Compositions of biocompounds derived from cacao and methods for the production thereof

Abstract

The present disclosure relates to compositions of biocompounds derived from cacao, mainly aromatic compounds such as flavanols and methylxanthines in the form of suspended particles, and to methods for producing the compositions from cacao beans, in particular unfermented cacao beans. The disclosed compositions have high concentrations of epicatechin, procyanidin B2 and theobromine and demonstrate high bioavailability in in vitro tests. These results suggest that the compositions developed are potentially useful for producing functional foods.

Description

[0001] COMPOSITIONS OF BIOCOMPOSITES DERIVED FROM COCOA AND METHODS FOR THEIR PRODUCTION

[0002] TECHNICAL FIELD

[0003] The present development is related to the field of food industry, particularly with biocomposite compositions derived from plant sources, and more particularly with biocomposite compositions derived from cocoa and methods for producing such compositions.

[0004] BACKGROUND

[0005] Cocoa is one of the most popular raw materials in the food industry around the world. This is mainly attributed to its high nutritional value and the organoleptic characteristics (color, flavor, aroma) of the products that can be obtained from it. Additionally, different studies have revealed the beneficial effects of its consumption on human health, such as a decreased risk of cardiovascular diseases, cancer prevention, and a reduction in cognitive decline (Fanning E., et al. 2023. Linking cocoa quality attributes to its origin using geographical indications. Food control; 151: 109825).

[0006] In addition to traditional products such as chocolate in its various forms, various researchers have proposed obtaining cocoa extracts that enhance the nutritional properties derived from it, especially those associated with cocoa polyphenols. An example of this is found in document US20070077318, which discloses a cocoa polyphenol extract, as well as a process for producing said extract. Said extract has a polyphenol content ranging from 10% to 30% by weight. The main polyphenol compounds present are monomeric flavonoids such as (+)-catechin and (-)-epicatechin, and oligomeric flavonoids such as proanthocyanidins, half of which are dimers. Other compounds that have been identified are quercetin and quercetin glycosides, quercetrin, luteolin, apigenin, and p-coumaric, caffeic, chlorogenic, and other acids.Additionally, the polyphenol extract disclosed in US20070077318 preferably contains less than 10% by weight of stabilizers, preferably less than 3% by weight.

[0007] Said document also discloses a process for obtaining cocoa extract, which comprises the following steps: a) subjecting unfermented cocoa beans to a bleaching step in water at a temperature in the range between 85-100 ° C for a period of time in the range between 3 to 15 minutes to give unfermented cocoa beans with reduced polyphenol oxidase activity; b) drying the unfermented cocoa beans with reduced polyphenol oxidase activity at a temperature below 85 ° C to obtain dry unfermented cocoa beans with a moisture content not exceeding 15%; c) subjecting the dried unfermented cocoa beans to a particle size reduction step to obtain an intermediate product of dried unfermented cocoa bean, where at least 99% by weight of the product has a particle size less than or equal to 300 pm;(d) extracting the polyphenols from the dried unfermented cocoa bean intermediate product to give a cocoa polyphenol extract and extracted solids; and (e) concentrating the cocoa polyphenol extract to produce a cocoa polyphenol concentrate wherein the concentration of polyphenols present is at least 10% by weight; and wherein the process further includes a defatting step that is carried out before step (d). In particular, in the process of US20070077318 different types of solvents may be used, preferably polar solvents such as water, ethanol or a mixture of both. Likewise, the extraction step may be repeated with the extracted solids three or four additional times depending on the desired extraction yield.

[0008] Document US9241500 discloses beverages made from macerated cocoa, wherein said beverages contain high concentrations of polyphenols or flavanols, particularly catechins and epicatechins. In some specific embodiments, the beverage disclosed in said document additionally comprises theobromine, caffeine, procyanidins, and / or gum arabic.Likewise, US9241500 discloses methods for preparing cocoa-derived beverages, which comprise the following steps: a) selecting a quantity of cocoa product in the form of one or more of cocoa bean extract, cocoa bean concentrate, cocoa powder, cocoa nibs or a cocoa seed composition, to be mixed with the desired amount of water; b) mixing the water and cocoa at a temperature above 154 ° C; c) adjusting the pH of the water or mixture with one or more of citric acid or ascorbic acid to separate insoluble particles from the mixture, and d) purifying or filtering the separated mixture, so that the mixture can be stored at room temperature for at least one month without separating. In some embodiments, the method of said document comprises an evaporation and spray drying step that allows obtaining a concentrate or solid product.

[0009] Finally, EP2170091 discloses cocoa extracts with a reduced particle size and refers to products such as foods, dietary supplements, and pharmaceutical compounds containing cocoa extracts with reduced particle sizes. Specifically, the extracts in said document contain cocoa-derived polyphenols, such as flavanols (such as catechin and epicatechin) and procyanidins, where said procyanidins range from 50 mg / g to 80 mg / g. In addition to the above, in particular embodiments, the beverage of EP2170091 comprises polyphenols such as sorbitol, mannitol, xylitol, maltitol, isomalt, lactitol, among others; as well as thickening agents such as polydextrose, cellulose and its derivatives, maltodextrin, gum arabic, among others. Additionally, the particle size in the cocoa extract is less than 15 pm, preferably less than 10 pm.

