Irritable chocolates

Abstract

The present invention relates to non-cocoa or non-cocoa chocolate analogs and methods of making the same. The invention also extends to a fermentation process for the production of nonraw cocoa / noncoriable chocolate analogues, as well as to food and cosmetics comprising such analogues.

Description

Technical Field

[0001] The present invention relates to non-cocoa or cocoa-free chocolate analogues and methods for their preparation. The invention also extends to fermentation methods for producing raw-cocoa / cocoa-free chocolate analogues, as well as ingredients, foods, and cosmetics containing such analogues. Background Art

[0002] Raw cocoa is the seed of the tropical cacao tree, Theobroma Cacao. Cocoa and chocolate are both made from raw cocoa, and there is a high demand for it. Moreover, cocoa is a relatively expensive raw material used in a wide variety of foods. Products made from raw cocoa and raw cocoa beans, such as chocolate and cocoa, have many well-documented health benefits. For example, studies have shown that dark chocolate can reduce the risk of cardiovascular disease. In addition, unsweetened cocoa is relatively low in calories and contains essential minerals that support the health of the heart, bones, and immune system.

[0003] The taste of cocoa is also cherished worldwide and is the basis for a variety of flavored beverages and confectionery products. The various cocoa flavors depend specifically on the type of cocoa bean and the growing region, the sugar content, the content of free amino acids, and the cocoa pigments.

[0004] However, several side effects associated with the use of cocoa have been reported. These side effects include triggering migraines in sensitive individuals, increasing the risk of bleeding and bruising in patients with bleeding disorders, worsening irregular heart rhythms, interfering with blood sugar control during and after surgery, or worsening the symptoms of gastroesophageal reflux disease (GERD) or irritable bowel syndrome (IBS). The theobromine content in cocoa is also associated with severe toxicity in dogs, which is why chocolate should not be given to pet dogs.

[0005] Chocolate is the fifth-highest food in terms of global carbon emissions. Governments, industry regulations, and customers are demanding low-carbon foods on the market. And, due to the increasing concern about climate change, the future of the cocoa and chocolate industries is at stake. Some models show that starting from 2030, 28% of cocoa imports will be affected, which will in turn lead to a significant decline in the global market supply of cocoa. In addition, given that cocoa beans are transported an average of about 3,000 miles before reaching local supermarket shelves, their price is rising sharply for consumers. Moreover, approximately 1.56 million children work on cocoa farms around the world, some as young as 5 years old. The reluctance of chocolate companies to increase the price they pay to cocoa growers complicates the situation. Coupled with the increasing demand in the consumer market, this means that a substitute for cocoa-based chocolate is needed.

[0006] To overcome these challenges, synthetic chocolates and cocoa flavors made from various alternative synthetic compounds have been developed. However, these chocolate analogs cannot mimic the exact taste of cocoa and chocolate, exhibiting strong off-flavors and foreign odors, and some also have poor safety characteristics.

[0007] Therefore, it is necessary to provide a substitute for such chocolates and cocoa that can be produced from locally sourced natural products and is very similar (in taste, aroma, and texture) to the original chocolate made from cocoa beans, but without the various associated drawbacks mentioned above. Summary of the Invention

[0008] The present inventors set out to develop a new type of raw-cocoa-bean-free chocolate / cocoa analog. After a thorough analysis of various locally sourced agricultural products (i.e., legumes), the present inventors identified several candidate legumes that can be used to successfully substitute cocoa in chocolate formulations and developed and optimized a new method for manufacturing a substitute chocolate / cocoa analog.

[0009] In one embodiment, the method involves subjecting the legumes to a microbial fermentation treatment using yeast and / or bacteria to produce a cocoa-bean-free chocolate analog that has a taste and aroma similar to that of ordinary (cocoa-bean) chocolate. In another embodiment, the process involves subjecting the legumes to two separate treatments. In one treatment, the legumes are exposed to microbial fermentation, and in the other treatment, the legumes are treated without any exogenously added microorganisms. Then, a certain proportion of the microbially fermented legumes is mixed with a certain proportion of the non-microbially treated legumes to obtain a cocoa-bean-free chocolate analog that has a taste and aroma similar to that of ordinary (cocoa-bean) chocolate.

[0010] Accordingly, in a first aspect of the present invention, there is provided a cocoa-bean-free chocolate analog comprising microbially fermented and / or non-microbially fermented legumes.

[0011] In a second aspect of the present invention, there is provided a cocoa-bean-free chocolate analog comprising microbially fermented legumes.

[0012] In a third aspect, there is provided a method for producing a cocoa-bean-free chocolate analog, the method comprising contacting legumes with at least one microorganism under conditions suitable for fermentation of the legumes by the at least one microorganism to ferment the legumes, thereby forming a cocoa-bean-free chocolate analog comprising microbially fermented legumes.

[0013] In a fourth aspect, there is provided a cocoa-bean-free chocolate analog obtained by or obtainable by the method of the third aspect.

[0014] It is understood that the cocoa - free chocolate analogues described herein may also be referred to as non - cocoa chocolate analogues.

[0015] Although alternatives to cocoa beans, such as Theobroma grandiflorum and Ceratonia siliqua, are already known for their cocoa - like qualities, these crops are relatively under - developed and subject to similar burdens from seasonal variations and global transportation costs. Advantageously, however, the inventors of the cocoa - free chocolate analogues of the first, second or fourth aspect have identified several low - cost, agriculturally mature and relatively close - to - European production - facility bean candidates. Furthermore, the chocolate analogues of the present invention highly resemble or mimic traditional chocolate made from cocoa beans in terms of flavor and aroma (i.e., the tone and tone style) as well as texture.

[0016] It is understood that beans are plants of the Fabaceae or Leguminosae family, or the fruits or seeds of such plants. Dry cereal seeds can also be referred to as beans. Beans can be selected from kidney beans, soybeans, peas, chickpeas, peanuts, lentils, lupins, Prosopis, carob, tamarind, alfalfa and clover.

[0017] Preferably, the fermented beans include vicilin, a globulin storage protein associated with beans. The globulin fraction, identified as vicilin - like globulin (VCG; also known as 7S storage protein), contains a 66 kDa common precursor that, after proteolysis, consists of 3 polypeptide subunits (47, 31 and 14.5 kD; Biehl et al., 1982, Spencer and Hodge, 1992; Voight, Biehl and Wazir, 1993). More recently, by protein electrophoresis (SDS PAGE) of raw cocoa protein, the 14.5 - 15 kD band commonly observed has been shown to be a complex consisting of 3 lower - molecular - weight polypeptides of 14.5, 15 and 15.5 kDa respectively (Kratzer et al., 2009). As is common with vicilin, the precursor can form trimers. Warwick and O'Connor (1995) constructed a homology model of raw cocoa vicilin of 138 kDa size, which is close to the previously observed 144 and 150 kDa raw cocoa trimer sizes (Macdonald et al., 1991; Voight, Biehl and Wazir, 1993). 11S beans were not found in raw cocoa (Voight, Biehl and Wazir, 1993).

[0018] During microbial or non-microbial fermentation, VCG is degraded by two key enzymes. First, aspartic endoprotease results in the release of hydrophobic oligopeptides, i.e., oligopeptides ending with hydrophobic residues. Second, endopeptidase (carboxypeptidase) further degrades these products to form hydrophilic peptides and hydrophobic free amino acids (Voight and Biehl, 1995). The activities of these enzymes change during the fermentation of legumes; aspartic endopeptidase peaks midway, and carboxypeptidase peaks at the end of 144 hours of fermentation (Amin, Jinap, and Jamilah, 1997). As a result, the properties of raw cocoa VCG also change during fermentation. For example, Kumari et al. (2016) observed 5 major protein bands after protein electrophoresis of unfermented raw cocoa protein, namely vicilin subunits (15, 31, and 47 kDa), albumin (21 kDa), and a putative kinase superfamily protein of 43 kDa. Within the first 72 hours of fermentation, the 15 kDa subunit was the main target of vicilin hydrolysis, while the 31 and 47 kDa subunits only started to break down after 96 hours. However, this hydrolysis was rapid and complete, and by 120 hours, both the 31 and 47 kDa bands were completely degraded, while the approximately 15 kDa band persisted. In addition, aspartic endoprotease and carboxypeptidase have completely different optimal pH values (pH 3.5 and 5.5 - 6 respectively), and during in vitro proteolysis of VCG isolates, "nutty" aroma precursors are more prevalent at pH 4.8 - 5.6, while lower pH values (4.4 - 5.2) are associated with "cocoa" aroma precursors and more complete proteolysis, with greater release of free amino acids (Voigt et al., 2018). However, as pointed out by Biehl et al. (1985), low pH values (pH < 5) during legume fermentation may be associated with flavor and aroma losses, so "complete proteolysis" is not required.

