Process for treating cacao nib or liquor with enzyme and process for producing of cacao and cacao nib or liquor composition

Abstract

The invention relates to a method for treating cocoa beans or liquor, which comprises preparing cocoa beans or liquor from cocoa beans fermented for 1 to 15 days in a known manner, mixing them with at least 0.01 to 5% by weight of a technical protease in an aqueous medium with a pH of 3 to 8, and incubating them for a time and at a temperature sufficient to hydrolyze the proteins and peptides contained in the mixture. The invention also relates to a composition comprising cocoa beans or liquor obtained from cocoa beans fermented for 1 to 15 days, 0.01 to 5% by weight of a technical protease and optionally 0.01 to 5% by weight of a technical invertase. The invention also relates to a method for producing cocoa by roasting the composition according to the invention. à

Description

The invention relates to a method for enzymatically treating cocoa nibs or liquor to improve the aroma precursor composition, which allows for the wide use of cocoa beans with different degrees of fermentation. The invention also relates to a method for producing cocoa and a cocoa nib or liquor composition. The quality of chocolate is directly influenced by the geographical and genetic origin of the cocoa bean and the conditions under which the cocoa bean is handled, from cultivation in the fields to processing in the factory. Cocoa beans, harvested at one time, undergo fermentation and a natural drying process in the country of origin, which causes the appearance of aroma precursors. Unfortunately, the collection of ripe beans and the conditions under which they are fermented are not always sufficiently controlled. Furthermore, of the three main types of cocoa trees of the Theobroma cacao species, named Criollo, Forastero and Trinitario, each type produces beans with a different composition. Finally, dried cocoa beans can also be compromised by shipping to countries that specialize in cocoa bean processing. The industry must therefore expect a high degree of variability in the composition of cocoa bean batches (see, for example, Smaffer M.: The Manufacturing Confectioner, 92-94, June 1994). The fermented and dried cocoa beans are then usually pressed into smaller or larger volumes, the outer shell is removed, and the beans are then roasted, if necessary in combination with alkalization, which causes the cocoa aroma and color to appear. The roasting step involves Maillard reactions between reducing sugars and protein degradation products, especially amino acids and hydrolysis products (Wood and Láss, Cocoa, Longman Scientific &Technical, Longman Group UK, England, 1985, ISBN 0-582-46352-1). Aroma precursors only appear during the natural fermentation of cocoa beans as a result of hydrolysis reactions. Experiments on in vitro incubation of unfermented cocoa beans or cocoa bean fragments have shown that these hydrolysis reactions depend on pH, temperature and the binding of enzymes and substrates in the beans. Hydrolysis reactions are also sensitive to the presence of polyphenols. It is therefore advantageous to extract the polyphenols from the beans with acetone and to incubate the resulting beans in the presence of acetic acid to separate the enzymes and substrates (Biehl B. et al.: J. Sci. Food. Agric., 33, 1280-1290, 1982). Furthermore, other studies have shown that cocoa beans contain at least three proteolytic activities in the presence of cocoa aroma precursors, namely an endoprotease activity with an optimum at pH 3.5, a carboxypeptidase activity with an optimum at pH 5.8, and an aminopeptidase activity with an optimum at pH 7. Experiments on the hydrolysis of purified cocoa bean globulin (a protein with two bands, a 31 kD and a 47 kD band on SDS-PAGE gel chromatography) with common proteases and / or purified cocoa endoprotease and carboxypeptidase have shown that it is advantageous to hydrolyze the globulin sequentially with cocoa endoprotease at pH 3.5 and then with cocoa carboxypeptidase or common carboxypeptidase at pH 5-6.In this case, a hydrolysis product and amino acid mixture are obtained which are suitable for absorbing cocoa aroma during the subsequent roasting of the mixture in the presence of reducing sugars (Voigt J. et al.: Food Chemistry, 50, 177-184, 1994). In addition, US Patent No. 2,965,490 describes a process for the in vitro hydrolysis of unfermented cocoa beans (green cocoa beans) in an attempt to replace the natural fermentation of the beans. For this purpose, the proteins of the green cocoa beans are hydrolyzed with acid or enzymatically, the carbohydrates in said beans are hydrolyzed enzymatically to simple sugars, and both hydrolysates are allowed to react under anhydrous conditions at a suitable temperature and for a sufficient time to form aromatic factors. However, it has been found that the polyphenols in the green beans significantly reduce the enzymatic hydrolysis of the stored proteins (see Comparative Example 1). In summary, the methods and studies described above have clearly attempted to replace the natural fermentation of cocoa beans with controlled enzymatic hydrolysis of green beans (a problem caused by the presence of polyphenols) or their purified proteins (from which