COCOA PRODUCT AND METHOD FOR MAKING THEREOF
20230404103 ยท 2023-12-21
Inventors
- Federico Mora (Morges, CH)
- ELSA VILANOVA (Lausanne, CH)
- MARINA RESINK-ROMBOUTS (Blonay, CH)
- Luigi Poisson (Courgevaux, CH)
- Stefan Spreng (Orbe, CH)
- Celine Sarrazin-Horisberger (Suchy, CH)
- SATOSHI OKAMURA (Lausanne, CH)
Cpc classification
A23G1/0009
HUMAN NECESSITIES
A23G1/002
HUMAN NECESSITIES
A23G1/0016
HUMAN NECESSITIES
A23G1/32
HUMAN NECESSITIES
A23G1/56
HUMAN NECESSITIES
International classification
A23G1/00
HUMAN NECESSITIES
A23G1/56
HUMAN NECESSITIES
A23G1/32
HUMAN NECESSITIES
Abstract
The present invention relates to a cocoa product containing elevated amounts of antioxidant molecules. Further aspects of the invention relate to the use of this product for making a beverage and into a capsule and to the process of making this product.
Claims
1. Cocoa product comprising a ratio of epicatechin in % by weight to theobromine in % by weight of at least 0.2.
2. A cocoa product according to claim 1 comprising between 0.8% to 4% by weight of epicatechin.
3. A cocoa product according to claim 1 comprising a ratio of procyanidin C1 in % by weight to theobromine in % by weight of at least 0.005.
4. A cocoa product according to claim 1 comprising between 0.01% to 1% by weight of procyanidin C1.
5. A cocoa product according to claim 1 comprising a ratio of procyanidin B2 in % by weight to theobromine in % by weight of at least 0.04.
6. A cocoa product according to claim 1 comprising between 0.15% to 1% by weight of procyanidin B2.
7. A cocoa product according to claim 1 comprising at least 145 g/kg alkyl and phenyl alkenals.
8. A cocoa product according to claim 1 comprising at least 10 g/kg of 2-isopropyl-5-methyl-2-hexenal and at least 10 g/kg of 4-methyl-2-phenyl-2-pentenal.
9. A cocoa product according to claim 1, wherein said ingredient is in the form of a liquid.
10. A cocoa product according to claim 1, wherein said cocoa product is mixed with an ingredient selected from the group consisting of roast and ground coffee, soluble coffee powder, coffee extract, dairy product, non-dairy or dairy creamer, chicory and combination thereof.
11-12. (canceled)
13. A process for manufacturing a cocoa product comprising the steps of a. providing roast and ground cocoa nibs; b. extracting first said roast and ground cocoa nibs with water at temperature between 95 C. and 115 C. at a draw-off ratio between 2 to 4, for 20 to 40 minutes; and c. further extracting the roast and ground cocoa nibs of step b) with water at temperature between 160 C. and 180 C. at a draw-off ratio between 2 and 4, for a duration of between 20 to 40 minutes.
14. A process according to claim 13 further comprising the step of drying the concentrate.
15. A process according to claim 14 wherein drying is performed by spray drying or freeze drying.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0023]
[0024]
[0025]
DETAILED DESCRIPTION OF THE INVENTION
[0026] Consequently, the present invention relates in part to a cocoa product comprising a ratio of epicatechin in % by weight to theobromine in % by weight of at least 0.2. In one embodiment, the cocoa product comprises a ratio of epicatechin in % by weight to theobromine in % by weight between 0.2 and 1.2.
[0027] Epicatechins are phytochemical compounds, classified as flavanol found in a variety of fruits, plant-based food and beverages. Several beneficial biological effects have been associated with epicatechins, such as increasing plasma antioxidant activity, brachial artery dilatation, fat oxidation, and promotion of gut health. Epicatechins can usually be found in cocoa in amount ranging from about 1.5 to 2.8 mg of epicatechin per gram of cocoa, but the more pure and less processed the cocoas is, the higher the concentration of flavanols inside cocoa.