[0019] Preferably, the fermented legumes contain convicilin. Convicilin is a globulin storage protein associated with legumes.

[0020] Preferably, the fermented beans (which can be microbially fermented and / or non-microbially fermented) are selected from a group of beans consisting of: adzuki bean (Phaseolus angularis); alfalfa (Medicago sativa); bambara groundnut (Vigna subterranean); black gram (Vigna mungo); chickpea (Cicer arietinum); cowpea (Vigna unguiculata); broad bean (Vicia faba); common bean (Phaseolus vulgaris); lima bean (Phaseolus lunatus); lentil (Lens culinaris or Lens esculenta); lupin (Lupinus albus, Lupinus angustifolius, or Lupinus luteus); moth bean (Vigna aconitifolia); mung bean (Vigna radiate); pea (Pisum sativum); pigeon pea (Cajanus cajan); sesame (Sesamum indicum); runner bean (Phaseolus coccineus); soybean (Glycine max); and field pea (Vicia sativa).

[0021] The present inventors determined the physico-chemical composition of the above beans based on data provided by the United States Department of Agriculture (USDA). The data for each bean is summarized in Table 2 of the Examples. However, an overview of the protein profiles and phenolic contents of the selected bean candidates is provided below.

[0022] Adzuki bean: Phaseolus angularis

[0023] Almost half of the adzuki bean (or red bean) protein is globulin, most of which (about 80%) is 7S vicilin. Catechin, quercetin glycosides, procyanidins, and protocatechuic acid are known to be the main polyphenols found in adzuki beans.

[0024] Alfalfa: Medicago sativa

[0025] Alfalfa contains three main storage proteins, mainly consisting of about 10% 7S-alfin, 30% 11S-medicagin, and 20% 2S albumin. Regarding the phenolic composition of alfalfa seeds, the main flavonoids found in the aerial parts of this plant are flavonoid triazines and glycosides of apigenin.

[0026] Bambara groundnut: Vigna subterranea

[0027] The total protein content of Bambara groundnut ranges from 18% to 27%. Vicilin accounts for 46% of the total protein, while legumin and albumin account for 29% and 20.5% respectively. Regarding the phenolic content of Bambara, flavonols (especially rutin) and phenolic acids such as chlorogenic acid and ellagic acid have been found in Bambara species, and their content is generally higher in red and brown beans compared to brown-eye and black-eye types. Phenols are concentrated in the hulls.

[0028] Black gram: Vigna mungo

[0029] Vicilin protein accounts for approximately 50% of the total soluble protein content of black gram (or mung bean). Data shows that in black gram mainly containing ferulic acid, the distribution of phenolic acids is mainly concentrated in the germ and seed coat. The distribution ranges of total polyphenols and total anthocyanins are 0.9 - 134.7 mg / 100 g and 5.9 - 86.8 mg / 100 g respectively, and the concentration is the highest in the seed coat.

[0030] Chickpea: Cicer arietinum

[0031] The protein composition of chickpea includes 56% globulin, 18% glutelin, 12% albumin and 2.8% prolamin. The complete 11s and 7s globulins have been identified, and the vicilin subunits are considered similar to the 7s subunits observed in pea and soybean extracts. 96 polyphenols have been identified in chickpea, among which catechins (and related pentosides), hexoside of sinapic acid, gallic acid and gambogic alcohol glycosides are the main contributors.

[0032] Cowpea: Vigna unguiculata

[0033] Globulin accounts for 51% - 72% of the total protein of cowpea, and its main component is 7s vicilin-like β-conglycinin. It shares approximately 62% of the amino acid sequence with the α subunit of β-conglycinin (from soybean). Analysis of four cowpea varieties shows that the total polyphenol (mainly flavonoids and phenolic acids) content ranges from 642 - 2376 μg / g, with Agrinawa having the highest content. Another important phenolic compound found in cowpea is catechin-3-O-glucoside. Interestingly, black-eyed peas have also been shown to contain proanthocyanidins.

[0034] Broad bean: Vicia faba

[0035] Also known as fava bean, approximately 80% of the broad bean protein is composed of globulin from 11s to 7s. The protein profile of broad bean is complex, containing vicilin and convicilin. It has been reported that broad bean is a good source of catechins, epicatechins and proanthocyanidins.

[0036] Common bean: Phaseolus vulgaris

[0037] The common bean is a highly diverse species that encompasses several well-known bean varieties such as French beans, kidney beans, black turtle beans, pinto beans, and navy beans. These beans are generally rich in globulins. The 7S protein in the common bean is called "phaseolin" and accounts for 50% of the total protein in French beans. Although most common bean varieties contain similar phenolic acids, there is diversity in their monomeric flavonoid profiles, which is partly related to their color. For example, black beans are a good source of anthocyanins, pinto beans are rich in kaempferol glycosides, red kidney beans are dominated by quercetin and kaempferol glycosides, while navy beans seem to have little detectable flavonoid content. The proanthocyanidin content of several cultivated common bean varieties is much higher than that of other common bean varieties, including the large white kidney bean (P. lunatus), the perennial bean (P. polyanthus), the scarlet runner bean (P. coccineus), and the zebra bean (P. zebra).

[0038] Lima bean: Phaseolus lunatus

[0039] The lima bean contains vicilin (phaseolin). Lima bean flour contains similar levels of p-coumaric acid and ferulic acid. Other phenolic acids and flavonoids have also been found in lima bean oil, including p-coumaric acid and its downstream metabolite rosmarinic acid.

[0040] Faba bean: Lens culinaris / Lens esculenta

[0041] Globulins and albumins account for 42% and 11% of the total protein content of the faba bean, respectively. However, the major native globulin, which accounts for 34% of the total protein in the faba bean, was found to be the legumin type. The faba bean also contains a wide range of flavan-3-ols, proanthocyanidins, and flavanol glycosides, especially catechin glycosides, quercetin diglycosides, and proanthocyanidin dimers.

[0042] Lupin: Lupinus albus / Lupinus angustifolius / Lupinus luteus

[0043] Lupins are high-protein seeds and also contain about 10% lipids. Blue lupins (L. angustifolius or narrow-leaved lupins) and yellow lupins (L. luteus) are usually grown as animal feed, while white lupins (L. albus) are mainly used for human consumption. In lupin species, the ratio of globulins to albumins is approximately 9:1, and they are divided into α-convicilin (11s, bean-like), β-convicilin (7s, legumin-like), and two minor proteins called γ-convicilin and Δ-convicilin. Regarding the phenolic composition of lupin seeds, the total polyphenol content consists of gallic acid equivalents per 100 grams and several phenylpropanoids (such as ferulic acid, catechol), benzoic acids (such as vanillin, vanillic acid), and flavonoids (such as apigenin-7-glucoside, apin) in blue, yellow, and white lupins. Additionally, lupin leaves and seeds are good sources of isoflavones, mainly the aglycone genistein and several related glycosides and 2'-hydroxy forms.

[0044] Moth bean: Vigna aconitifolia

[0045] Globulins account for 63.9% of the total soluble protein content in moth beans, while glutelins, albumins, and prolamins account for 27.8%, 5.1%, and 3.2% respectively. The total polyphenol content in 22 moth bean samples ranges from 57 ± 23 mg GAE / 100 g to 1034 ± 12 mg GAE / 100 g, with catechin, tannic acid, and gallic acid being the main components.

[0046] Mung bean: Vigna radiata

[0047] Mung beans contain 7S and 11S globulins (3% and 8% respectively), although their main storage protein is 8S legumin. Mung beans also contain various flavonoids and phenolic acids, with the glycosylated form of the flavonoid apigenin being the main form.

[0048] Pea: Pisum sativum

[0049] Protein profiles of different pea varieties show that their globulin content accounts for 49.2% - 81.8% of the total protein, with legumin having the highest content, accounting for 26.3% - 52.0% of the total globulin content. Vicilin content ranges from 5.9% - 24.5%, while convicilin only accounts for 3.9% - 8.3% of the total globulin. Although hydroxybenzoic acids dominate the phenolic composition of pea cotyledons, the polyphenols in the seed coat are mainly flavonoids (such as luteolin, apigenin) and flavonol (such as quercetin) glycosides, as well as lower levels of flavan-3-ol monomers and proanthocyanidins. Pea leaves contain various quercetin glycosides.

[0050] Pigeon pea: Cajanus cajan

[0051] The complete pigeon pea contains legumin. The phenolic constituents of pigeon pea consist of the glycosylated flavones orientin and vitexin, as well as cajanin and cajanin.

[0052] Sesame: Sesamum indicum

[0053] Although approximately 67% of sesame protein is globulin, 7S globulin accounts for only 5% of the total protein content. The main polyphenols in sesame are lignans, especially sesamin, sesamol, sesamolin and sesamol. There are also flavonoids and phenolic acids in sesame seeds, such as catechin and 1,2-dihydroxybenzoic acid.