[0010] Likewise, EP2170091 discloses a process for obtaining cocoa extracts from unfermented cocoa beans with an initial moisture content of approximately 7% to 8% by weight. In this process, the beans are cleaned in a scalper and subsequently in an air-fluidized bed density separator. The cleaned beans are treated in an infrared heating apparatus. The depth of the beans in the vibrating bed of the apparatus is approximately 2-3 beans deep. The surface temperature of the apparatus is set at approximately 165 °C, thereby producing an internal bean temperature (IBT) of approximately 135 °C in a time ranging from 1 to 1.5 minutes. This treatment allows the cocoa pods and nibs to be rapidly separated. Subsequently, the pieces separated by the vibrating screen are reintroduced into the product stream before the winnowing stage.The resulting beans after micronization should have a moisture content of approximately 39% by weight. At this point, the beans are heated to approximately 135°C (275°F) and immediately cooled to approximately 90°C (190°F) in approximately 3 minutes to minimize further moisture loss. The beans are winnowed to break them up, loosen the pods, and separate the lighter pods from the nibs; while at the same time minimizing the amount of nib lost in the pod waste stream. The resulting cocoa nibs are then pressed with a thyme to extract the cocoa butter from the cocoa solids.

[0011] Finally, the cocoa solids are placed in contact at room temperature for 0.5 to 3.5 hours with an aqueous organic solvent comprising approximately 75% ethanol / 25% water (v / v) or approximately 80% acetone / 20% water (v / v). The micelle is separated from the cocoa residue and concentrated by evaporation to a total solids content of 30 to 50%. The concentrated extract is subsequently spray-dried.

[0012] Although compositions based on cocoa extracts have been published in the state of the art, there is still a need to generate versatile alternatives that allow us to take advantage of the nutritional potential of cocoa, as well as to develop processes that allow the production of such compositions.

[0013] BRIEF DESCRIPTION

[0014] The present development is directed to a composition of biocompounds derived from cocoa, comprising flavanols, in a total concentration between 150 and 800 mg / g; and methylxanthines, in a total concentration between 25 and 140 mg / g; wherein the flavanols comprise (-)-epicatechin between 25 and 200 mg / g and procyanidin B2 between 25 and 110 mg / g; wherein the methylxanthines are selected from the group consisting of caffeine, theobromine or combinations thereof; and wherein the biocompounds are in the form of suspended particles.

[0015] Likewise, the present development covers a process for producing a composition of biocomposites derived from cocoa, comprising: a) cold extracting biocomposites from unfermented cocoa beans, by means of high intensity dynamic flow ultrasound at a temperature between 10 ° C and 45 ° C, a frequency of 18 to 40 KHz and an amplitude between 25 dpi and 100 dpi, until obtaining a cocoa extract; and b) formulating a colloidal system from the mixture of the cocoa extract from step a) and a stabilizing agent in a concentration between 0.05% and 40% by high intensity continuous flow ultrasound and evaporation, until obtaining a liquid composition of biocomposites in the form of suspended microparticles.

[0016] BRIEF DESCRIPTION OF THE FIGURES

[0017] FIG. 1 Structural formulas of (-)-epicatechin (A), (+)-catechin (B) and procyanidin B2 (C).

[0018] FIG. 2 Particle size distribution analysis of purple cocoa extract in powder formulation carried in mannitol (A) and in liquid formulation (B).

[0019] FIG. 3 Release and dissolution profile of cocoa biocompounds in the developed extracts and in the starting raw material.

[0020] FIG. 4 Bioavailability of bioactive compounds present in the cocoa extract from Caco-2 cells.

[0021] FIG. 5 Antioxidant capacity of cocoa-coffee blends evaluated using the DPPH (a) and ORAC (b) methods. Values ​​1, 2, and 3 correspond to the average of the three analyses performed.

[0022] FIG. 6 Inhibitory concentration (IC50) of DPPH of cocoa and coffee mixtures.

[0023] FIG. 7 Combination index (CI) of cocoa and coffee blends. CI values ​​determine the type of interaction between two antioxidants as follows: Values ​​from 0.3 to 0.7 = synergistic interaction; 0.7 to 0.85 moderate synergistic interaction; 0.85 to 0.90 = slightly synergistic interaction; 0.90 to 1.10 = almost additive interaction; 1.10 to 1.20 = slightly antagonistic interaction; 1.20 to 1.45 = moderately antagonistic interaction; 1.45 to 3.3 = antagonistic interaction.

[0024] DETAILED DESCRIPTION

[0025] For the purposes of interpreting the terms used throughout this document, their usual meaning in the technical field should be taken into account, unless a specific definition is incorporated or the context clearly indicates otherwise. Additionally, terms used in the singular form also include the plural form.

[0026] Composition of biocomposites derived from cocoa

[0027] In a first aspect, the development described here corresponds to a composition of biocomposites derived from cocoa, wherein said biocomposites are in the form of suspended particles. In particular embodiments, the disclosed composition comprises flavanols and methylxanthines.