polyphenols have been removed). Furthermore, the quality and quantity of aroma precursors required to produce a good cocoa aroma during roasting is still unknown. The present invention aims to eliminate the differences in the aroma precursor composition of fermented cocoa beans and to transform it into an optimal aroma precursor composition in the cocoa nib or fermentation broth. For this purpose, in the process according to the invention, the seed core or juice obtained from cocoa beans fermented for 1-15 days is mixed with at least one protease in an aqueous medium at a pH of 3-8, and the mixture is incubated for a period of time at a suitable temperature to hydrolyze the proteins and peptides. Preferably, the mixture is incubated for a time and at a temperature suitable to obtain 10 pmol of hydrophobic amino acid mixture / g dry matter and / or at least 1.4 times more hydrolysis products compared to the amount in the starting cocoa beans. The process according to the invention allows the hydrolysis of proteins stored in cocoa beans, which initially participate in the natural fermentation of the beans, to be enhanced until a sufficient amount, correct amino acid and hydrolysis product composition is obtained. During the subsequent roasting of the blend according to the invention, it is then possible to obtain a good cocoa aroma, which may be even stronger and more concentrated than the aroma obtained after roasting conventionally fermented cocoa beans. HU 219 572 B It is also possible to use beans with different degrees of fermentation, such as under-fermented beans, which do not yet contain enough aroma precursors, or over-fermented beans, which do not contain the correct aroma precursor composition, or barely fermented beans, such as sour beans, which contain a small amount of aroma precursor composition. Similarly, cocoa beans can come from different plants and geographical locations. The invention thus makes it possible to homogenize the aroma precursor composition of a mixture of beans of different origins and fermentation states. Furthermore, the hydrolysis of cocoa nibs or liquors by technical enzymes is surprising due to their sensitivity to the polyphenols contained in the beans. It has been shown that beans fermented naturally for at least one day have a sufficiently low level of polyphenols so that the technical enzymes are not significantly inhibited in the process according to the invention. On the other hand, green cocoa beans have a level of polyphenols that is too high to be used in the present process. In particular, the addition of an invertase makes it possible to advantageously increase the natural glucose and fructose content of the mixture, which promotes the formation of aroma during the subsequent roasting of the mixture. Finally, the possible use of various technical enzymes in the process according to the invention opens up the possibility of creating different cocoa aroma variations, which consumers are likely to appreciate. Figure 1 shows the amount of glucose produced by invertase in a mixture of insufficiently fermented cocoa bean pulp and water as a function of the duration of treatment and the amount of invertase added. The invention is described in detail below. A unit of enzymatic activity is defined in the remainder of this specification as pmol of product converted in 1 minute. Similarly, percentages are given by weight unless otherwise indicated. The term "underfermented or insufficiently fermented cocoa beans" refers to cocoa beans that have been conventionally fermented for 1-4 days and then dried, in which the proteins give two visible bands on a polyacrylamide gel by SDS-PAGE chromatography, corresponding to 21 kD and 31 kD / 47 kD storage proteins (see WO 91 / 00913 and WO 91 / 00914). In addition, the beans do not have sufficient amino acids and peptides to produce cocoa aroma during subsequent roasting. The term “Well-fermented cocoa beans” refers to cocoa beans that have been traditionally fermented and dried for 2-10 days, in which the proteins give a visible band on a polyacrylamide gel by SDS-PAGE chromatography, corresponding to the 21 kD stored protein, and the band of the 31 kD / 47 kD proteins has been partially or completely degraded. In addition, these beans contain sufficient amino acids and peptides to produce the cocoa aroma during subsequent roasting. The term “overfermented cocoa beans” refers to cocoa beans that have been traditionally fermented and dried for 7-15 days and in which proteins, visible and distinguishable bands corresponding to the 21 kD and 31 kD / 47 kD proteins, cannot be detected by SDS-PAGE chromatography on polyacrylamide gels. Furthermore, although these beans contain sufficient amino acids, they do not contain sufficient peptides to produce cocoa aroma during subsequent roasting. Finally, for the process according to the invention, the cocoa nib is obtained by the usual pressing of fermented or unfermented cocoa beans, and the cocoa liquor is obtained by pressing said beans or nib to a smaller or larger volume until the cocoa butter is released from the cells. For the purpose of the present invention, the liquor or liquor is prepared from cocoa beans fermented for 1-15 days. Therefore, cocoa beans with different degrees of fermentation, such