[0054] Runner bean: Phaseolus coccineus

[0055] The main storage protein of runner bean is the complete lectin. On the other hand, caffeic acid and chlorogenic acid are the main phenolic compounds found in runner bean oil, while the seed coat is an excellent source of high molecular weight proanthocyanidins.

[0056] Soybean: Glycine max

[0057] The legumin storage protein of soybean is called β-conglycinin, which exists in the form of a trimer of three glycosylated peptides. More than 70% of the protein content in soybean consists of 7S and 11S (conglycinin) globulins, which are valued in the food industry for their nutritional value and gelling properties. Soybean is also a rich source of isoflavones genistein, daidzein and glycitein, which mainly exist in the form of 7-O-glycoside genistein, daidzin and glycitein malonate conjugates.

[0058] Common vetch: Vicia sativa

[0059] The protein of common vetch consists of approximately 50.8% globulin and 43.6% albumin. The globulin group is mainly dominated by a legume-like 10 and 6S storage protein called α-vicinins. Phenolic compounds such as kaempferol glycosides, myricetin and naringenin have been reported to be present in the roots and leaves of common vetch. In addition, the total polyphenol content of common vetch seeds is more than three times that of soybean.

[0060] Table 2 summarizes the physicochemical properties of the candidate legumes based on the data provided by the United States Department of Agriculture (USDA).

[0061] Advantageously, the present inventors evaluated the protein, polyphenol, and lipid profiles of 19 candidate legumes and assigned a similarity to the cocoa score for each legume based on a summary scoring system. In the case of characterizing the relative abundance and protein profile of 7S VCG, the legumes were scored according to their relative similarity to cocoa. Other factors were also considered, such as polyphenol components rich in flavan-3-ols and proanthocyanidins, or high fat content. Table 3 summarizes the profiles and scores of each candidate legume.

[0062] Through significant inventive activity and based on the analysis of the present inventors, six of the above-listed legumes were considered to exhibit characteristics substantially similar to raw cocoa beans / cocoa beans (i.e., legumes with a total score ≥ 4 in Table 3), including broad bean, white lupin, pea, moth bean, runner bean, and sesame.

[0063] Furthermore, in a preferred embodiment, the legume capable of being fermented is selected from a group of legumes consisting of: broad bean (vicia faba); white lupin (lupinus albus); pea (pisum sativum); moth bean (vigna aconitifolia); runner bean (phaseolus coccineus); and sesame (sesamum indicum). Most preferably, the fermented legume is broad bean.

[0064] However, Example VII explains how various legumes other than broad bean, such as lupin, green pea, moth bean, adzuki bean, and hemp seed, can also be used to develop cocoa-like analogs and are therefore preferred.

[0065] In one embodiment, at least one microorganism for microbial fermentation can be selected from yeast and / or bacteria. In one embodiment, the microbial fermentation substantially comprises yeast or consists only of yeast. Preferably, at least 70%, 80%, or 90% of the microorganisms in the fermentation comprise yeast cells. More preferably, at least 95%, 96%, 97%, 98%, 99%, or 100% of the microorganisms in the fermentation comprise yeast cells.

[0066] In another embodiment, the microbial fermentation substantially comprises bacteria or consists only of bacteria. Preferably, at least 70%, 80%, or 90% of the microorganisms in the fermentation comprise bacterial cells. More preferably, at least 95%, 96%, 97%, 98%, 99%, or 100% of the microorganisms in the fermentation comprise bacterial cells.

[0067] In yet another embodiment, the microbial fermentation comprises yeast and bacteria. Preferably, the ratio of yeast to bacteria is about 50:50. However, other ratios are also contemplated, including 10:90, 20:80, 30:70, 40:60 yeast cells to bacteria cells, or alternatively, 10:90, 20:80, 30:70, 40:60 bacteria cells to yeast cells.

[0068] In some embodiments, when yeast cells are used for microbial fermentation, only one type of yeast is used. Similarly, when bacterial cells are used for microbial fermentation, in some embodiments, only one type of bacteria is used. However, in other embodiments, the microbial fermentation can include using one or more types of yeast and / or one or more types of bacteria. For example, two, three, four or more different types of yeast or bacteria can be used.

[0069] The bacteria can be Gram-positive or Gram-negative. Preferably, the family of bacteria can be selected from a group of bacterial families consisting of: Lactobacillus; and Acetobacter. Preferably, the species of bacteria can be selected from a group of bacterial species consisting of: Lactobacillus fermentum; Lactobacillus plantarum; and Acetobacter pasteurianus. Example six describes the effects of these different microorganisms on the obtained chocolate analogues.

[0070] Preferably, the genus of yeast can be selected from a group of yeast genera consisting of: Pichia; Hanseniaspora; and Saccharomyces. Preferably, the species of yeast can be selected from a group of yeast species consisting of: Pichia kudriavzevii; Hanseniaspora opuntiae; and Saccharomyces cerevisiae.

[0071] Preferably, the microbial fermentation method comprises growing at least one microorganism and legumes in a growth medium (referred to as "synthetic pulp" in the examples and Figure 1 ). The growth medium is a solution of chemicals and nutrients for fermenting legumes. It can be understood that the legumes, the growth medium and at least one microorganism together form a microbial fermentation mixture.

[0072] Furthermore, it can be understood that in one embodiment, for example Figure 1 B and Figure 1 C show, the method comprises subjecting legumes, preferably broad beans, to microbial fermentation treatment in a growth medium. Two types of fermentation culture solutions can be prepared, one containing yeast and bacteria (Option 1), and the other containing only yeast or only bacteria (Option 2). Figure 1 Figure B shows an exemplary legume fermentation process, in which the fermentation culture solution according to Option 1 is used to produce a cocoa-free chocolate analogue, Figure 1C shows an exemplary legume fermentation process in which the fermentation culture medium according to Option 2 is used to produce a cocoa-free chocolate analogue.

[0073] However, in another embodiment, as Figure 1 shown in D, the method preferably comprises two separate treatments of legumes, such as broad beans. The method preferably comprises a first treatment and a second treatment. In the first treatment, the legumes are preferably subjected to microbial fermentation (Option 1 or Option 2) in a growth medium to produce microbially fermented legumes. In the second treatment, the legumes are separately subjected to non-microbial fermentation, preferably in a culture solution, in the absence of any exogenously added microorganisms, to produce non-microbially treated legumes. The method then comprises combining a proportion of the microbially fermented legumes and a proportion of the non-microbially fermented legumes to produce a cocoa-free chocolate analogue.

[0074] The ratio of microbially fermented legumes to non-microbially fermented legumes is between about 10%:90% and 90%:10%, or about 50%:50%.

[0075] In one embodiment, the growth medium or culture solution may comprise one or more sugars. Preferably, the one or more sugars may be selected from the group consisting of: sucrose; glucose; fructose; high-viscosity carboxymethyl cellulose; low-viscosity carboxymethyl cellulose; and / or pectin.

[0076] Suitable concentrations of sucrose can be between about 0.1% (w / v) and 10% (w / v), preferably between about 0.5% (w / v) and 7% (w / v), more preferably between about 1% (w / v) and 5% (w / v), and even more preferably between about 2% (w / v) and 3% (w / v). Most preferably about 2.5% sucrose. Suitable concentrations of glucose can be between about 0.1% (w / v) and 10% (w / v), preferably between about 1% (w / v) and 8% (w / v), more preferably between about 2% (w / v) and 6% (w / v), and even more preferably between about 3% (w / v) and 5% (w / v). Most preferably about 4% (w / v) glucose. Suitable concentrations of fructose can be between about 0.1% (w / v) and 10% (w / v), preferably between about 1% (w / v) and 8% (w / v), more preferably between about 3% (w / v) and 7% (w / v), and even more preferably between about 4% (w / v) and 6% (w / v). Most preferably about 5% (w / v) fructose. Suitable concentrations of high-viscosity carboxymethyl cellulose can be between about 0.005% (w / v) and 5% (w / v), preferably between about 0.005% (w / v) and 2% (w / v), more preferably between about 0.005% (w / v) and 1% (w / v), and even more preferably between about 0.05% (w / v) and 0.5% (w / v). Most preferably about 0.1% (w / v) high-viscosity carboxymethyl cellulose. Suitable concentrations of low-viscosity carboxymethyl cellulose can be between about 0.005% (w / v) and 5% (w / v), preferably between about 0.005% (w / v) and 2% (w / v), more preferably between about 0.005% (w / v) and 1% (w / v), and even more preferably between about 0.05% (w / v) and 1% (w / v). Most preferably about 0.8% (w / v) low-viscosity carboxymethyl cellulose.