[0028] Theobromine is a bitter xanthine alkaloid found in cocoa, coffee, tea leaves and numbers of other foods. The variation of the amount of theobromine was found to be quite stable in different cocoa products (see Table 2), independently of the processing method, while the variation of the amounts of epicatechin was important.
[0029] The inventors have found that the cocoa product according to the present invention has significant concentration of epicatechins despite the fact that the cocoa product has been heavily processed. It can be seen from
[0030] In one embodiment the cocoa product contains between 0.8% to 4% by weight of epicatechin.
[0031] In one aspect of the present invention, the cocoa product contains epicatechin in an amount of between 8 mg and 40 mg epicatechin per gram of cocoa. The amounts of epicatechin found in the cocoa product according to the present invention are surprisingly higher than the usual amounts found in pure cocoa products, such as in dark chocolate. Without to be bound by the theory, the inventors believe that epicatechin is actually enriched in the cocoa product.
[0032] The elevated levels of epicatechin found in the cocoa product according to the present invention advantageously confers increased antioxidant capabilities to the cocoa product as can be seen in
[0033] Procyanidins are a type of polyphenolic substances (in particular they are a subgroup of the proanthocyanidin class of flavonoids) found in various part of plants such as flowers, fruits, berries, seeds and bark. It is for example found in cocoa beans, red wine and apples. Procyanidins have antioxidant properties. Procyanthocyanidins have a complex structure being oligomers (dimers to pentamers) or polymers (six or more units, up to 60) of catechins or flavanols which are joined by carbon-carbon bonds. Procyanidins consists exclusively of catechin and/or epicathechin: procyanidin B2 consists of two epicatechin monomers and procyanidin C1 consists of three epicatechin monomers.
[0034] In an embodiment the cocoa product has a ratio of procyanidin C1 in a % by weight to theobromine in a % by weight of at least 0.005. In another embodiment the ratio of procyanidin C1 in a % by weight to theobromine in a % by weight is between 0.005 and 0.3.
[0035] In one embodiment the cocoa product contains between 0.01% to 1% by weight of procyanidin C1. Preferably, the cocoa product has between 0.01% to 0.1% by weight of procyanidin C1, more preferably between 0.05% to 0.08% by weight of procyanidin C1.
[0036] In an embodiment the cocoa product has a ratio of procyanidin B2 in a % by weight to theobromine in a % by weight of at least 0.04. In another embodiment the ratio of procyanidin B2 in a % by weight to theobromine in a % by weight is between 0.04 and 0.3.
[0037] In one embodiment the cocoa product contains between 0.15% to 1% by weight of procyanidin B2. Preferably, the cocoa product has between 0.15% to 0.5% by weight of procyanidin B2, most preferably between 0.15% to 0.3% by weight of procyanidin B2.
[0038] The inventors have found that the amount of procyanidin B2 and procyanidin C1 in the cocoa product of the present invention are significantly higher as compared to cocoa powders prepared from commercially available cocoa products (Table 2). These elevated amounts of procyanidin B2 and procyanidin C1 are contributing to the increased antioxidant capabilities demonstrated by the cocoa product of the present invention (Table 5 and
[0039] In one aspect of the present invention, the cocoa product has a ratio of epicatechin in % by weight to theobromine in % by weight of at least 0.2, a ratio of procyanidin C1 in a % by weight to theobromine in a % by weight of at least 0.005 and a ratio of procyanidin B2 in a % by weight to theobromine in a % by weight of at least 0.04.
[0040] In another aspect, the cocoa product contains between 0.8 to 4% by weight of epicatechin, between 0.01% to 1% by weight of procyanidin C1 and between 0.15% to 1% by weight of procyanidin B2.