as underfermented, well-fermented, over-fermented or weakly fermented beans, are suitable for use - as a mixture or otherwise. Cocoa beans from different genotypes of cocoa trees, such as Theobroma cacao Criollo, Forastero and / or Trinitario, can be used in the present invention - as a mixture or otherwise. The liquor or liquor can be finely ground; however, it should be noted that if the liquor or liquor is enzymatically treated at temperatures higher than 35-45 °C, the liquor or liquor pieces melt, so that the innermost material in the mixture becomes accessible to the enzymes. The seed coat or juice (liquor) is then mixed with at least one protease in an aqueous medium at a pH of 3-8. The protease can be used alone or in combination and can be selected, for example, from the group of endoproteases, carboxypeptidases and aminopeptidases. The pH of the aqueous medium can be adjusted by adding an inorganic or organic acid or a buffer, which is selected from the buffers commonly used in food processing, preferably, for example, buffers based on citrate, phosphate or acetate. In particular, a pH of 3-5 can be preferred, as well as an activator of endogenous proteases in the bean; it is therefore advantageous to choose a technical protease that is active in a wide acidic range. The mixture may contain 10-80% by weight, especially 20-70% by weight, of cocoa bean pulp or juice, and 1-100 units of protease per gram of the mixture, preferably 0.01-5% by weight, for example of a commercial technical protease preparation. To carry out the method of the invention, the mixture is then incubated for a sufficient time and at a suitable temperature to hydrolyze the proteins and peptides contained therein. Preferably, the mixture is treated for a time and temperature sufficient to produce at least 10 pmol hydrophobic amino acids / g dry matter and / or at least 1.4 times more, and even 1.7 times or 2 times more, hydrolysis products compared to those initially present in the treated cocoa beans. HU 219 572 Β is incubated. In this context, the amount of the same hydrolysis products (peptides and amino acids) found in the seed guts obtained before and after the treatment according to the invention can be compared. It is also possible to take into account the appearance of new hydrolysis products compared to those in the untreated seed gut according to the invention, since they are enclosed, but appear significantly, for example, during the natural fermentation of green cocoa beans. In particular, the amounts of four hydrolysis products with retention times of approximately 9, 17, 24 and 29.5 min were compared by chromatography on a C18 reverse phase HPLC column (No. 218TP54 Vydac; USA) at a constant flow rate of 1 ml / min using an aqueous solution of 0.1% trifluoroacetic acid and 2% acetonitrile as eluent for 10 min, followed by a linear gradient of 0.1% trifluoroacetic acid and acetonitrile increasing from 2% to 52.7% for 50 min.The eluted hydrolysis products were detected at 215 and 280 nm. Similarly, only those amino acids which predominate in the seed coat treated according to the invention can be considered as hydrophobic amino acids, in particular, for example, phenylalanine, leucine and alanine. To obtain sufficient amounts of hydrophobic amino acids and hydrolysis products, the mixture can be incubated at 10-60 °C for a period of 30 minutes to 20 hours. However, the hydrolysis time and temperature should be selected depending on the role of the medium, the type of enzyme, the fermentation level of the beans and the origin of the beans. According to a preferred embodiment of the method according to the invention, the seed core and liquor are prepared from cocoa beans fermented for 1-15 days by mixing them with an aqueous medium with a pH of 3-6, incubating the mixture at 40-60 °C for 10 minutes to 20 hours to promote the action of cocoa endoproteases, then adjusting the pH of the medium to between 4-8, adding at least one technical protease and incubating at 10-60 °C for 5 minutes to 20 hours to hydrolyze the proteins and peptides, but preferably to such an extent that at least 10 pmol hydrophobic amino acids / g dry matter and / or at least 1.4 times more hydrolysis products (peptides and amino acids) are obtained compared to those originally present in the cocoa beans. According to another preferred embodiment of the method according to the invention, the seed core and liquor are prepared from cocoa beans fermented for 1-15 days by mixing them with an aqueous medium with a pH of 4-8, adding at least one technical protease and incubating at 10-60 °C for 5 minutes to 20 hours to hydrolyze the proteins and peptides, but preferably until at least 10 pmol hydrophobic amino acids / g dry matter and / or at least 1.4 times more hydrolysis products (peptides and amino acids) are obtained compared to those originally present in the cocoa beans. Preferably, the pH range is chosen such that the cocoa endoprotease as well as the technical protease remain active. Finally, according to a third preferred embodiment of the process according to the invention, at least one enzyme selected from the group consisting of invertases, glycosidases, cellulases, pectinases and oxidases is added to the reaction mixture. Preferably, at least one of