[0077] Suitable concentrations of pectin can be between about 0.01% (w / v) and 10% (w / v), preferably between about 0.01% (w / v) and 5% (w / v), more preferably between about 0.1% (w / v) and 3% (w / v), and most preferably between about 0.5% (w / v) and 2% (w / v). Most preferably about 1% (w / v) pectin.

[0078] The growth medium or culture solution may contain one or more salts. Preferably, one or more salts may be selected from the group consisting of: calcium lactate pentahydrate; magnesium sulfate heptahydrate; and manganese sulfate monohydrate. The suitable concentration of calcium lactate pentahydrate may be between about 0.001% (w / v) and 2% (w / v), preferably between about 0.001% (w / v) and 1% (w / v), more preferably between about 0.01% (w / v) and 0.75% (w / v), and most preferably between about 0.05% (w / v) and 0.5% (w / v). Most preferably about 0.1% (w / v) of calcium lactate pentahydrate. The suitable concentration of magnesium sulfate heptahydrate may be between about 0.001% (w / v) and 1% (w / v), preferably between about 0.001% (w / v) and 0.5% (w / v), and most preferably between about 0.01% (w / v) and 0.1% (w / v). Most preferably about 0.05% (w / v) of magnesium sulfate heptahydrate. The suitable concentration of manganese sulfate monohydrate may be between about 0.001% (w / v) and 1% (w / v), preferably between about 0.001% (w / v) and 0.5% (w / v), and most preferably between about 0.01% (w / v) and 0.1% (w / v). Most preferably about 0.02% (w / v) of manganese sulfate monohydrate.

[0079] The growth medium or culture solution may contain one of one or more protein hydrolysates. Preferably, one or more protein hydrolysates are peptone. The suitable concentration of peptone may be between about 0.001% (w / v) and 7% (w / v), preferably between about 0.001% (w / v) and 5% (w / v), more preferably between about 0.01% (w / v) and 2% (w / v), and most preferably between about 0.05% (w / v) and 1% (w / v). Most preferably about 0.5% (w / v) of peptone.

[0080] The growth medium or culture solution may contain yeast extract. The suitable concentration of yeast extract may be between about 0.001% (w / v) and 7% (w / v), preferably between about 0.001% (w / v) and 5% (w / v), more preferably between about 0.01% (w / v) and 2% (w / v), and most preferably between about 0.05% (w / v) and 1% (w / v). Most preferably about 0.5% (w / v) of yeast extract.

[0081] The growth medium or culture solution may contain a surfactant. Preferably, the surfactant is Tween 80. A suitable concentration of Tween 80 may be between about 0.001% (w / v) and 2% (w / v), preferably between about 0.001% (w / v) and 1% (w / v), more preferably between about 0.01% (w / v) and 0.75% (w / v), and most preferably between about 0.05% (w / v) and 0.5% (w / v). Most preferably, Tween 80 is about 0.1% (w / v).

[0082] The growth medium or culture solution may contain citric acid. A suitable concentration of citric acid may be between about 0.0001% (w / v) and 6% (w / v), preferably between about 0.005% (w / v) and 5% (w / v), more preferably between about 0.05% (w / v) and 3% (w / v), and most preferably between about 0.5% (w / v) and 2% (w / v). Most preferably, citric acid is about 1% (w / v).

[0083] In a preferred embodiment, the growth medium or culture solution contains sucrose, glucose, fructose, citric acid, high-viscosity carboxymethyl cellulose, low-viscosity carboxymethyl cellulose, pectin, yeast extract, peptone, calcium lactate pentahydrate, Tween 80, magnesium sulfate heptahydrate, and manganese sulfate monohydrate.

[0084] In embodiments of microbial fermentation using yeast or bacteria (but not both together), the fermentation may be carried out in the following growth medium: microorganisms and legumes (i.e., leguminous plants) are added to the growth medium and co-cultured.

[0085] Yeast and Lactobacillus - LB broth (Lennox), which contains 5 g / L of NaCl, 10 g / L of tryptone, and 5 g / L of yeast extract.

[0086] Acetobacter - AA broth prepared by mixing the following ingredients: 1% (w / v) D-glucose, 1.5% (w / v) bacterial peptone, and 0.8% (w / v) yeast extract. After sterilization, 0.5% (v / v) ethanol and 0.3% (v / v) acetic acid are added to the mixture.

[0087] The growth medium or culture solution is preferably prepared by mixing reagents and adjusting the pH.

[0088] Preferably, the growth medium or culture solution has an acidic pH value. Preferably, the growth medium or culture solution has a pH value of 1 - 6, 2 - 5, or 3 - 4. In a preferred embodiment, the pH value of the growth medium is about 3.6.

[0089] As described in the examples, the inventors have found that analogues with good chocolate aroma are associated with faba bean flour treated at a lower pH. Referring to Figure 5 , the optimal pH value for chocolate aroma is shown to be 4.8 - 5.1. Additionally, Figure 6 shows that in chocolate analogues with a lower pH value, chocolate aroma begins to form on the 3rd - 4th day after baking and grinding, forms on the 7th day, and continues to become more intense for up to 1 month, and then remains stable for at least 4 months.

[0090] Preferably, the pH value of the chocolate analogue is between about 4 and about 7, more preferably between about 4.3 and about 6.8, and even more preferably between about 4.4 and about 6.7.

[0091] Preferably, the pH value of the chocolate analogue is between about 4.4 and about 6.3, more preferably between about 4.7 and about 5.9, and even more preferably between about 4.7 and about 5.5.

[0092] Preferably, the pH value of the chocolate analogue is between about 4.4 and about 5.5, more preferably between about 4.7 and about 5.2, and even more preferably, the pH value of the chocolate analogue is between about 4.8 and about 5.1.

[0093] Then, preferably, the culture medium or culture solution is autoclaved and then it can be stored at 4°C.

[0094] For microbial fermentation, preferably, the beans are contacted with at least one microorganism for one or more days at a temperature suitable for maintaining the growth of one or more microorganisms so that microbial fermentation occurs. Preferably, the microbial fermentation is allowed to proceed for at least two days, three days, four days, or five days.

[0095] In embodiments where at least one microorganism is yeast, the microbial fermentation can be carried out at a temperature of 21°C - 37°C, 23°C - 35°C, 24°C - 34°C, 26°C - 33°C, 27°C - 32°C. Preferably, the microbial fermentation mixture is cultured at a temperature of about 30°C, which is optimal for yeast cell growth.

[0096] However, in embodiments where at least one microorganism is bacteria, the microbial fermentation can be carried out at a temperature of 24°C - 50°C, 26°C - 48°C, 28°C - 46°C, 30°C - 44°C, or 32°C - 44°C, 34°C - 44°C, 36°C - 44°C, 38°C - 44°C, or 40°C - 44°C. Preferably, the microbial fermentation mixture is cultured at a temperature of about 42°C, which is optimal for bacterial cell growth.

[0097] It is understood that when yeast and bacteria are grown together in combination, a suitable temperature will be used, such as 24°C - 44°C, 26°C - 43°C, or 28°C - 42°C.

[0098] It is understood that any temperature described herein can be combined with any time described herein. Thus, for example, microbial fermentation can include culturing yeast and bacteria at 24°C - 44°C for at least 1 or 2 days. Alternatively, microbial fermentation can include culturing yeast at 21°C - 37°C for at least 1 or 2 days. Alternatively, microbial fermentation can include culturing bacteria at 24°C - 50°C for at least 1 or 2 days, and so on.

[0099] As described in the examples, the inventors demonstrated two different embodiments for the microbial fermentation of legumes to produce raw cocoa bean chocolate analogues, as Figure 1 shown in Figure 1 B and

[0100] Figure 1 Thus, in one embodiment (as shown in 7 B), the microbial fermentation method can include simultaneously microbial fermenting legumes in the presence of yeast and bacteria. In this embodiment, preferably the yeast includes Pichia kudriavzevii, Hanseniaspora opuntiae, and / or Saccharomyces cerevisiae, and most preferably Pichia kudriavzevii, Hanseniaspora opuntiae, and Saccharomyces cerevisiae. Preferably, the bacteria includes Lactobacillus fermentum, Lactobacillus plantarum, and / or Acetobacter pasterianus. And most preferably Lactobacillus fermentum, Lactobacillus plantarum, and Acetobacter pasterianus. Preferably, the yeast and bacteria are mixed or blended and aggregated together, preferably at a concentration of at least 1×10 8 or 1×10

[0101] In the first embodiment (co-fermentation of yeast and bacteria), the method includes contacting the legumes with yeast and bacterial cells at a temperature of 21°C - 37°C for at least 24 hours. Preferably, the microbial fermentation mixture is cultured at a temperature of 21°C - 37°C, 23°C - 35°C, 24°C - 34°C, 26°C - 33°C, or 27°C - 32°C. Preferably, the microbial fermentation mixture is cultured for at least 36 or 48 hours.