[0028] In some embodiments, the concentration of flavanols in the cocoa-derived biocomposite composition is between 150 and 800 mg / g of composition. In particular embodiments, the concentration of flavanols in the composition is 150 to 700 mg / g, 175 to 700 mg / g, 175 to 600 mg / g, 200 to 600 mg / g, 200 to 400 mg / g, 225 to 400 mg / g, or 225 to 300 mg / g.

[0029] In some embodiments, the flavanols present in the composition are selected from, but are not limited to, catechin, epicatechin, procyanidin, or combinations thereof. In particular embodiments, the epicatechin present in the composition corresponds to (-)-epicatechin. In other particular embodiments, the procyanidin present in the composition corresponds to procyanidin B2.

[0030] In particular embodiments, the concentration of (-)-epicatechin in the composition is between 25 and 200 mg / g of composition. In more particular embodiments, the concentration of (-)-epicatechin in the composition is between 40 and 200 mg / g, 40 and 180 mg / g, 60 and 180 mg / g, 60 and 160 mg / g, 80 and 160 mg / g, 80 and 140 mg / g, 100 and 140 mg / g, or 120 and 140 mg / g.

[0031] In some embodiments, the composition described herein has a procyanidin B2 concentration between 25 and 110 mg / g of composition. In particular embodiments, the procyanidin B2 concentration is in a range of 40 to 110 mg / g, 40 to 100 mg / g, 50 to 100 mg / g, 50 to 90 mg / g, 60 to 90 mg / g, or 70 to 90 mg / g. In some embodiments, the methylxanthines in the cocoa-derived biocomposite composition are present in concentrations between 25 and 140 mg / g of composition. In particular embodiments, the concentration of methylxanthines in the composition is between 40 and 140 mg / g, 40 and 130 mg / g, 50 and 130 mg / g, 50 and 120 mg / g, 60 and 120 mg / g, 70 and 120 mg / g, or 70 and 110 mg / g.

[0032] In particular embodiments, methylxanthines are selected from, but not limited to, caffeine, theobromine, or combinations thereof.

[0033] In a particular embodiment of the invention, the particles of the composition are in sizes between 100 nm and 8,000 nm; 200 nm and 8,000 nm, 200 nm and 6,000 nm, 200 nm and 4,000 nm, 400 nm and 8,000 nm, 400 nm and 6,000 nm, 200 nm and 2,000 nm, or 100 nm to 1,000 nm.

[0034] In another particular embodiment, the composition of biocomposites derived from cocoa is a liquid or powder composition.

[0035] In some embodiments, the cocoa-derived biocomposite composition further comprises a stabilizing agent. For the purposes of this disclosure, the term "stabilizing agent" refers to a substance or mixture of substances that prevents the particles in the suspension from agglomerating. In particular embodiments, the stabilizing agent used in the composition described herein includes, but is not limited to, mannitol, maltodextrin, cocoa, natural gums, natural saponins, or a combination thereof.

[0036] In some embodiments, the cocoa-derived biocomposite composition further comprises a dispersion vehicle. A "dispersion vehicle" is any compound or mixture thereof that promotes the uniform distribution of the biocomposite particles in the composition. In particular embodiments, the dispersion vehicle is selected from the group comprising propylene glycol, glycerin, vegetable oils, water, fractionated oils, or a combination thereof. Method for producing the cocoa-derived biocomposite composition

[0037] In a second aspect, the present development relates to a process for obtaining a composition of biocomposites derived from cocoa, comprising: a) cold-extracting biocomposites from cocoa beans until obtaining a cocoa extract; and b) formulating a colloidal system from said cocoa extract until obtaining a liquid biocomposite composition, wherein said biocomposites are in the form of suspended particles. In particular embodiments, the cocoa beans used in the process correspond to unfermented cocoa beans.

[0038] In some embodiments, the extraction of the biocompounds in step a) of the process described herein is carried out by ultrasound. In particular embodiments, said ultrasonic extraction corresponds to high dynamic flux density ultrasound extraction. In more particular embodiments, the high dynamic flux density ultrasound extraction is carried out at temperatures between 10 and 45 ° C, 15 and 45 ° C, 10 and 40 ° C, 15 to 40 ° C, 15 to 35 ° C or 20 and 35 ° C. In other particular embodiments, the high dynamic flux density ultrasound extraction is carried out at a frequency of 18 to 40 KHz, 20 to 40 KHz, 18 to 35 KHz, 20 to 38 KHz, 22 to 38 KHz, 22 to 36 KHz, 24 to 36 KHz or 24 to 34 KHz. In other particular embodiments, high dynamic flux density ultrasound extraction is performed with amplitudes between 25 and 100 dpi, 20 to 90 dpi, 25 to 90 dpi, 25 to 80 dpi, 30 to 80 dpi, 30 to 70 dpi, 35 to 70 dpi or 40 to 70 dpi.

[0039] In some embodiments, in step a) of the disclosed process, the cocoa beans are present in a solvent, such as an ethanol / water solution. In particular embodiments, the ethanol is present at a concentration between 30 and 100% v / v, 35 and 100% v / v, 35 and 90% v / v, 40 and 90% v / v, 45 and 90% v / v, 45 and 80% v / v, or 50 to 80% v / v. In other particular embodiments, the solid-to-liquid ratio of the cocoa beans and the solvent is in a range between 5 and 20%, 5 and 18%, 7 and 18%, 7 and 16%, 9 and 16%, 9 and 14%, or 11 and 14%.