the aforementioned enzymes is added as a commercial enzyme preparation in an amount of 0.01-5% or in an amount of 1-100 units / g of mixture. The reaction mixture is one of the mixtures described above, which is incubated for a certain period of time (containing a technical protease or the like). It is advantageous to combine the technical protease and / or cocoa endoprotease treatment with a technical invertase. This treatment advantageously allows the formation of hydrophobic amino acids and other hydrolysis products which, when combined with glucose and fructose, give an even more chocolatey aroma (which produces a rich taste) during subsequent roasting. Surprisingly, the cocoa endoprotease, technical invertase and protease can be used in a single step (second preferred embodiment of the invention), but the pH range must be chosen so as to be favorable for the activity of all enzymes (endoprotease, technical protease and invertase). In the case where invertase is not used, it may also be advantageous to add at least 0.1% reducing sugar to the final mixture before roasting, such as glucose and / or fructose in an amount of 0.1-5%. After carrying out the process according to the invention, the mixture can finally be preserved by inactivating the enzymes, for example by heat (80-120 ° C for 1-60 minutes) or by treatment at high hydrostatic pressure (greater than 500,000 kPa). It is also possible to reduce the moisture content of the mixture, for example by freeze-drying, spray-drying, vacuum evaporation or evaporation of the aqueous solution at moderate temperatures. Preferably, the mixture is dried to a moisture content of less than 10%, in particular to 5%. The mixture can then be subjected to roasting and / or alkalization, as described in European Publication No. 0,226,727. Preferably, after carrying out the process according to the invention, the pH of the mixture is adjusted to between 5-6, then dried and roasted. The invention therefore relates to mixtures comprising a core and liquor from cocoa beans fermented for 115 days, a technical protease and optionally a technical invertase. If the enzymes are still active, the compositions may contain at least 1 enzymatic activity unit / g of composition. In the event that these enzymes are inactive, for example after heat denaturation treatment, they can nevertheless still be identified using suitable antibodies indicating the previous use of this enzyme. Furthermore, these compositions may comprise cocoa nibs or liquor obtained from a mixture of beans obtained from cocoa trees of different fermentation stages and / or different genotypes. These compositions are one of the compositions that fall within the scope of the invention. The invention also relates to dried preparations with added glucose and / or fructose and cocoa obtained by roasting said compositions. HU 219 572 Β cocoas (powder, gut, liquor and the like). These preparations differ significantly from those known from the prior art, as they are derived from mixtures containing beans in different fermentation states and / or beans from different genotypes of cocoa trees, and active or inactive technical proteases and, optionally, invertases. The invention is described in detail by the following examples. The examples are preceded by a description of various tests and a presentation of the figure. However, the examples given are only intended to illustrate the subject matter of the invention and in no way limit the scope of the invention. Amino Acid Analysis The inactivated mixture according to the invention is placed in an ultrasonic bath for 10 minutes, homogenized in a Polytron homogenizer (Kinematica AG; Switzerland) twice for 1 minute at 9600 rpm, centrifuged at 10,000 g for 10 minutes, the supernatant is collected, filtered, and then its amino acid content is analyzed using the ACCQTag® kit (Waters, USA). For this, 20 μΐ of sample and standard are mixed with 140 μΐ of borate buffer, 40 μΐ of 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) reagent is added, the mixture is stirred and left to stand at room temperature for 1 min, then heated at 55 °C for 7 min, then injected onto a Cl8 reversed phase HPLC column (Nova-Pak Cl 8.4 pm; USA) and the amino acids are eluted at 36.5 °C, with a flow rate of 1 ml / min with the following gradient: 100% solvent A for 10 min, 98% solvent A for 10.5 min, 93% solvent A for 26 min, 90% solvent A for 30 min, 67% solvent A for 43 min, 30% solvent A for 53 min, 100% w / w water for 54 min and 100% acetonitrile / water solvent mixture (65% / 35%) for 60 minutes (Solvent A=phosphate buffer obtained from Waters). Derivatized and eluted amino acids are detected at 395 nm using an Applied Biosystem model 980 detector. The amino acid content of the mixture is then determined in pmol / g dry matter and compared to standard samples derivatized and eluted under the same conditions. Analysis of hydrolysis products Methanol is added to the inactivated mixture according to the invention to a concentration of 70%, then 1.5 ml of 70% methanol and 100 mg of polyvinylpyrrolidone (PVPP) are added, the mixture is kept at room temperature for 1 hour with gentle stirring, centrifuged twice for 10 minutes at 20,000 g, the supernatant is collected, the methanol is removed by suction under