[0102] Preferably, the method then includes discarding the growth medium one or more days after the initial microbial fermentation. Preferably, the method includes further culturing the legumes and at least one microorganism at a temperature of 24°C - 50°C for one or more days. Preferably, the microbial fermentation mixture is cultured at a temperature of 26°C - 48°C, 28°C - 46°C, 30°C - 44°C, 32°C - 44°C, 34°C - 44°C, 36°C - 44°C, 38°C - 44°C, or 40°C - 44°C. Preferably, the microbial fermentation mixture is cultured at a temperature of 42°C. Preferably, the microbial fermentation mixture is cultured for at least two or three days. Preferably, the microbial fermentation mixture is cultured for three days. It will be understood that any temperature described herein can be combined with any time described herein. At the end of the microbial fermentation process, the method preferably includes drying the legumes. The dried legumes can then be processed to produce a suitable substrate with which food can be prepared, as described hereinafter.

[0103] In a second embodiment (as Figure 1 shown in C), the method can include first microbially fermenting the legumes in the presence of only yeast and then microbially fermenting in the presence of bacteria (and yeast). In this embodiment, preferably the yeast includes Pichia kudriavzevii, Hanseniaspora opuntiae, and / or Saccharomyces cerevisiae, and most preferably is Pichia kudriavzevii, Hanseniaspora opuntiae, and Saccharomyces cerevisiae. Preferably, the bacteria includes Lactobacillus fermentum, Lactobacillus plantarum, and / or Acetobacter pasteurianus, and most preferably is Lactobacillus fermentum, Lactobacillus plantarum, and Acetobacter pasteurianus. Preferably, the yeast and cells are kept separate in separate pools, each at a concentration of at least 1×10 7 or 1×10 8 cells / mL.

[0104] In the second embodiment (initial yeast-only fermentation followed by yeast and bacteria fermentation), the method includes contacting the legumes with yeast cells at a temperature of 21°C - 37°C for at least 24 hours. Preferably, the microbial fermentation mixture is cultured at a temperature of 21°C - 37°C, 23°C - 35°C, 24°C - 34°C, 26°C - 33°C, or 27°C - 32°C. Preferably, the microbial fermentation mixture is cultured at a temperature of 30°C. Preferably, the microbial fermentation mixture is cultured for at least 36 or 48 hours.

[0105] Preferably, the method then includes discarding the growth medium one or more days after the initial microbial fermentation. Preferably, the method includes contacting the fermented beans with bacterial cells at a temperature of 24°C - 50°C for at least 24 hours. Preferably, the microbial fermentation mixture is incubated at a temperature of 26°C - 48°C, 28°C - 46°C, 30°C - 44°C, 32°C - 44°C, 34°C - 44°C, 36°C - 44°C, 38°C - 44°C, or 40°C - 44°C. Preferably, the microbial fermentation mixture is incubated at a temperature of 42°C. Preferably, the mixture is incubated for at least two or three days. Preferably, the microbial fermentation mixture is incubated for three days. It will be understood that any temperature described herein can be combined with any time described herein.

[0106] At the end of the microbial fermentation process, the method preferably includes drying the beans. The dried beans can then be processed to produce a suitable substrate with which food can be prepared, as described hereinafter.

[0107] In a third embodiment (not shown in the figures), the method may include first subjecting the beans to microbial fermentation in the presence of only bacteria and then in the presence of yeast (and bacteria). In this embodiment, preferably the yeast comprises Pichia kudriavzevii, Hanseniaspora opuntiae, and / or Saccharomyces cerevisiae, and most preferably is Pichia kudriavzevii, Hanseniaspora opuntiae, and Saccharomyces cerevisiae. Preferably, the bacteria comprise Lactobacillus fermentum, Lactobacillus plantarum, and / or Acetobacter pasteurianus, and most preferably are Lactobacillus fermentum, Lactobacillus plantarum, and Acetobacter pasteurianus. Preferably, the yeast and bacteria are kept separate in separate pools, each at a concentration of at least 1×10 7 or 1×10 8 cells / mL.

[0108] In the third embodiment (initial bacterial fermentation followed by bacterial and yeast fermentation), the beans are contacted with bacterial cells at a temperature of 24°C - 50°C for at least 24 hours. Preferably, the microbial fermentation mixture is incubated at a temperature of 26°C - 48°C, 28°C - 46°C, 30°C - 44°C, 32°C - 44°C, 34°C - 44°C, 36°C - 44°C, 38°C - 44°C, or 40°C - 44°C. Preferably, the microbial fermentation mixture is incubated at a temperature of 42°C. Preferably, the mixture is incubated for at least two or three days. Preferably, the microbial fermentation mixture is incubated for three days.

[0109] Preferably, the method subsequently includes discarding the growth medium one or more days after the initial microbial fermentation. Preferably, the method includes contacting the microbially fermented legumes with yeast cells at a temperature of 21°C to 37°C for at least 24 hours. Preferably, the microbially fermented mixture is incubated at a temperature of 21°C to 37°C, 23°C to 35°C, 24°C to 34°C, 26°C to 33°C, or 27°C to 32°C. Preferably, the microbially fermented mixture is incubated at a temperature of 30°C. Preferably, the microbially fermented mixture is incubated for at least 36 or 48 hours. It should be understood that any temperature described herein can be combined with any time described herein. At the end of the microbial fermentation process, the method preferably includes drying the legumes. The dried legumes can then be processed to produce a suitable substrate, with which food can be prepared, as described later.

[0110] In embodiments where the method includes non-microbial fermentation, the method includes using a medium with a pH between 3 and 5 (preferably about 3.6), so culturing in this medium will tend to acidify the legumes (preferably broad beans). In addition, when the legumes (e.g., broad beans) are completely submerged when they start to germinate, the available oxygen will be rapidly consumed for respiration. Under anaerobic conditions, fermentation metabolism begins through the oxidation of sugars, resulting in the production of ethanol, lactic acid, and possibly acetic acid. The accumulation of lactic acid and acetic acid can further lower the pH of the legumes (preferably broad beans).

[0111] Preferably, the non-microbially fermented mixture is incubated at a first temperature of 20°C to 38°C, 23°C to 37°C, 24°C to 36°C, 25°C to 35°C, 26°C to 34°C, 27°C to 33°C, 28°C to 32°C, or 29°C to 31°C. Preferably, the non-microbially fermented mixture is incubated at a temperature of 30°C. Preferably, the mixture is incubated for at least one or two days. Preferably, the non-microbially fermented mixture is incubated for two days.

[0112] Preferably, the non-microbially fermented mixture is incubated at a second temperature of 26°C to 48°C, 28°C to 46°C, 30°C to 44°C, 32°C to 44°C, 34°C to 44°C, 36°C to 44°C, 38°C to 44°C, or 40°C to 44°C. Preferably, the non-microbially fermented mixture is incubated at a temperature of 42°C. Preferably, the mixture is incubated for at least one, two, or three days. Preferably, the non-microbially fermented mixture is incubated for three days.

[0113] In a preferred embodiment, the method includes drying the beans after microbial or non - microbial fermentation. Preferably, the beans are dried by heating in an oven. Preferably, the beans are dried in the oven at a temperature of about 21°C - 37°C for one day or more. Preferably, the beans are dried in the oven at a temperature of 23°C - 35°C, 25°C - 40°C, 28°C - 38°C or 30°C - 36°C. During this period, the beans are mixed on the 1st, 2nd and 3rd days of drying to minimize bean caking. Preferably, the legumes are dried in an oven at 35°C. Preferably, the drying is carried out for two, three, four, five, six or seven days. Preferably, the beans are dried in the oven for seven days.

[0114] In one embodiment, the cocoa - free chocolate analogue containing microbial / non - microbial beans can be in solid, liquid or powder form.

[0115] In one embodiment, the cocoa - free chocolate analogue containing microbially and / or non - microbially fermented beans can be contained or converted into a paste. Figure 2 An embodiment showing how to prepare a paste from fermented beans is presented.

[0116] Thus, preferably, the method includes roasting microbially and / or non - microbially fermented beans. Preferably, the method includes crushing and winnowing the roasted beans to form a plurality of grits. Preferably, the method includes refining and concentrating the plurality of grits to form a non - cocoa chocolate liquor. Preferably, the method includes adding one or more additives to the non - cocoa chocolate liquor. It is noted that when using authentic cocoa beans, grinding roasted beans produces a viscous liquid / paste called "liquor (cocoa liquor, cocoa mass)". This is because cocoa beans contain approximately 40% fat (called "cocoa butter"). However, broad beans have a very low fat content. Therefore, when the processed broad beans of the present invention are roasted and ground, a powder (not a liquid) is produced. To produce a "liquid" from broad bean powder, a fat with properties similar to cocoa butter (such as shea butter) can be added.