[0040] In some embodiments, step a) of the production process is performed 2 to 6 times. In some embodiments, the liquid composition obtained in step b) has a solids content between 10 and 60%, 10 to 50%, 15 to 50%, 15 to 45%, 20 to 45%, or 25 to 45%.

[0041] In some embodiments, the liquid composition obtained in step b) of the process described herein is dried until a powder composition is obtained. In particular embodiments, the drying is carried out by freeze-drying or spray-drying. In other particular embodiments, the drying is carried out at a temperature between 50 and 200 °C, 50 to 180 °C, 60 to 180 °C, 60 to 160 °C, 80 to 160 °C, 80 to 140 °C or 100 to 140 °C.

[0042] EXAMPLES

[0043] The technological development is presented in detail through the following examples, which are provided solely for illustrative purposes and not intended to limit its scope.

[0044] Example 1. Preparation of compositions from biocomposites derived from cocoa

[0045] Extraction is a fundamental process for obtaining biocompounds derived from cocoa and constitutes the starting point for the subsequent formulation of ingredients that can be incorporated into food products and supplements.

[0046] A cold extraction model was developed using cavitation through high-intensity ultrasound that allows the concentration of biocompounds such as flavanols and methylxanthines from unfermented cocoa beans, which are subsequently formulated using different stabilizers and drying methods that will allow the production of powdered and liquid ingredients suitable for use in different food and supplement formulations.

[0047] To this end, two experimental runs were conducted. In the first run, 12 experiments were performed, evaluating the impact of ultrasound amplitude and the plant material load used in an extraction cycle. In the second run, seven ingredient formulations were evaluated using different stabilizers, vehicles, and drying methods.

[0048] Each run of the first experiment was carried out with 2 kg of solvent in a dynamic flow ultrasound at 20 Khz frequency, with a cooling jacket to maintain the system between 5 °C and 35 °C, the extraction process was carried out for 5 minutes at the amplitude described in Table 1. After the time, each sample was filtered, and the percentage of total non-volatile soluble solids in the filtered extract was measured using a moisture balance.

[0049] Table 1. Effect of amplitude and plant material load on extracted solids

[0050] Solids extracted % of material loading

[0051] Experiment Amplitude obtained total vegetable ethanol (%)

[0052] (% w / w)

[0053] 1 70 90 8 2.01

[0054] 2 70 80 8 1.98

[0055] 3 70 60 8 1.95

[0056] 4 70 50 8 1.92

[0057] 5 70 96 10 2.50

[0058] 6 70 80 10 2.43

[0059] 7 70 60 10 2.41

[0060] 8 70 50 10 2.38

[0061] 9 70 90 12 3.01

[0062] 10 70 80 12 2.97

[0063] 11 70 60 12 2.83

[0064] 12 70 50 12 2.75

[0065] As can be seen in the table above, the highest amount of total extracted compounds from the plant material was obtained when 12% solids were used and amplitudes greater than 80 dpi, indicating that the highest process yields are achieved in this range. However, the results with 8% plant material and lower amplitudes achieve significant extractability for the required process.

[0066] For the second run of experiments, 12% solids, 80 dpi amplitude, and 70% ethanol were used as the basis for each extraction cycle. Each experiment started with a 10-kg batch, employing between 1 and 4 extraction cycles for a percentage of plant material used between 12% and 48% relative to the batch size. The ultrasound conditions were the same as those of the previous experiment. The bioactive compounds of interest were analyzed for each run: theobromine, caffeine, catechin, procyanidin P2, and epicatechin using HPLC-DAD (Figure 1).

[0067] Table 2. Formulation of extracts in solid (powder) and liquid form

[0068] Experimental run #1 was carried out with process control, which did not include the incorporation of stabilizers. Only one extraction cycle was performed, and the drying process was carried out by lyophilization (-50°C). Under these conditions, the greatest stability of the formula was guaranteed by using a process that allowed for greater conservation of the biocompounds, due to having the lowest degree of processing and the least exposure to temperature and energy. However, it was the experiment with the lowest yield and highest manufacturing cost. When comparing the results obtained in products dried by spray dryer, formulated with mannitol and cocoa, it was found that it is possible to obtain a composition similar to the lyophilized product, increasing yields and productivity.In the case of liquid ingredients, these did not achieve the biocomposite composition obtained in powder formulations; however, performance improved significantly, opening up different fields of application.

[0069] Additionally, with the powder formulation conveyed in mannitol and the liquid formulation, a particle size distribution of the cocoa biocompounds below 16 microns was obtained (Table 3 and Figure 2), allowing their easy incorporation into liquid, semi-solid, fatty matrices and powder products.

[0070] Table 3. Results of the particle size distribution analysis of the purple cocoa extract.