vacuum, 0.35 ml of water is added to the pellet, the solution is centrifuged for 5 minutes at 20,000 g, then filtered, and the content of the hydrolysis product is analyzed by elution on a Cl8 reverse phase HPLC column (No. 218TP54 Vydac; USA) at a flow rate of 1 ml / min. The eluent used was 0.1% trifluoroacetic acid and 2% acetonitrile in water for 10 min, followed by a linear gradient of 0.1% trifluoroacetic acid and increasing acetonitrile concentration from 2% to 52.7% over 50 min. The hydrolysis products were detected at 215 and 280 nm. The invention relates to the determination of the increase in the amount of hydrolysis products (peptides and amino acids) in the mixture treated according to the invention compared to that found in the untreated mixture, by comparing the area under the elution peaks of the four hydrolysis products, with retention times of 9, 17, 24 and 29.5 minutes. These areas are generally found during the traditional fermentation of cocoa beans from Theobroma cacao Trinitario, Criollo and Forastero cocoa trees. However, it cannot be excluded that other hydrolysis products may also appear during the traditional fermentation of cocoa beans from some specific genotypes of cocoa trees. Sugar analysis The inactivated mixture according to the invention is incubated for 30 minutes at 60 °C with stirring, then left to stand at room temperature for 15-30 minutes, centrifuged twice at 20,000 g for 5 minutes, the supernatant is filtered through a Cl8 Waters Sep-Pak (Waters, USA) cartridge, the filtrate is filtered again through a Satorius Minisart filter [SRP15, No. 1755K, Sartorius Werke GmbH, Göttingen (Germany)], and its glucose content is determined by the “God-Perfid” spectrophotometric method using the Boehringer-Mannheim No. 124028 kit (Germany) or alternatively by HPLC. Perception analysis According to the invention, the hydrolyzed seed coat or liquor is heat inactivated, the pH is adjusted to between 5 and 6, dried under vacuum with gentle heating (up to 60 °C) to remove the water until the moisture content is below 10%, and then roasted in an oven at 130 °C for 15 minutes. The aromas resulting from the roasting are then evaluated by a panel of people who are accustomed to perceiving such aromas. All aromas are compared to those produced from cocoa nibs or liquors from under-fermented Sanchez and Sulawesi cocoa beans and well-fermented Ghana cocoa beans (these beans are known to those skilled in the art) simply dried and roasted under the same conditions. Each sample is evaluated based on the following sensations: “cocoa aroma” (derived from Ghana beans), “sour” (acidity is characterized by the basic taste produced by dilute aqueous solutions of most acids), “bitter” (bitterness is characterized by the basic taste produced by dilute solutions of various substances, such as quinine, perceived on the tip of the tongue and the back of the palate), “astringent” (the concept of astringency is extended to the internal effects of polyphenols, which manifest themselves in a natural physical sensation, from the suppression of the smoothness of taste to the medical sensation of astringency, which includes the contraction of tissues and / or goosebumps), “fruity” (this taste belongs to the aroma (bouquet) and evokes a ripe fruit: apple, banana, pear and the like), “floral” (corresponds to an olfactory reminiscent of flowers in general: rose, jasmine, hyacinth, lilac and the like), “smoked” (the taste and smell of smoked ham;the defect usually results from drying the cocoa beans after fermentation using charcoal), “sour” and “raw” (characteristic of insufficiently roasted cocoas, where the aroma has not developed; related to astringency and acidity; reminiscent of the taste of unripe peanuts); HU 219 572 B Comparative example 1 Theobroma cacao Trinitario ICS-95 green cocoa beans are prepared in the usual way, 100 mg of this and, where necessary, 100 mg of PVPP, which forms a complex with the polyphenols, are mixed with 1 ml of 200 mM citrate buffer 5 (pH 4). The mixture is incubated for 2 hours at 50 °C, the pH is adjusted to pH 7 with 1 ml of 200 mM citrate buffer, optionally 40 µl of phenoxypeptidase A (0.06 units / ml, Sigma) are added, incubated for 1 hour at 25 °C, and then the reaction is stopped by heating the mixture for 5 minutes at 95 °C. The degree of hydrolysis of the cocoa proteins is then analyzed by determining the free amino acid content in the treated kernel and the area under the elution peak of the four hydrolysis products in the starting kernel and the treated kernel (the products are associated with the natural fermentation of the beans: amino acids and peptides) using the method described above. The results given in Table 1 show that the autolysis of the seed coat at pH 4 and pH 7 does not allow to obtain a significant increase in the aroma precursors compared to those initially present in the green cocoa bean. On the other hand, the autolysis of the seed coat containing PVPP 10 at pH 4 followed by enzymatic hydrolysis of the proteins at pH 7 allows to obtain a significant increase in the aroma precursors. The polyphenols present in the green cocoa bean therefore have a remarkable inhibitory effect. Table 1 Enzyme treatment PVPP addition Amino acids (pmol / g dry matter) Hydrolysis products