[0117] Preferably, the method includes repeating the above steps on the non - cocoa chocolate solution, thereby producing a non - cocoa chocolate analogue paste.

[0118] Preferably, the method includes tempering the non - cocoa chocolate analogue paste. Preferably, the method includes molding the tempered non - cocoa chocolate analogue paste.

[0119] In one embodiment, baking fermented or non-fermented legumes comprises heat-treating the legumes at at least 150 °C for at least 1 minute. Preferably, baking fermented legumes comprises heat-treating the legumes at at least 170 °C, 190 °C or 210 °C, more preferably at least 220 °C, 240 °C or 250 °C. Preferably, baking fermented legumes comprises heat-treating the legumes at below 350 °C, 330 °C or 310 °C, more preferably at below 300 °C, 290 °C or 280 °C. Preferably, baking fermented legumes comprises heat-treating the legumes at 150 to 350 °C, more preferably between 180 and 280 °C. Preferably, the baking is carried out for at least 2, 3 or 4 minutes, more preferably at least 5, 6 or 7 minutes. Preferably, roasting is carried out for 5 to 20 minutes between 180 and 280 °C. It will be understood that any temperature described herein can be combined with any time described herein.

[0120] In another embodiment, crushing of the legumes is carried out in a mechanical or electric cracker. In another embodiment, winnowing of the legumes is carried out in a mechanical or electric winnower.

[0121] In one embodiment, crushing and winnowing of the legumes are carried out simultaneously. In one embodiment, crushing and winnowing of the legumes are carried out sequentially. In a preferred embodiment, crushing of the legumes is carried out before winnowing of the legumes.

[0122] In one embodiment, multiple kibbles are preheated before the refining and concentration steps. In another embodiment, multiple kibbles are not preheated before the refining and concentration steps.

[0123] In one embodiment, refining and concentration of the fermented legumes comprises heat-treating the legumes in a concentrator.

[0124] In another embodiment, tempering of the fermented legumes comprises heat-treating the legumes in a temperer.

[0125] In another embodiment, the non-cocoa chocolate analogue paste is stored in a temperature range of 5 °C - 25 °C; 7 °C - 23 °C or 7 °C - 33 °C. Preferably, the non-cocoa chocolate analogue paste is stored in a temperature range of 10 °C - 20 °C.

[0126] However, in an alternative embodiment, the cocoa-free chocolate analogue comprising microbial and / or non-microbial legumes can comprise a powder. Figure 3 Embodiments are shown of how to prepare a powder from fermented legumes. However, in an alternative embodiment, the cocoa-free chocolate analogue comprising microbial and / or non-microbial legumes can comprise a liquid.

[0127] In an alternative embodiment, the cocoa-free chocolate analogue comprises microbially and / or non-microbially fermented broad beans, which are preferably combined with a cocoa-free chocolate analogue that comprises non-microbially fermented non-broad beans.

[0128] However, in another alternative embodiment, the cocoa-free chocolate analogue comprises microbially and / or non-microbially fermented broad beans, which are preferably combined with a cocoa-free analogue that comprises biochemically fermented non-broad beans.

[0129] The non-broad bean legumes can be any of the legumes described herein.

[0130] Preferably, the method comprises roasting the microbially or non-microbially fermented legumes. Preferably, the method comprises crushing and winnowing the legumes to form a plurality of grits. Preferably, the method comprises refining and concentrating the plurality of grits to form a non-cocoa chocolate liquor. Preferably, the method comprises pressing the non-cocoa chocolate liquor to form a non-cocoa chocolate cake. Preferably, the method comprises grinding the non-cocoa chocolate cake, thereby producing a non-cocoa chocolate analogue powder.

[0131] In one embodiment, roasting the microbially or non-microbially fermented legumes comprises heat-treating the legumes at at least 150 °C for at least 1 minute. Preferably, roasting the fermented legumes comprises heat-treating the legumes at at least 170 °C, 190 °C or 210 °C, more preferably at least 220 °C, 240 °C or 250 °C. Preferably, roasting the fermented legumes comprises heat-treating the legumes at below 350 °C, 330 °C or 310 °C, more preferably below 300 °C, 290 °C or 280 °C. Preferably, roasting the fermented legumes comprises heat-treating the legumes between 150 °C and 350 °C, more preferably between 180 °C and 280 °C. Preferably, roasting is carried out for at least 2, 3 or 4 minutes, more preferably at least 5, 6 or 7 minutes. Preferably, roasting is carried out at 180 °C to 280 °C for 5 to 20 minutes. It should be understood that any of the temperatures described herein can be combined with any of the times described herein.

[0132] In another embodiment, crushing of the legumes is carried out in a mechanical or electric cracker. In another example, winnowing of the legumes is carried out in a mechanical or electric winnower.

[0133] In one embodiment, crushing and winnowing of the legumes are carried out simultaneously. In one example, crushing and winnowing of the legumes are carried out sequentially. In a preferred embodiment, crushing of the legumes is carried out before winnowing of the legumes.

[0134] In one embodiment, the plurality of grits are preheated before the refining and concentrating step. In another embodiment, the plurality of grits are not preheated before the refining and concentrating step.

[0135] In one embodiment, the non-cocoa chocolate powder cake is first coarsely ground and then finely ground.

[0136] In another embodiment, the non-cocoa chocolate analogue powder is stored in a temperature range of 5°C - 25°C, 7°C - 23°C or 7°C - 33°C. Preferably, the non-cocoa chocolate analogue paste is stored in a temperature range of 10°C - 20°C.

[0137] The present inventors have shown that various different solid or liquid foods can be prepared using the cocoa-bean-free chocolate analogues described herein.

[0138] Thus, in a fifth aspect, there is provided a product ingredient comprising a cocoa-bean-free chocolate analogue according to the first, second or fourth aspect.

[0139] Thus, in a sixth aspect, there is provided a food or beverage comprising a cocoa-bean-free chocolate analogue according to the first, second or fourth aspect, or an ingredient according to the fifth aspect.

[0140] The cocoa-bean-free chocolate analogue can be in the form of a powder, pulp, liquid, solid or paste.

[0141] Roasted and ground broad beans may produce a dry powder. If shea butter is used, this dry powder can be added to a fat (such as shea butter) to form a solid that melts when heated to 31°C - 38°C. This liquid is equivalent to the liquid produced when cocoa beans are ground, but for cocoa beans, no fat needs to be added.

[0142] The food is preferably a confectionery product. For example, the food can be a bar, a molded shape, a bean, a truffle, a sauce, a coated fruit, a nut and other inclusions, a baked filling, a product coating such as a doughnut or a muffin, a coating on yogurt, ice cream or other desserts. The beverage can be a hot drink or a cold drink.

[0143] In a seventh aspect, there is provided a cosmetic comprising a cocoa-bean-free chocolate analogue according to the first, second or fourth aspect, or an ingredient according to the fifth aspect.

[0144] The cosmetic may be a skin care product such as a moisturizer, an exfoliant, a lip balm, eyeshadow, soap, a body cream or butter, or a hair mask.

[0145] All features described herein (including any accompanying claims, abstract and drawings) and / or all steps of any method or process disclosed, can be combined with any of the above aspects in any combination, except combinations where at least some of the features and / or steps are mutually exclusive. Description of the Drawings

[0146] To better understand the present invention and to show how embodiments of the present invention may be implemented, examples will now be given with reference to the accompanying drawings.

[0147] Figure 1 Schematic diagrams of legume fermentation according to two embodiments of the present invention are shown. First, as Figure 1 shown in A, a fermentation inoculum is prepared from a single colony of yeast or bacteria. The colony is grown in a selective medium, and a preparation of the growing microbial cells is pooled in a vial and stored until the fermentation process. Two types of fermentation inocula are prepared, one containing both yeast and bacteria (Option 1), and a second fermentation inoculum containing only yeast or only bacteria (Option 2). Figure 1 B shows an exemplary legume fermentation process according to the present invention, in which the fermentation inoculum according to Option 1 is used, Figure 1 C shows an exemplary legume fermentation process according to the second method. Figure 1 D shows another exemplary legume fermentation process according to the present invention, in which two separate reactions are carried out, namely the microbial treatment and the non-microbial treatment of the legumes. First, the legumes are treated microbially (Option 1 or Option 2) to produce microbially fermented legumes, and then the legumes are treated separately without any microorganisms to produce non-microbially treated legumes. Then, a certain proportion of the microbially fermented legumes and a certain proportion of the non-microbially treated legumes are mixed or blended together to obtain a cocoa-free chocolate analogue.

[0148] Figure 2 A schematic diagram of one embodiment of a method for making a chocolate analogue paste is shown.