[0041] Alkyl and phenyl alkenals are volatile compounds that can participate to taste and aroma. Alkyl and phenyl alkenals provide intense cocoa-like, floral, honey, and herbaceous notes.
[0042] Alkyl and phenyl alkenals are ,-unsaturated aldehydes with an alkyl group on the -atom and an alkyl group (2-alkyl-2-alkenals) or a phenyl group (2-phenyl-2-alkenals) on the -carbon. These unsaturated aldehydes can be cis, trans, or mixtures of the two isomers and are generated via the aldol condensation of two saturated aldehydes issued by the Strecker degradation of amino acids. The Strecker degradation is an oxidative deamination of amino acids via interaction with -dicarbonyl compounds.
[0043] Alkyl alkenals are 3-methyl-2-butenal and 2-isopropyl-5-methyl-2-hexenal. Phenyl alkenals are 2-phenyl-2-butenal, 4-methyl-2-phenyl-2-pentenal and 5-methyl-2-phenyl-2-hexenal.
[0044] In one embodiment the cocoa product comprises at least 145 g/kg alkyl and phenyl alkenals, preferably at least 200 g/kg alkyl and phenyl alkenals, more preferably at least 300 g/kg alkyl and phenyl alkenals.
[0045] In another embodiment the cocoa product comprises between 145 g/kg and 8000 g/kg alkyl and phenyl alkenals, preferably between 200 g/kg and 8000 g/kg alkyl and phenyl alkenals, more preferably between 300 g/kg and 8000 g/kg alkyl and phenyl alkenals.
[0046] In one aspect of the present invention, the cocoa product comprises between 145 g/kg and 7000 g/kg alkyl and phenyl alkenals, preferably between 200 g/kg and 7000 g/kg alkyl and phenyl alkenals, more preferably 300 g/kg and 7000 g/kg alkyl and phenyl alkenals.
[0047] The inventors have surprisingly found that the quantities of alkyl and phenyl alkenals in the cocoa product according to the present invention are significantly increased as compared to comparative samples (Table 4 and
[0048] Alkyl alkenals can be selected from the group of 3-methyl-2-butenal and 2-isopropyl-5-methyl-2-hexenal and phenyl alkenals can be selected from the group of 2-phenyl-2-butenal, 4-methyl-2-phenyl-2-pentenal and 5-methyl-2-phenyl-2-hexenal.
[0049] In one embodiment the cocoa product comprises at least 10 g/kg of 2-isopropyl-5-methyl-2-hexenal and at least 10 g/kg of 4-methyl-2-phenyl-2-pentenal, preferably at least 20 g/kg of 2-isopropyl-5-methyl-2-hexenal and at least 20 g/kg of 4-methyl-2-phenyl-2-pentenal, more preferably at least 30 g/kg of 2-isopropyl-5-methyl-2-hexenal and at least 30 g/kg of 4-methyl-2-phenyl-2-pentenal, even more preferably at least 40 g/kg of 2-isopropyl-5-methyl-2-hexenal and at least 40 g/kg of 4-methyl-2-phenyl-2-pentenal.
[0050] In one embodiment the cocoa product comprises between 10 g/kg and 1000 g/kg of 2-isopropyl-5-methyl-2-hexenal and between 10 g/kg and 1000 g/kg of 4-methyl-2-phenyl-2-pentenal, preferably between 20 g/kg and 1000 g/kg of 2-isopropyl-5-methyl-2-hexenal and between 20 g/kg and 1000 g/kg of 4-methyl-2-phenyl-2-pentenal, more preferably between 30 g/kg and 1000 g/kg of 2-isopropyl-5-methyl-2-hexenal and between 30 g/kg and 1000 g/kg of 4-methyl-2-phenyl-2-pentenal, even more preferably between 40 g/kg and 1000 g/kg of 2-isopropyl-5-methyl-2-hexenal and between 40 g/kg and 1000 g/kg of 4-methyl-2-phenyl-2-pentenal.