characteristic areas (arbitrary units) Total Hydrophobic 1st peak 2nd peak 3rd peak 4th peak none - 15.9 2.5 604 551 716 - present - 26.8 8.3 695 1224 696 - present + 34.9 12.1 1559 2847 2854 1804 (The retention times of hydrolysis products 1-4 are 9, 17.24 and 29.5 minutes, respectively.) Comparative example 2 The bolls of Theobroma cacao Trinitario ICS-95 cocoa beans are prepared in a conventional manner and are usually fermented for 1, 2, 3, 4, 5 or 7 days and then dried. The degree of hydrolysis of the boll proteins is then analyzed using the method described above, determining the area under the elution peak of the four hydrolysis products present in the fermented bolls (the products are associated with the natural fermentation of the beans). The results are given in Table 2 below. Analysis of endosperm proteins by SDS-PAGE chromatography on polyacrylamide gels shows intensive degradation of the 31 kD and 47 kD bands when the beans are fermented for at least 3 days. On the other hand, the 21 kD band is not degraded when the beans are fermented for 1-7 days. The different seed cores are roasted in an oven at 130 °C for 15 minutes. Sensory analysis of the aroma given off by the seed cores shows that a fermentation of the beans for 3 days is sufficient to obtain an acceptable cocoa aroma. The samples were fermented for 1, 2, 3, 4, 5 or 7 days and then dried. 1 ml of 200 mM citrate buffer (pH 4) was then mixed with 100 mg of each type of seed coat, the mixture was incubated for 2 hours at 50°C, the pH was adjusted to pH 7 with 1 ml of 200 mM citrate buffer and 40 μΐ of carboxypeptidase A (0.06 units / μΐ, Sigma) was added, incubated for 1 hour at 25°C and the reaction was stopped by heating at 95°C for 5 minutes. The extent of hydrolysis of the cocoa proteins was then analyzed using the method described above, determining the free amino acid content and the area under the elution peaks of the four predominant hydrolysis products of the seed coats (the products are related to the natural fermentation of the beans). The results given in Table 2 show that the hydrolysis product content in the kernel obtained from underfermented or well-fermented cocoa beans can be increased by at least 1.4 times. Furthermore, the hydrophobic amino acid content of all treated kernels and the kernels fermented for 3-7 days is always greater than 10 pmol / g dry matter. Example 1 The Theobroma cacao Trinitario ICS-95 cocoa bean is traditionally produced and is customarily used for its 50 Table 2 Fermentation days Enzymatic treatment Areas characteristic of hydrolysis products (arbitrary units) Increase in hydrolysis products Peak 1 Peak 2 Peak 3 Peak 4 1 none 565 1,017,833 - 1 present 1,181 22,626 1,191 - x2.06 2 none 1,072 2,393 1,190 - 2 present 1,880 4,044 1,968 - x1.70 HU 219 572 B Table 2 (continued) Fermentation days Enzymatic treatment Areas characteristic of hydrolysis products (arbitrary units) Increase in hydrolysis products 1st peak 2nd peak 3rd peak 4th peak 3 none 1 600 3 643 1 595 170 3 present 2 083 5 074 2 618 890 xl,52 4 none 1 583 3444 1794 202 4 present 3 107 6 051 2 836 596 xl,79 5 none 1663 3 383 1249 270 5 present 2 230 4 719 2 486 - xl,43 7 none 2 061 3 971 1202 267 7 present 2 862 5 555 2 563 - xl,46 (The retention times of hydrolysis chambers 1-4 are 9, 17.24 and 29.5 minutes.) Example 2 Theobroma cacao Trinitario ICS-95 cocoa beans were conventionally prepared and fermented for 1, 2, 3, 4, 5 or 7 days and then dried. After this, 100 μΐ of 200 mM citrate buffer (pH 4) was mixed with 100 mg of each type of cocoa bean, the mixture was incubated for 2 hours at 50°C, the pH was adjusted to pH 7 with 100 μΐ of 200 mM citrate buffer, and 40 μΐ of carboxypeptidase A (0.06 units / μΐ, Sigma) was added, followed by incubation for 1 hour at 25°C and the reaction was stopped by heating at 95°C for 5 minutes. The degree of hydrolysis of the cocoa proteins was then analyzed using the method described above, determining the free amino acid content and the area under the elution peak of the four predominant hydrolysis products of the seed cores (the products are related to the natural fermentation of the beans). Similar results were obtained as in Example 1. Example 3 35 We produce liquor from under-fermented cocoa beans from different genotypes of Theobroma cacao. The proteins of these beans give 3 visible bands on polyacrylamide gel SDS-PAGE chromatography, corresponding to 21 kD and 31 kD / 47 kD storage proteins. The hydrolysis product content of a portion of the CSF is determined using the method described above. 