[0149] Figure 3 A schematic diagram of one embodiment of a method for manufacturing a chocolate analogue powder is shown.

[0150] Figure 4 Photographs of dried broad beans after 3 hours of rehydration treatment are shown. (a) 0 hours, (b) 24 hours of cultivation, (c) 48 hours of cultivation, (d) 72 hours of cultivation, (e) 120 hours of cultivation, (f) dried beans after 120 hours of fermentation, (g) unfermented dried beans.

[0151] Figure 5 A bar graph showing the percentage frequency of chocolate aroma within each interval, and the optimal pH value of roasted broad bean powder that produces the best chocolate aroma is shown.

[0152] Figure 6 A diagram showing the aroma characteristics of beans over time after roasting and grinding is shown.

[0153] Figure 7Shows the flavor profile of a paste (powder + shea butter 1:1 w / w) scored by five independent assessors; broad bean flour is representative of the powders made from ten independent bean preparations. It can be seen that the broad bean flour of the present invention shows flavor characteristics very similar to those of cocoa powder sold in supermarkets (Tesco) and Peruvian cocoa powder.

[0154] Figure 8 Shows the flavor profile of a paste (bean flour, sugar and shea butter) made from broad bean flour that has been processed, roasted and ground, including a comparison using broad beans (NK0034) treated with our non-microbial process. The flavor was evaluated by 5 independent individuals.

[0155] Figure 9 Shows the flavor profile of a chocolate analogue according to the present invention (referred to herein as "NuKoKo chocolate") and genuine supermarket Tesco brand milk chocolate and dark chocolate (Mollys). The flavor was evaluated by 5 independent individuals.

[0156] Figure 10 Shows the flavor profile of a paste (bean flour, sugar and shea butter) made from processed, roasted and ground broad beans and a range of other beans and seeds, all of which are treated using the non-microbial method of the present invention. (A) Hemp seeds, (B) Adzuki beans, (C) Yardlong beans, (D) Lupins, (E) Peas. Broad beans (NK0034) are shown for comparison (A, B, C, D, E). (F) All non-broad bean and seed groups for comparison. The flavor was evaluated by 3 independent individuals. Detailed Description

[0157] Example

[0158] Fermentation is an important step necessary for raw cocoa / cocoa beans to develop their unique chocolate flavor and aroma. To develop non-cocoa chocolate alternatives, the present inventors first identified various beans that the inventors believed had the key characteristics that could provide a unique consistency, flavor, color and aroma to traditional chocolate, and subsequently selected a subset of beans that met the desired characteristics. The inventors also developed and highly optimized a fermentation process applied to the selected beans (such as broad beans), whether or not certain microorganisms such as yeast and bacteria were present, which made the cocoa analogue powder made from these beans have the same characteristics (in terms of taste and aroma) as ordinary cocoa powder. The fermented beans can then be processed to prepare a chocolate analogue, from which various foods can be formed.

[0159] In a first embodiment, as Figure 1As shown in B and 1C, the process involves the microbial fermentation treatment of legumes (such as broad beans). Two types of fermentation inoculants were prepared, one containing both yeast and bacteria (Option 1), and the second fermentation inoculant containing only yeast or only bacteria (Option 2). Figure 1 B shows an exemplary legume fermentation process in which the fermentation inoculant according to Option 1 is used to produce a cocoa - free chocolate analogue, Figure 1 C shows an exemplary legume fermentation process in which the fermentation inoculant according to Option 2 is used to produce a cocoa - free chocolate analogue.

[0160] In a second embodiment, as Figure 1 shown in D, two separate treatments are carried out on legumes (such as broad beans). First, the legumes are treated with microorganisms (Option 1 or Option 2) to produce microbially fermented legumes. Second, the legumes are treated separately without any microorganisms to produce non - microbially treated legumes. Subsequently, a certain proportion of microbially fermented legumes is mixed with a certain proportion of non - microbially treated legumes to obtain a cocoa - free cocoa powder analogue.

[0161] Materials and Methods

[0162] Selection of Candidate Beans

[0163] The present inventors initially established specific selection criteria for legumes considered for further testing phases, which included crops with low cost, agricultural maturity, and relatively close cultivation to production facilities (especially in Europe). Based on the inventors' extensive research, the following attributes were determined to be necessary for legume candidates to exhibit chocolate characteristics (flavor and aroma) after fermentation. These attributes include:

[0164] 1) Sufficient content of 7S VCG (pea protein 7S vicilin) protein or its homologues.

[0165] 2) An easily utilizable carbohydrate source that can overcome the drawback of reducing sugar content during fermentation.

[0166] 3) High content of flavan - 3 - ols and proanthocyanidins, which can enhance the flavor and color of chocolate.

[0167] For candidate legumes that only meet the first attribute, the legumes are pre - enriched with a suitable food - grade sugar, flavan - 3 - ols, and proanthocyanidin source (such as apple juice or grape juice) before fermentation.

[0168] Bean Fermentation

[0169] Materials

[0170] Unless otherwise stated, the chemicals and plastic consumables used for fermenting the selected beans were purchased from Sigma-Aldrich and Scientific Laboratory Supplies Limited. All solutions were prepared using reverse osmosis (RO) water.

[0171] Sterilization

[0172] The solutions and consumables used in this invention were heat sterilized by autoclaving at 121 °C (130 kPa) for 15 minutes.

[0173] Step 1: Preparation of Synthetic Pulp (Microbial Fermentation Growth Medium)

[0174] ● Prepare synthetic pulp by mixing the following components: 2.5% (w / v) sucrose (catalog number: S0389-1KG)

[0175] ● 4% (w / v) glucose (catalog number: G7021-5KG)

[0176] ● 5% (w / v) fructose (catalog number: F0127-1KG)

[0177] ● 1% (w / v) citric acid (catalog number: C0759-1KG)

[0178] ● 0.14% (w / v) high-viscosity carboxymethyl cellulose (catalog number: C5678-1KG)

[0179] ● 0.77% (w / v) low-viscosity carboxymethyl cellulose (catalog number: C5013-1KG)

[0180] ● 1.09% (w / v) pectin (catalog number: P9135-500G)

[0181] ● 0.5% (w / v) yeast extract (catalog number: 70161-500G)

[0182] ● 0.5% (w / v) peptone (catalog number: 91249-500G)

[0183] ● 0.1% (w / v) calcium lactate pentahydrate (catalog number: C8356-250G)

[0184] ● 0.1% (v / v) Tween 80 (catalog number: P4780-100ML)

[0185] ● 0.05% (w / v) magnesium sulfate heptahydrate (catalog number: M2773-1KG)

[0186] ● 0.02% (w / v) manganese sulfate monohydrate (catalog number: M7899-500G)

[0187] Then adjust the pH value of the mixture of the above components to 3.6 to produce a growth medium suitable for yeast and bacteria, enabling the fermentation of beans by microorganisms. Then sterilize the mixture according to the above method and store it at 4°C.

[0188] Step 2: Preparation of Selective Agar and Broth (Inoculum Growth Medium)

[0189] Use the following agar media:

[0190] Acetic Acid (AA) Agar

[0191] AA agar is prepared by mixing the following components: 1% (w / v) D-glucose, 1.5% (w / v) bacteriological peptone, 0.8% (w / v) yeast extract, and 2% (w / v) agar. Then sterilize the mixture as described above. After sterilization, add 0.5% (v / v) ethanol and 0.3% (v / v) acetic acid to the mixture and adjust the final pH value of the mixture to 4.5.

[0192] Acetic Acid (AA) Broth

[0193] AA broth is prepared by mixing the following components: 1% (w / v) D-glucose, 1.5% (w / v) bacteriological peptone, and 0.8% (w / v) yeast extract. Then sterilize the mixture as described above. After sterilization, add 0.5% (v / v) ethanol and 0.3% (v / v) acetic acid to the mixture.

[0194] De Man, Rogosa, Sharpe (MRS) Agar:

[0195] MRS agar (Catalog No.: 69964-500G) is prepared according to the manufacturer's instructions. Summarized as follows, add 61.15 grams of MRS agar to 800 ml of reverse osmosis water containing 1 ml of Tween 80 (Catalog No.: P8074). Then boil the suspension until the medium is completely dissolved and adjust the total volume to 1000 ml. Then sterilize the mixture as described above.

[0196] Yeast Peptone Glucose (YPG) Agar:

[0197] YPG agar is prepared by mixing the following components: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / w) glucose. Adjust the pH value of the mixture to 5.6, then add 2% (w / v) agar. Then sterilize the solution as described above.