[0071] " For r meter / Formulation z Powder formulation „ . . ,

[0072] (vehicuhzad ,o in m 1 ani .t.ol) Liquid formulation

[0073] Concentration (%) 0.0106 0.0115

[0074] Width 4,385 7,001

[0075] Uniformity 1,455 1,983

[0076] Specific Surface Area z

[0077] (m O 2 / nkg \)

[0078] D [3.2] (pm) 2.67 1.81

[0079] D [4.3] (pm) 6.53 4.96

[0080] Dv 10 (pm) 1.28 0.953

[0081] Dv 50 (pm) 3.19 1.06

[0082] Dv 90 (pm) 15.3 14.0 Considering that the average particle size obtained from both the powder and liquid extract was between 1.5 and 3.2 pm, these formulations were evaluated to determine if there were improvements in the release and dissolution profile of the biocompounds compared to the base raw material, ground and defatted unfermented cocoa beans. For this, a dissolution test was performed using the conditions described by the USP with a type II dissolver at 75 rpm, 37.0 ± 0.5 ° C, using 1000 mg of sample and 1000 mL of dissolution medium with 0.1 M HCl. As a control, the starting raw material used was 1) unfermented, dried and ground cocoa beans, 2) unfermented, dried, defatted and ground cocoa beans, 3) the powder extract formulated in 10% mannitol, and 4) the liquid extract. To perform the dissolution profile, 1 mL aliquots were taken at 1, 5, 10, 15, 20, 25, 30, 45, and 60 min.In response, the absorbance of the aliquot was monitored at 280 nm as an indirect indicator of the total biocompound content, since both flavanols and methylxanthines have their maximum absorption at this wavelength.

[0083] As observed in Figure 3, the formulations significantly improved both the release rate and the amount of bioactive components released into the tested medium. Both the mannitol-vehicled powder formulation and the liquid formulation achieved a release above 95% in less than 5 minutes, while the starting raw materials reached maximum release close to 60 minutes with a percentage below 90%. This effect is mainly attributed to the particle size obtained for the extracts.

[0084] Example 2. Formulation of cocoa extract in powdered beverages and evaluation of flavanol content

[0085] The most popular way to consume cocoa is as chocolate and confectionery. However, in many countries, one of the main ways to consume cocoa is through soluble powder preparations containing cocoa (known as cocoas) that can be dissolved in water or milk. The objective of this study was to apply cocoa extract with a high flavanol content in cocoa-based powdered beverages. These beverages can contain the extract alone or in a blend with other natural ingredients such as seeds, cereals, fruits, mushrooms, natural extracts, vitamins, minerals, and others, which can be used to make nutritional claims.

[0086] For these trials, the extract was applied at a concentration of 5-30% in the formulations, which were a powdered mixture for preparing cocoa drinks with cocoa extract containing flavanols and other natural ingredients such as seeds, cereals, fruits, mushrooms, natural extracts, vitamins, minerals, among others.

[0087] For preparation, 8 g of powdered product were added to 200 mL of low-fat milk, aiming for a serving of product (1 cup per day for adults) to have at least a concentration of 100-300 mg of total flavanols. The flavanol content in the powdered and prepared products was evaluated in milk and water; and at room temperature and hot (90 °C) following the method proposed by Tsanova-Savova and collaborators (Tsanova-Savova S., et al. 2005. (+)-Catechin and (-) -epicatechin in Bulgarian fruits. J. Food Compos. Anal.; 18: 691-698.) with some modifications. HPLC analyses were performed on an Agilent 1260 high-performance liquid chromatograph, with an automatic autosampler and fluorescence and diode array detectors (FLD and DAD). 0.5 g of the solid and liquid samples were extracted with 10 mL of a 60:40 isopropanol / water solution pH 9.0 and subsequently subjected to an ultrasonic bath at room temperature for 60 min.

[0088] These samples were subsequently vortexed for 1 minute and allowed to stand at -20 °C for 60 minutes. These samples were then centrifuged at 4,000 rpm for 8 minutes. One milliliter of the resulting supernatant was filtered through a 0.45 pm acrodisc. An aliquot of this filtrate was mixed with the mobile phase (water with 0.1% acetic acid), with a dilution factor (DF) depending on the color of the solution (dark samples: DF = 5 or 10, less dark samples: DF = 4). This dilution was made in an amber chromatography vial for injection into the HPLC / DAD / FLD. Detection was performed at 280 nm. The intensity of the acquired signals was amplified, and these were subsequently stored and processed using the OpenLab CDS Chemstation Edition software.For the quantification of flavanols and xanthines in the samples, external standardization (a standard mixture of the four compounds) was applied, allowing the determination of the exact amount of analytes per gram of sample, applying linear calibration between values ​​of 0.5-20 ppm for catechin; 5-200 ppm for epicatechin; 10-275 ppm for theobromine; 5-125 ppm for caffeine. Analyses were performed in triplicate for each sample and reported in dry cocoa solids.

[0089] The cocoa flavanols in the extract applied to these beverages were primarily absorbable monomers such as (-)-epicatechin, (+)-catechin, and procyanidin B2. These flavanols also had a significant polymer content, as shown in Table 4, which are present in significant proportions in the beverage composition.