1 ml of 200 mM citrate buffer (pH 4) is mixed with 100 mg of a portion of the CSF, the mixture is incubated for 2 hours at 50 °C, the pH is adjusted to pH 7 with 1 ml of 200 mM citrate buffer and 40 μΐ carboxypeptidase A (0.06 units / μΐ, Sigma) is added, then incubated for 1 hour at 25 °C and the reaction is stopped by heating at 95 °C for 5 minutes. The degree of protein hydrolysis is finally analyzed by determining the hydrolysis product content of the CSF (see method above). The results, which are given in Table 3, show that the treatment according to the invention results in a significant increase in the aroma precursor content in the liquor obtained from the underfermented bean mixture. Table 3 Enzyme treatment Amino acids (pmol / g dry matter) Areas characteristic of hydrolysis products (arbitrary units) Total Hydrophobic Peak 1 Peak 2 Peak 3 Peak 4 None 22.2 7.7 - 1451 613 - Yes 35.9 14.7 771 2359 1115 724 (The retention times of hydrolysis products 1-4 are 9, 17, 24 and 29.5 minutes, respectively.) Example 4 500 g of underfermented cocoa beans obtained from different genotypes of Theobroma cacao are enzymatically treated as described in Example 3. The pH of the inactivated mixture is then adjusted to between 5 and 6, 0.3% glucose and 0.6% fructose are added, and the mixture is then dried under vacuum with gentle heating (60 °C) by suction of water until the moisture content is below 5%. It is then roasted in an oven at 130 °C for 15 minutes and the aromas emitted by the roasted mixture are analyzed using the sensory test described above. The results show that the core obtained from underfermented beans, treated and roasted according to the invention, has a strong aroma characteristic of the cocoa aroma of well-fermented and roasted beans. Example 5 Overfermented cocoa beans from different genotypes of Theobroma cacao are routinely used to produce liquor. Proteins from these beans are not detectable, and no protein bands corresponding to the 21 kD and 31 kD / 47 kD storage proteins are visible in SDS-PAGE chromatography on polyacrylamide gels. HU 219 572 B The hydrolysis product content of the CSF was determined by the methods described above. 1 ml of 200 mM citrate buffer (pH 4) was mixed with 100 mg of CSF, the mixture was incubated at 50 °C for 2 hours, the pH was adjusted to pH 7 with 1 ml of 200 mM citrate buffer, and 40 μΐ 5 carboxypeptidase At (0.06 units / μΐ, Sigma) was added, followed by incubation at 25 °C for 1 hour, and the reaction was stopped by heating at 95 °C for 5 minutes. The extent of protein hydrolysis is finally analyzed by determining the hydrolysis product content of the liquor (see methods above). The results, which are given in Table 4, show that the treatment according to the invention allows a significant increase in the aroma precursor content in the liquor obtained from the overfermented bean mixture. Table 4 Enzyme treatment Amino acids (pmol / g dry matter) Areas characteristic of hydrolysis products (arbitrary unit) All Hydrophobic Peak 1 Peak 2 Peak 3 Peak 4 no 49.5 19.2 960 2192 727 - yes 59.6 25.8 2102 4508 3026 - (The retention times of hydrolysis steps 1-4 are 9, 17, 24 and 29.5 minutes, respectively.) Example 6 500 g of overfermented cocoa beans obtained from different genotypes of Theobroma cacao are enzymatically treated as described in Example 5. The pH of the inactivated mixture is then adjusted to between 5 and 6, 0.3% glucose and 0.6% fructose are added, and the mixture is then dried under vacuum with gentle heating (60 °C) by suction of water until the moisture content is reduced to below 5%. It is then roasted in an oven at 130 °C for 15 minutes and the aromas emitted by the roasted mixture are analyzed using the sensory test described above. The results show that the core obtained from overfermented beans, treated and roasted according to the invention, has a strong aroma characteristic of cocoa aromas of well-fermented and roasted beans. Example 7 Various seed cores were prepared from conventionally fermented cocoa beans, 100 mg of each seed core was mixed with 100 μΐ of an aqueous solution of Maxin vert L10000 invertase (Gist Brocades) containing different units of activity, the mixtures were incubated for 30 minutes or 120 minutes, then the reaction was stopped by heating at 95 °C for 1 minute, and the glucose content of each mixture was determined by the method described above. The results given in Figure 1 show that 1 unit / ml of invertase is sufficient to hydrolyze more than 80% of the sugars into glucose and fructose. Example 8 175 g of juice obtained from underfermented Sanchez cocoa beans was incubated for 2 hours at 50 °C in 350 ml of water, the pH of which was adjusted to 4.5 with acetic acid solution (to activate endoproteases), then the pH was adjusted to 7 with potassium carbonate solution, and the mixture was incubated for 15 minutes at 50 °C with Maxinvert L10000 invertase (20 units / g) and carboxypeptidase A (0.06 units / ml), or the mixture was incubated for 15 minutes at 50 °C with 20 units / g of Maxinvert L10000 invertase and 1% of Corolase PP protease (Roehm, DE), or for 60 minutes at 50 °C with 20 units / g of Maxinvert L10000 invertase and 1% of Promod Incubate with 192P protease (Biocatalyst, UK) or 1% Promod 279P protease (Biocatalyst). The reaction is stopped by heating the mixture at 95 °C for 5 minutes. Finally, the degree of protein hydrolysis is analyzed by determining the hydrolysis product content of the liquor (see methods above). After the reaction is complete, the pH of the mixture is adjusted to 5.2, the mixture is dried, roasted, and subjected to sensory analysis. The results obtained are presented in Table 5, which shows that aroma precursors are formed when protease and invertase preparations are used. Example 9 Similar results to those obtained in Example 8 are obtained when under-fermented Indonesian Sulawesi cocoa beans are treated with protease under the same conditions and when the invertase is replaced by the addition of 0.6% fructose