[0051] The advantage of the present invention is that the cocoa product contains elevated amount of antioxidant compounds and elevated amount of aromatic, volatile compounds which altogether confer interesting antioxidant properties associated with an intense, natural cocoa flavour and aroma to the cocoa product.
[0052] The cocoa product according to the present invention can be in liquid, concentrated or extract form, or in dry, powder form. It can advantageously be used to prepare beverages, either liquid, ready-to-drink or powdered beverages, by mixing the cocoa product with other ingredients. For example, the cocoa product can be mixed with roast and ground coffee, soluble coffee powder, coffee extract, dairy product, non-dairy or dairy creamer or chicory. The cocoa product can also be used in a capsule or in a beverage system. The cocoa product of the present invention has the advantage that in the powdered form, it is easily dissolvable in hot or cold liquid.
[0053] In another aspect the present invention relates to a process for manufacturing a cocoa product comprising the steps of [0054] a. providing roast and ground cocoa nibs; [0055] b. extracting first said roast and ground cocoa nibs with water at temperature between 95 C. and 115 C. at a draw-off ratio between 2 to 4, for 20 to 40 minutes; [0056] c. further extracting the roast and ground cocoa nibs of step b) with water at temperature between 160 C. and 180 C. at a draw-off ratio between 2 and 4, for a duration of between 20 to 40 minutes; and optionally [0057] d. evaporating the extract to obtain a concentrated cocoa composition.
[0058] The process may further comprise the step of drying the concentrate. Preferably, drying is performed by spray drying or freeze drying.
[0059] In one embodiment the extract obtained in step d) can be dried using drying methods well-known in the art, such as freeze-drying or spray-drying. If freeze drying is used to dry the extract, it may be desirable no to evaporate the extract, accordingly, in one embodiment, the extract obtained in step c. is dried by freeze drying.
[0060] In the context of the present invention, the draw-off ratio refers to the ratio of water to cocoa nibs. A draw off ratio of 2 means that 2 kg of water are used to extract 1 kg of cocoa nibs.
[0061] The term cocoa nibs refers to roasted cocoa beans that have been crushed or milled into small pieces. The roast and ground cocoa nibs provided in step a) of the process have preferably a particle size comprised between 2 and 5 mm, in order to have efficient extraction.
[0062] The inventors have found that the process of the present invention unexpectedly preserves or even increases the amount of antioxidant compounds, such as epicatechin. The process also advantageously favoured the formation of volatile compounds responsible for cocoa flavours.
[0063] Those skilled in the art will understand that they can freely combine all features of the present invention disclosed herein. In particular, features described for the product of the present invention may be combined with the method of the present invention and vice versa. Further, features described for different embodiments of the present invention may be combined. Where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification.
[0064] Further advantages and features of the present invention are apparent from the figures and non-limiting examples.
EXAMPLES
Example 1: Method for Preparation of the Cocoa Product and of Comparative Samples Ingredient
[0065] Cocoa products according to the present invention were prepared as follows. 4.4 kg of cocoa nibs was filled in a 7 liters cylindrical extraction cell and extracted with water in two steps. The first step involved extraction for 36 minutes with water at a temperature of 110 C. and at a draw-off ratio (kg water/kg of cocoa nibs) of 3. The second step involved further extraction for 36 minutes with water at 170 C. at a draw-off ratio of 3. The resultant cocoa extract was then concentrated and freeze dried to afford a water-soluble cocoa powder.
[0066] The comparative samples were prepared to obtain a lyophilized water-soluble cocoa powder. 15 g of commercially available cocoa powder (non-alkalized or alkalized cocoa product) was suspended in 100 ml water and boiled under reflux for 4 hours until reaching the end point in total dissolved solids. After centrifugation (1000 rpm, 15 min at 20 C.), the supernatant was frozen at 20 C. prior to lyophilization, resulting in a water-soluble cocoa powder. [0067] Sample A=non-alkalized commercial natural cocoa powder [0068] Sample B=alkalized commercial cocoa powder [0069] Sample C=medium alkalized commercial cocoa powder [0070] Samples D, E and F=cocoa products according to the invention prepared as described above.