[0090] Table 4. Degree of polymerization of flavanols in a portion (8 g) of powdered product

[0091] Flavanols and procyanidins mg / serving

[0092] DPI Monomers 86.4

[0093] DP2 Dimers 78.96

[0094] DP3 Trimers 45.28

[0095] DP4 51,04 tetramers

[0096] DP5 Pentamers 42,48

[0097] DP6 Hexamers 32,48

[0098] DP7 heptamers 22.88

[0099] DP8 Octamers 15.84

[0100] Nonamers DP9 9.92

[0101] Decamers DP10 4.64

[0102] Total flavanols and procyanidins 392

[0103] The flavanol content, especially the monomers and procyanidin B2 in the beverages prepared in water or milk, cold (25-30 °C) or hot (90-95 °C) presented a content between 200-300 mg as shown in Table 5. It was evident that the impact of the temperature of the water or milk in the preparation of cocoa does not significantly affect the final concentration of flavanols in the prepared beverage. Table 5. Flavanol content in prepared product

[0104] Flavanols

[0105] (+)-Catechin (-)-Epicatechin Procyanidin Sample to

[0106] Cocoa in cold water 0.15 ± 0.01 0.56 ± 0.06 0.69 ± 0.09 289 ± 8.96

[0107] Cocoa in milk

[0108] 0.12 ± 0.002 1.00 ± 0.02 0.41 ± 0.03 321 ± 5.97 cold

[0109] Cocoa in water

[0110] 0.14 ± 0.004 0.58 ± 0.02 0.53 ± 0.01 259 ± 7.28 hot

[0111] Cocoa in milk

[0112] 0.13 ± 0.001 0.55 ± 0.008 0.42 ± 0.02 228 ±6.35 hot

[0113] Example 3. Sensory, microbiological and physicochemical stability analysis of cocoa extracts in powdered beverages

[0114] The sensory, microbiological and physicochemical stability of cocoas packed in a PET / PET met / PE material was evaluated in 240 g portions under accelerated temperature conditions (30 °C) and relative humidity (75%) in a climatic chamber according to the parameters established by the Quality area of ​​Compañía Nacional de Chocolates SAS.

[0115] According to the results obtained during the stability analysis, the product maintains its quality characteristics (Aw, moisture content, and microbial counts) for 12 months under appropriate storage conditions (Table 6). Furthermore, the concentrations of biocompounds present in the product also remain stable over time (Table 7). Table 6. Physicochemical and microbiological analysis of product stability

[0116] Physicochemical Time

[0117] - Sensory storage counts

[0118] Aw %Humidity (CFU / mL)

[0119] (months)

[0120] Molds and <10 yeasts

[0121] 0 0.290 2.823 Complies

[0122] Salmonella Absence

[0123] Colifornics / i'. coli <10 / <10

[0124] 6 0.360 2.969 Molds and yeasts <10 Compliant

[0125] 12 0.371 3.499 Molds and yeasts <10 Compliant

[0126] Table 7. Analysis of the flavanol content of the product during storage

[0127] Procyanidin Time

[0128] Theobromine Caffeine (+)-Catechin (-)-Epicatechin storage Bl and B2

[0129] (mg / g) (mg / g) (mg / g) (mg / g)

[0130] (months) (mg / g)

[0131] 13.61 ±

[0132] 0 4.42 ± 0.000 3.05 ± 0.021 5.24 ± 0.113 10.24 ± 0.197

[0133] 0.148

[0134] 13.81 ±

[0135] 6 4, 58 ± 0.417 2.84 ± 0.247 5.28 ± 0.466 10.05 ± 1.103

[0136] 1,294

[0137] 12.98 ±

[0138] 12 4.44 ± 0.091 3.02 ± 0.035 5.57 ± 0.120 10.94 ± 0.169

[0139] 0.318

[0140] Example 4. In vitro bioavailability analysis of cocoa extract

[0141] To determine the bioavailability of the phenolic compounds present in the cocoa extract, an in vitro model of the intestinal barrier developed with a human colorectal carcinoma cell line (Caco-2, ATCC®HTB37) was used. These cells were cultured at 37 °C in a humidified atmosphere with 5% CO2 in EMEM medium supplemented with 1% (v / v) fetal bovine serum and 2 mM L-glutamine. The cells used in the assays correspond to passage number 15.

[0142] For the generation of the intestinal barrier, Caco-2 cells were seeded at a density of 3 x 10 5 cells / well in 300 pL of culture medium in a 12-well, semi-permeable Transwell® Advanced Screening System (HTS) (0.4 pm pore diameter polyester membranes - apical chamber) (Costar, Coming, Birmingham, UK). In the lower compartment (basolateral chamber), 600 pL of culture medium was added.

[0143] Once confluent, the seeded Caco-2 cells were allowed to differentiate for 21 days prior to experiments, replacing the culture medium every 48–72 hours. The integrity of the cell monolayer was assessed by determining the transepithelial electrical resistance (TEER) using a commercial measurement system (Millicell ERS; Millipore Co., Bedford, MA) that includes Ag-AgCl electrodes. Final values ​​were expressed in Q / cm 2 were calculated according to the following equation:

[0144] TEER = (R - Rb) xA

[0145] Where R is the electrical resistance of the filter inserted with the cell layer, Rb is the resistance of the filter alone, and A is the growth area of ​​the filter in cm 2 . Only those inserts that presented a TEER value greater than 600 / cm 2 were used in the bioavailability experiments.