and 0.3% glucose during the enzymatic treatment. Example 10 In a manner similar to Example 8, liquor from underfermented Sanchez cocoa beans was incubated with 20 units / g invertase and various concentrations of Promod 192P protease simultaneously or sequentially at pH 4.5 until the endoprotease and Promod 192P protease were acting. The reaction was stopped by heating the mixture at 95°C for 5 minutes (except for Test 5). The hydrolysis of the proteins was then analyzed by determining the hydrolysis product content of the liquor (see methods above). The pH of the mixture was adjusted to 5.2, the mixture was dried, roasted and subjected to sensory analysis as described above. The reaction conditions are given in Table 6. The results, which are shown in Table 7, show that (1) the enzyme concentration can be reduced, (2) that all reactions can be carried out in a single step, (3) that inactivation of the mixture before drying can be avoided, (4) that the water content can be reduced during the incubation period. HU 219 572 B After the reaction, the pH of the mixtures was adjusted to 5.2, the mixtures were dried, roasted, and then subjected to sensory testing as described above. The results showed that the off-flavors, which are characteristic of insufficiently fermented cocoa beans, had disappeared. 5 Furthermore, the cocoa aroma developed in the treated mixtures is identical to that intended to be produced. In particular, the samples obtained from test 6 (reduced water content) are particularly advantageous. Table 5 Enzymes Amino Acids Hydrophobic Hydrolysis Products Formed (pmol / g dry matter) amino acid% (area: in arbitrary units) glucose (Phe, Leu, (mg / g) Total Hydrophobic Ala) Peak 1 Peak 2 Peak 3 No treatment 36.6 10.6 29.0 412 651 377 0.0 Promod 192P 49.8 15.4 30.9 607 1012 494 5.2 Promod 279P 53.4 16.3 30.5 575 958 470 5.6 Corolase PP 44.3 15.6 35.2 1053 1423 1088 1.7 Carboxypeptidase 37.6 12.9 34.3 759 1194 646 2.3 (The retention times of hydrolysis steps 1-3 are 9, 17 and 24 minutes, respectively.) Table 6 Test Enzyme Promod 192P Endoprotease Promod pH 4.5 192PpH4.5 Invertase pH 4.5 Cocoa liquor (g) Added water (ml) Heating before drying Final PH 1. Control: no treatment 2. 1.0% 2 hours 1 hour 20 units / g 175 350 yes 5.2 3. 1.0% 3 hours 20 units / g 175 350 yes 5.2 4. 0.2% 3 hours 20 units / g 175 350 yes 5.2 5. 1.0% 2 hours 1 hour 20 units / g 175 350 no 5.2 6. 1.0% 2 hours 1 hour 20 units / g 175 175 yes 5.2 Table 7 Test Amino acids (pmol / g dry matter) Hydrophobic Hydrolysis products (area: in arbitrary units) Amino acid % formed Glucose Total Hydrophobic (Phe, Leu, Ala) Peak 1 Peak 2 Peak 3 (mg / g) 1. 36.6 10.6 28.9 412 651 377 0.0 2. 60.9 18.5 30.4 736 1277 560 4.6 3. 69.3 20.7 29.8 1025 1692 751 4.3 4. 46.2 14.0 30.4 547 942 427 5.0 5. 104.6 31.6 30.2 1322 2241 1167 5.4 6. 65.3 20.6 31.5 798 1444 606 3.8 (The retention times of hydrolysis products 1-3 are 9, 17 and 24 minutes, respectively.)

Claims

PATENT CLAIMS 1. Method for treating cocoa nibs or liquor produced from cocoa beans by fermentation for 1-15 days, characterized in that it is mixed with 0.01-5% by weight of at least one technical protease in an aqueous medium at a pH of 3-8, and the proteins and peptides in the mixture are hydrolyzed.

2. The method according to claim 1, characterized in that the mixture is incubated until at least 10 pmol hydrophobic amino acids / g dry matter and at least 1.4 times more hydrolysis products are formed compared to those in the starting cocoa beans.

3. The method according to claim 1, characterized in that a seed core or liquor is prepared from cocoa beans fermented for 1-15 days, mixed with an aqueous medium with a pH of 3-6, the mixture is incubated at 40-60 °C for a period of between 10 minutes and 20 hours, the pH of the mixture is 4-8, at least one technical protease is added and hydrolyzed at 10-60 °C for 5 minutes to 20 hours.

4. The process according to claim 1, characterized in that a seed core or liquor is prepared from cocoa beans fermented for 1-15 days, mixed with an aqueous medium with a pH of 4-8, at least one technical protease is added and hydrolyzed at 10-60 °C for 5 minutes to 20 hours.

5. The method according to claim 1, characterized in that the cocoa nib or liquor is produced from a mixture of beans from cocoa trees of different fermentation stages and / or different genotypes. 15 6. The method according to any one of claims 1-5, characterized in that 1-100 units / g of mixture of technical protease is added to the 10-80% by weight cocoa nib or liquor.

7. The method according to any one of claims 1-5, characterized in that a protease and at least one other technical enzyme selected from invertases, glucosidases, cellulases, pectinases and oxidases are added to the mixture.

8. The method according to any one of claims 1-5, characterized in that at least 0.1 wt% of reducing sugar is also added to the mixture.

9. A cocoa bean or liquor composition comprising a bean or liquor prepared from cocoa beans fermented for 1-15 days, 0.01-5% by weight of technical protease and optionally 0.01-5% by weight of technical invertase.

10. A method for producing cocoa by roasting fermented cocoa beans, characterized in that a composition according to claim 9 is roasted during the production.