Example 2: Analysis of Chemical Compounds
[0071] 2.1 Analysis of epicatechin, procyanidins and theobromine
[0072] The analysis of epicatechin, procyanidins and theobromine was performed on a QTRAP 6500 LC-MS/MS system (AbSciex), operating in multiple reaction monitoring (MRM) mode.
[0073] Prior to the instrumental analysis, the soluble cocoa powders (25 mg) were each dissolved in 25 mL of water, followed by membrane filtration.
[0074] To perform the chromatography, an Agilent 1290 Infinity II system (Agilent) was used, equipped with a binary G7104A pump, an G7167B auto sampler cooled to 4 C., and a G7116B column oven heated to 30 C. The samples (5 L) were injected in a triplicate determination onto a Kinetex Phenyl-Hexyl 100 mm2.1 mm1.7 m column (Phenomenex), using 0.1% aqueous formic acid (A) and 0.1% formic acid in acetonitrile (B) as mobile phase. At flow rate of 0.4 mL/min, the following gradient was applied: 0% B for 1 min, up to 35% B in 10 min, then in 2 min to 100% B and kept for 3.5 min before going to starting conditions in 0.5 min and kept for 3 min.
[0075] The LC-system was coupled with a QTRAP 6500 mass spectrometer (AbSciex), using Analyst (version 1.7.1) as software. Thereby, the source conditions for application of the positive ESI mode were the following: gas 1: 55 psi, gas 2: 65 psi, curtain gas: 35 psi, source temperature: 550 C., ion spray voltage floating: 5500 V. For the MS/MS detection in multiple reaction monitoring (MRM) mode, the following mass transitions were applied, with each one quantifier (Q) and one qualifier mass transition per compound:
TABLE-US-00001 TABLE 1 Applied MS/MS detection parameters in multiple reaction monitoring (MRM) mode with Q1 = Precursor Ion m/z, Q3 = Fragmentation Ion m/z, DP = Declustering Potential, EP = Entrance Potential, CE = Collision Energy, CXP = Collision Cell Exit Potential. Name Q1 Q3 DP EP CE CXP Epicatechin Q 291 139 41 10 23 6 Epicatechin 291 123 41 10 23 14 Procyanidin B2 Q 579 127 80 10 29 6 Procyanidin B2 579 139 80 10 35 14 Procyanidin C1 Q 867 579 80 10 23 20 Procyanidin C1 867 245 80 10 61 30 Caffeine Q 195 138 56 10 43 12 Caffeine 195 123 56 10 43 12 5-chlorogenic acid 355 267 106 10 27 14 Theobromine Q 181 163 41 10 25 14 Theobromine 181 108 41 10 35 12
[0076] Data treatment was performed by MultiQuant (AbSciex, version 3.0.2), and the absolute values were determined based on an external calibration, diluting stepwise stock solutions of the individual analytes (epicatechin: 70 mg/L, procyanidin B2: 38 mg/L, procyanidin C1: 39 mg/L and theobromine: 67 mg/L).
TABLE-US-00002 TABLE 2 Epicatechin, procyanidins and theobromine - amounts in % by weight Samples Epicatechin Procyanidin C1 Procyanidin B2 Theobromine A 0.53 <0.0004 0.07 3.6 B 0.20 <0.0004 0.002 3.6 D 1.06 0.06 0.22 3.9 E 1.20 0.07 0.27 3.8 F 1.19 0.07 0.25 3.8
[0077] 2.2. Quantification of Alkyl and Phenyl Alkenals
[0078] Metabolite profiling of the samples was conducted by gas chromatography coupled to quadrupole time-of-flight mass spectrometry (GC-QTOF-MS).
