thankful to Dr. Patricio Meza Mosquera for the support in the cacao bean collection.

Author contributions

LHVC: Supervision, methodology, original draft preparation, writing-review & editing, resources. KEAV: original draft preparation; writing-review & editing, resources. FGIF: writing methodology, reviewing and editing. JFVC: writing methodology, reviewing and editing. NRM: methodology, writing-reviewing and editing. RP: methodology, data curation, writing-reviewing and editing.

Data availability

Data is provided within the manuscript.

Declarations

Competing interests

The authors declare no competing interests.

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