[0146] To perform the bioavailability experiment, 20 pL of the analytical extracts were added to 300 pL of fresh culture medium in the apical chamber. Transport from the apical chamber to the basolateral chamber was assessed after 120 min of incubation at 37 °C in a humidified atmosphere with 5% CO r. Subsequently, the culture medium from the apical and basolateral chambers was collected and dried using a SpeedVac concentrator. The dried residue was suspended in methanol, filtered through 0.45 pm microfilters, and stored at -20 °C until chromatographic analysis. Phenolic transport was assessed by collecting the culture medium from the apical and basolateral chambers, which was subsequently quantified to determine its (+)-catechin, (-)-epicatechin, procyanidin B2, and chlorogenic acid contents by HPLC-DAD-ESI-MS / MS analysis.The results of absorption efficiency were expressed as a percentage of the compounds available for absorption in the apical chamber, according to the following equation:

[0147] Absorption efficiency (%) = (Concentration in the culture medium in the basolateral chamber at 120 min / Concentration in the culture medium in the apical chamber at 0 min) x 100

[0148] As observed in Figure 4, the in vitro model revealed that (+)-catechin, (-)-epicatechin and procyanidin B2 of the extract have a high bioavailability, with absorption values ​​equivalent to 87.9%, 97.4% and 67.1%, respectively.

[0149] Example 5. Evaluation of the antioxidant capacity of cocoa extract in a mixture with a coffee extract

[0150] The presence of natural compounds in functional foods can generate various beneficial effects for consumers. For example, thanks to their antioxidant properties, the phenolic compounds in cocoa extract promote cellular protection against oxidative stress generated by free radicals, which reduces the risk of cardiovascular disease and atherosclerosis (Martínez F. et al. 2020. Use of standardized units for a correct interpretation of IC50 values ​​obtained from the inhibition of the DPPH radical by natural antioxidants. Chemical Papers; 74: 3325–3334).

[0151] To determine the antioxidant potential of the cocoa extracts obtained, mixtures of powdered cocoa and coffee extracts were made with the proportions described in Table 8. Table 8. Mixtures of cocoa and coffee extracts

[0152] Cocoa Mix (%) Coffee (%)

[0153] MI 0 100

[0154] M2 25 75

[0155] M3 50 50

[0156] M4 75 25

[0157] M5 100 0

[0158] Samples of the mixtures (10 mg) were diluted in 10 mL of deionized water, vortexed, and centrifuged at 10,500 rpm for 5 minutes at room temperature. The supernatants were filtered through a 0.45 pm filter and subsequently diluted for further assays.

[0159] To determine the radical scavenging capacity of the samples, assays were carried out with 2,2-diphenyl-l-picrylhydrazyl (DPPH) according to the methodology described by Migues et al. (Migues I., et al. 2018. Phenolic profiling and antioxidant capacity of Eugenia uniflora L. (Pitanga) samples collected in different Uruguayan locations. Foods; 7(5): 67-78) and with peroxide radical according to Paulsen et al. (Paulsen E., et al. 2018. Postharvest biology and technology effect of temperature on glucosinolate content and shelf life of ready-to-eat broccoli florets packaged in passive modified atmosphere. Postharvest Biol. Technol.; 138: 125-133). Standard curves were generated using 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and the results were expressed as millimolar concentration of Trolox equivalents per gram of dry weight (mM TE / g dw) using the equations described in Table 9.

[0160] Table 9. Equations for calculating antioxidant capacity based on DPPH Indicator Equation Reference

[0161] Jiménez M., et al. 2012. Optimization of the DPPH method to evaluate %I %I = (ABS mezcia / ABSDPPH) x 100 antioxidant activity of coffee brew. Annals of Veterinary Medicine of Murcia; 28: 67-78.

[0162] Sabahannur S., et al. 2018. Changes in phenol level and antioxidant IC50 IC50 = ((DPPH (mMTE / g dw) x 50) / %I) activity of cocoa beans during fermentation and roasting. Journal of Food Research; 7(4): 23-29.

[0163] Muhammad DRA, et al. 2017. Interaction between natural

[0164] CI = ((IC50 of the mixture x portion of cocoa in the mixture) / IC50 of cinnamon and cocoa in the mixture) + (IC50 of the mixture x portion of coffee in the mixture) / IC50 of coffee)) in binary and complex mixtures. Food

[0165] Chemistry; 231: 356-364.

[0166] Surprisingly, the radical scavenging activity (AC) of the blends increased as a function of the cocoa proportion, indicating a higher antioxidant activity of the cocoa phenolic compounds (Table 10 and Figure 5). Furthermore, a comparison of the results obtained with sample M5 and samples M2-M4 reveals that the addition of cocoa extract, even in a lower proportion than coffee (25:75), increases the AC value of the blend and suggests a greater potential of cocoa as an antioxidant source. This latter attribute is especially important in ingredients intended to be used as sources of bioactive compounds in the design of functional foods, since the high scavenging activity of cocoa compounds would allow obtaining functional effects similar to those of coffee, but at lower doses. Table 10. Antioxidant activity based on DPPH and ORAC (mM TE / g dw) and interaction of the extracts present in the blends.