[0079] The absolute amounts (in g/kg) of identified volatile alkenals were determined in the cocoa product according to the invention (samples D, E, and F) as well as in the reference extracted samples (samples A and B). For this purpose, the standard addition method was applied, whereby the standard was added directly to the aliquots of analyzed sample.
[0080] Sample Preparation
[0081] The soluble cocoa powders (500 mg, each) were provided into three silanised glass vials (20 mL vials used for headspace/SPME analysis) and solubilized with 5 mL of water, each, and sealed with a screwing septum cap.
[0082] For the quantitative evaluation the samples were spiked with 20 l of standard solution of the respective alkenal molecules at three levels (around 1, 2, and 3 g/mL), and sealed with a screwing septum cap.
[0083] Extraction of Aroma
[0084] The sample was equilibrated for 60 min at room temperature. Aroma compounds were then extracted from the headspace by solid phase microextraction (SPME) at 50 C. during 30 min (2 cm fiber, 50/30 m StableFlex, coated with PDMS/DVB/Carboxen; Supelco, Buchs, Switzerland), and thermally desorbed into the split-splitless injector (in split-mode; split of 2) heated at 240 C. for 3 min.
[0085] GC/MS Analysis
[0086] Qualitative and quantitative metabolite analysis was carried out using an Agilent 7200 QTOF-GC/MS system consisting of an Agilent 7890B gas chromatograph with a 7200 UHD accurate-mass QTOF mass spectrometer (Agilent, Basel, Switzerland).
[0087] Separation was carried out on a 60 m0.25 mm0.25 m polar DB-624UI column (Agilent, Basel, Switzerland), and Helium was used as carrier gas with a constant flow of 1.0 mL/min. Following oven program was applied: initial temperature of 40 C. was held for 2 min, raised at 5 C./min to 200 C., then at 20 C./min to 240 C., and final temperature held for 10 min.
[0088] Detection was performed by electron impact mode with ionization energy of 70 eV at the full mass scanning range from 30 to 250 amu at a spectra acquisition rate of 5 spectra/s.
[0089] Data Consolidation
[0090] Chromatograms were processed using the Agilent MassHunter software (Agilent, Basel, Switzerland), and the components were identified by comparison of their mass spectra with those in the NIST MS 14.0 Library and confirmed by comparison of their retention indices. The exact m/z and tolerance applied for the peak extraction and quantification of alkenals are given in Table 3.
TABLE-US-00003 TABLE 3 Exact m/z and tolerance applied for peak extraction and quantification of alkenals. Compound m/z (amu) m/z tolerance (amu) 2-isopropyl-5-methyl-2-hexenal 139.1090 +/0.02 2-phenyl-2-butenal 146.0749 +/0.02 4-methyl-2-phenyl-2-pentenal 174.1014 +/0.02 5-methyl-2-phenyl-2-hexenal 117.0635 +/0.02
[0091] The results in Table 4 reveal significantly higher concentrations of the identified alkenals in the cocoa products (samples D, E, F) according to the present invention as compared to the reference samples (samples A and B) extracted at boiling temperature (around 98 C.).
TABLE-US-00004 TABLE 4 Concentrations of alkyl and phenyl alkenals in [g/kg] Concentration [g/kg] 2-isopropyl- 4-methyl-2- 5-methyl-2- 5-methyl-2- 2-phenyl-2- phenyl-2- phenyl-2- Samples hexenal butenal pentenal hexenal A 2.3 29.0 1.1 41.6 B <1 31.7 <1 13.0 D 43.5 166 53.3 137 E 65.0 191 54.1 152 F 87.5 130 96.9 228
[0092] The quantities of the lavender and herbaceous, cocoa smelling 2-isopropyl-5-methyl-2-hexenal were determined at a range of 44 to 88 g/kg in the three analyzed invention samples. In contrast, an amount of only 2.3 g/kg was found in the natural, non-alkalized reference (sample A), and the quantity was below the detection/quantitation level in the alkalized reference (sample B).
[0093] Similarly, 4-methyl-2-phenyl-2-pentenal (floral, honey, powdery, and cocoa-like) was almost uniquely found in the invention samples, with concentrations ranging between 53.3 and 96.9 g/kg, but merely around detection level in the comparative samples A and B. This volatile component is formed in an aldol condensation of phenylacetaldehyde and methylpropanal.
[0094] The floral, cocoa, and honey-like 2-phenyl-2-butenal, a product from the condensation reaction of phenylacetaldehyde and acetaldehyde, was determined at 130 to 191 g/kg in the invention samples, and from 29 to 31.7 g/kg in the comparative samples A and B.
[0095] 5-methyl-2-phenyl-2-hexenal (cocoa, nutty, green) is generated from phenylacetaldehyde and 3-methylburtanal by aldol condensation. The concentrations were quantified from 137 to 228 g/kg in the invention samples and were significantly lower in the comparative samples at 13.0 and 41.6 g/kg.
[0096] In conclusion, the results demonstrated that the extraction conditions applied in the frame of present invention strongly favor the generation of identified alkenals from the aldol condensation of Strecker aldehydes. Particularly 2-isopropyl-5-methyl-2-hexenal and 4-methyl-2-phenyl-2-pentenal seem to be unique marker for the cocoa extraction as described in the invention application. In addition, 2-phenyl-2-butenal and 5-methyl-2-phenyl-2-hexenal were by factor 4 to 17 higher in the invention samples as compared to the colder extracted reference cocoa products, and hence are additional indicator for the specific conduct of the invention.
[0097] 2.3 Determination of the Antioxidant Capacity
[0098] Samples of cocoa products were prepared as described in the Example 1.
[0099] Antioxydants were quantified by assessing the sample capacity to bleach the colour of a free radical ABTS (Di-ammonium 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) solution. The methods used is based on the work of Serpen et al., 2008 and Serpen et al., 2007.
[0100] In practice, samples are diluted 1:100 (weight/weight) with cellulose powder. A stock solution of ABTS is made by dissolving 192 mg of ABTS and 33 mg de K2S2O8 in 25 mL of ultra-pure water. The working solution of ABTS should be freshly prepared everyday by diluting 700 L of the ABTS stock solution into 100 mL EtOH 50%.
[0101] In a tube, 20 mg of the diluted samples are mixed with 2 mL of ethanol (50%) and 10 mL of the ABTS working solution. Samples are kept gently agitated for 20 min at room temperature and subsequently centrifuged 2 min at 9200 g. 35 min precisely after the addition of the ABTS solution, the absorbance of supernatants is measured at 734 nm. All sample are analysed in triplicate. Calibration curves is built by following the same protocol but replacing sample with a Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid) solution (0.002-0.02 mol/mL). Antioxidant capacity of samples is expressed in mol/mg Trolox equivalent (TE).
TABLE-US-00005 TABLE 5 Antioxidant capacity (mol TE/kg) Samples (mol TE/kg) A 608 B 763 D 1111 E 1018 F 841
[0102] As can be seen in Table 5 and
Example 3: Use of the Cocoa Product
[0103] Cocoa Beverage Composition
[0104] The cocoa powder prepared as describe in Example 1 was mixed in different ratios with soluble coffee powder, starting from 5% up to 60% cocoa powder. Upon addition of hot water (85 C.), the beverage powder easily and completely dissolved to afford an intense cocoa-coffee drink.
[0105] Cocoa Containing Capsules
[0106] 1.5 g capsules to be used in system were prepared by mixing different ratios (from 30% up to 60%) of cocoa powder prepared as described in Example 1 with soluble coffee. The beverage was prepared and delivered an intense cocoa-coffee drink.