[0167] DPPH Mixture %I ICso CI Interaction Type ORAC

[0168] 1,109 ± 4,114 ± 15,030 ± 4,469 1,699 ± 6,532 ±

[0169] MY

[0170] 0.243 to 1.776 aa 0.505 to 0.309 a

[0171] 1,513 ± 6,477 ± 12,018 ± 1,237 ± Moderately 7,225 ±

[0172] M2

[0173] 0.173 b 1.533 b 1.739b 0.179 b antagonistic 0.663 ab

[0174] 1,743 ± 7,944 ± 11,235 ± 1,477 1,043 ± 7,771 ±

[0175] M3 Almost Additive

[0176] 0.194 be 1.747 be be 0.137 be 0.644 be

[0177] 1.939 ± 9.059 ± 10.802 ± 1.108 0.894 ± Slightly 8.312 ±

[0178] M4

[0179] 0.135 of 1.109 of bed 0.092 cd synergistic 0.574 cd

[0180] 2,428 ± 12,461 ± 9,976 ± 1,341 0,725 ± 9,603 ±

[0181] M5

[0182] 0.358 e 3.203 e ede 0.097 e 0.542 e

[0183] The values ​​correspond to the mean and standard deviation of the three analyses. Means with letters in the same column indicate statistically significant differences (p <0.05). %I, percentage of inhibition of the DPPH radical; ICso, half-maximal inhibitory concentration, which indicates the amount of biocompounds that can inhibit 50% of the DPPH radical; CI, combination index to determine the type of interaction between the extracts in the mixtures.

[0184] In addition, the IC50 values ​​of the M1 and M5 mixtures appear to indicate that a lower amount of cocoa extract in the mixture would allow achieving 50% inhibition of the radical, while a five-fold higher amount of coffee extract would be necessary to obtain similar results. Additionally, it is important to highlight that no statistically significant differences (p>0.05) were found between the mixtures, which presented a greater inhibitory capacity than the coffee extract (Figure 6). Indeed, the IC50 values ​​of the mixtures were lower the higher the proportion of cocoa in them.

[0185] Regarding the interaction between the phenolic compounds of cocoa and coffee extracts, based on the ranges established by Rahadian and collaborators (Rahadian, D., et al. 2017. Interaction between natural antioxidants derived from cinnamon and cocoa in binary and complex mixtures. Food Chemistry; 231: 356-364), it is possible to suggest that, the higher the proportion of cocoa extract in the mixture, the interaction tends to be synergistic, while the higher the proportion of coffee, the interaction tends to be antagonistic (Figure 7).

Claims

CLAIMS 1. A cocoa biocompound composition, comprising: flavanols, in a total concentration between 150 and 800 mg / g; and methylxanthines, in a total concentration between 25 and 140 mg / g; wherein the flavanols comprise (-)-epicatechin between 25 and 200 mg / g, and procyanidin B2 between 25 and 110 mg / g; wherein the methylxanthines are selected from the group consisting of caffeine, theobromine or combinations thereof; and wherein the biocompounds are in the form of suspended particles.

2. The composition of biocomposites derived from cocoa according to Claim 1, wherein the particles of the composition have a size between 100 nm and 8,000 nm.

3. The composition of biocomposites derived from cocoa according to Claim 1, wherein the composition is a liquid or powder composition.

4. The composition of biocomposites derived from cocoa according to Claim 1, wherein the composition further comprises a stabilizing agent.

5. The composition of biocomposites derived from cocoa according to Claim 4, wherein the stabilizing agent is selected from the group comprising mannitol, maltodextrin, cocoas, natural gums and natural saponins.

6. The composition of biocomposites derived from cocoa according to Claim 3, wherein the liquid composition further comprises a dispersion vehicle.

7. The composition of cocoa-derived biocomposites according to Claim 6, wherein the dispersion vehicle is selected from the group comprising propylene glycol, glycerin, vegetable oils, water, fractionated oils or a combination thereof.

8. A process for producing a cocoa-derived biocomposite composition, comprising: a) cold-extracting biocomposites from unfermented cocoa beans by means of high-intensity dynamic flow ultrasound at a temperature between 10 ° C and 45 ° C, a frequency of 18 to 40 KHz and an amplitude between 25 dpi and 100 dpi, until a cocoa extract is obtained; and b) formulating a colloidal system from the cocoa extract from step a) and a stabilizing agent at a concentration between 0.05% and 40% by continuous flow high-intensity ultrasound and evaporation, until a liquid biocomposite composition in the form of suspended particles is obtained; wherein step a) is carried out between 2 to 6 times; and wherein the composition obtained in step b) has a solids content between 10% and 60%.

9. The process according to Claim 8, wherein in step a) an ethanol / water solution between 30% and 100% ethanol is used as solvent, and a solid-liquid ratio between 5% and 20%.

10. The process according to Claim 8, wherein the liquid composition of step b) is subjected to spray drying or lyophilization until a powder composition is obtained.

11. The process according to Claim 10, wherein the drying step is carried out at a temperature between 50 °C and 200 °C.

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