[0198] Microorganisms

[0199] The inventors have identified six microorganisms (yeasts and bacteria) that show the best fermentation results for the selected legumes. These microorganisms include: Pichia kudriavzevii, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, Lactobacillus plantarum, and Acetobacter pasteurianus. The inventors further observed that each microorganism was selected on a specific agar medium, as shown in the following table:

[0200] Table 1 Selective Agar for Each Microorganism

[0201]

[0202]

[0203] Step 3: Preparation of Fermentation Inoculum

[0204] To prepare the fermentation inoculum, the following steps are carried out under aseptic conditions:

[0205] 1. Prepare the selective agar as described above and pour it into sterile 9 cm Petri dishes (20 ml each), cool and harden.

[0206] 2. Inoculate the glycerol stock of each microorganism on its respective selective agar plate (as shown in Table 1) and incubate at 28 °C for 72 hours.

[0207] 3. For each isolate, 15 ml tubes containing 3 ml of Luria broth Lennox were prepared, but Acetobacter pasteurianus required 15 ml tubes containing 3 ml of AA broth. The tubes were then sterilized as described above. For AA broth, 0.5% (v / v) ethanol and 0.3% (v / v) acetic acid were added to the tubes after sterilization.

[0208] 4. Select a colony for each isolate from the agar plate and transfer it to 3 ml of the corresponding growth medium and incubate in an orbital shaker at 28 °C, 200

[0209] rpm for 72 hours.

[0210] 5. Harvest the cells by centrifugation at 4000 x rpm for 8 minutes at 4 °C.

[0211] 6. Discard the supernatant and add 5 ml of sterile 10 mM MgCl2 to the tube.

[0212] 7. Invert the tube 10 times to resuspend the precipitated cells, then centrifuge at 4000 x rpm for 8 minutes at 4 °C.

[0213] 8. Repeat steps 6 - 7 two more times for a total of three washes.

[0214] 9. Finally, resuspend the cells in 1 mL of sterile 10 mM MgCl2 and measure the optical density at 600 nm (OD600) of each isolate (1 OD600 = 1×10 9 cells / mL) to determine the cell concentration.

[0215] 10. Once the microorganisms were purified, two different fermentation inocula were prepared as follows:

[0216] Option 1 (Yeast and Bacteria Combination)

[0217] Pool all isolates of purified yeast (Pichia kudriavzevii, Hanseniaspora opuntiae, and Saccharomyces cerevisiae) and bacteria (Lactobacillus fermentum, Lactobacillus plantarum, and Acetobacter pasteurianus), with a concentration of 1×10 8 cells / mL (dilute the cells with sterile 10 mM MgCl2 if necessary).

[0218] Option 2 (Only Yeast or Only Bacteria)

[0219] Mix the yeast isolates (Pichia kudriavzevii, Hanseniaspora opuntiae, and Saccharomyces cerevisiae) together and mix the bacterial isolates (Lactobacillus fermentum, Lactobacillus plantarum, and Acetobacter pasteurianus) together. Each combination is pooled into a separate pool, with a concentration of 1×10 8 cells / mL (dilute the cells with sterile 10 mM MgCl2 if necessary).

[0220] For each option, prepare 1 mL of inoculum and store it at 4°C for no more than 24 hours.

[0221] Step 4: Bean Fermentation

[0222] To ferment the beans, two different protocols were designed depending on whether the fermentation inoculum of Option 1 ( Figure 1 Protocol 1 in B) or the fermentation inoculant of Option 2 ( Figure 1 Protocol 2 in C) was used. The protocol included the following steps under sterile conditions:

[0223] Sterilization and rehydration of the beans

[0224] The selected beans (selection criteria are described below) were fermented as follows. The experiment was repeated for each fermentation. Weigh 125 g of beans using an analytical balance and transfer them to a 500 mL reagent glass bottle. The weight of the beans and the volume of the solution can be adjusted as needed to produce more or less material.

[0225] 1. Prepare a bean sterilization solution (70% ethanol, 0.05% Triton X-100 (Catalog No.: T8787-250ML)) (prepare 100 mL for each fermentation replicate experiment).

[0226] 2. Add the sterilization solution to the beans and stir for 30 minutes at 200 rpm and 25 °C using an orbital shaker.

[0227] The following steps are carried out under sterile conditions.

[0228] 3. Use a sterile 25 mL disposable polystyrene serological pipette to discard the sterilization solution.

[0229] 4. Subsequently, wash the beans by adding 250 mL of sterile reverse osmosis water to the bottle, invert the bottle 10 times and discard the wash solution to mix.

[0230] 5. Repeat step 5 two more times for a total of three washes.

[0231] 6. Add sterile reverse osmosis water (250 mL) to the bottle and incubate the beans at 25 °C for 3 hours to rehydrate them. The rehydration time can be increased and the beans can be further rehydrated at 4 °C or 25 °C to promote the germination process.

[0232] 7. Alternatively, the beans can be pre-enriched with a suitable food-grade sugar and a source of flavan-3-ols / proanthocyanidins (such as apple juice or grape juice) before fermentation by incorporating them into the rehydration solution.

[0233] Protocol 1 (Combined Microbial Fermentation)

[0234] 1. As described above, first rehydrate the beans in water.

[0235] 2. Discard the excess rehydration water and transfer the beans to a sterile SacO2 microbox (Catalog No.: TP1600+TPD1600#30WH).

[0236] 3. Add 100 mL of the synthetic pulp prepared as described above to the beans in the microbox, and then add 1 mL of a mixed fermentation inoculum containing yeast isolate and bacterial isolate (the fermentation inoculum of Option 1).

[0237] 4. Mix the beans, synthetic pulp and fermentation inoculum, seal the fermentation chamber with a filter lid and incubate at 30 °C.

[0238] 5. After 48 hours, discard the synthetic pulp and mix the beans.

[0239] 6. Then incubate the beans at 42 °C for another 72 hours for a total incubation of 120 hours ( Figure 1 B).

[0240] Protocol 2 (Sequential Microbial Fermentation: Yeast First then Bacteria)

[0241] 1. As described above, the beans are first rehydrated in water.

[0242] 2. Discard the excess rehydration water and transfer the beans to a sterile SacO2 microchamber (Catalog No.: TP1600+TPD1600#30WH).

[0243] 3. Add 100 mL of the synthetic pulp prepared as described above to the beans in the microchamber, and then add 1 mL of a mixed fermentation inoculum containing only yeast isolate.

[0244] 4. Mix the beans, synthetic pulp, and fermentation inoculum, seal the fermentation chamber with a filter lid, and incubate at 30 °C.

[0245] 5. After 48 hours, discard the synthetic pulp, add 1 mL of a mixed fermentation inoculum containing only bacterial isolate (the fermentation inoculum of Option 2) to the beans, and then stir the beans.

[0246] 6. Then incubate the beans at 42 °C for an additional 72 hours ( Figure 1 C).

[0247] Step 5: Drying Beans

[0248] After the fermentation process is complete, place the beans on a foil tray and put them in an oven at 35 °C for 7 days to reduce the water content of the beans to less than 7% and control the acidity of the beans. During this period, stir the beans on the 1st, 2nd, and 3rd days of drying to minimize bean caking.

[0249] Method for Neutralizing the Acidity of Chocolate Analogue, Enhancing Color and Generating Mild Flavor

[0250] "Alkalization treatment (Dutching)" is carried out by washing the cocoa powder with a potassium carbonate solution. This solution neutralizes the acidity of the powder to a pH of about 7, giving it a rich dark brown color. The powder treated by alkalization is often milder and has a more earthy flavor than the natural powder.

[0251] Adding Lipid Portion to Cocoa Analogue

[0252] In traditional chocolate made from cocoa beans, although the lipid component of the cocoa beans is crucial for the physical properties of chocolate, it does not change significantly during the fermentation process and does not seem to directly affect the flavor and aroma of chocolate. In view of the above, and considering that beans generally have a lower fat content compared to cocoa beans, lipid components (including standard cocoa butter substitutes and / or extenders) are added to the cocoa analog of the present invention to produce a chocolate analog. Other available fats include shea butter, palm, and coconut, as well as rapeseed oil-based fats.

[0253] Adding Sweetener to Chocolate Analogue

[0254] Use beet or cane sugar, as well as coconut sugar. Additionally, non-sugars can be used, which can provide more health benefits from other food fibers.

[0255] Adding Flavoring to Chocolate Analogue

[0256] Some brands of caramel or vanilla can be added to the base flavor. Any type of spice can be added to the final consumer product, such as orange, mint spice, etc.

[0257] Results

[0258] Example 1: Selection of Candidate Beans

[0259] The inventors have identified a number of candidate legumes that meet the initial requirements of low cost, agricultural maturity, and being grown relatively close to the production facility; these legumes include: adzuki bean (Phaseolus angularis); alfalfa (Medicago sativa); bambara groundnut (Vigna subterranean); black gram (Vigna mungo); chickpea (Cicer arietinum); cowpea (Vigna unguiculata); broad bean (Vicia faba); common bean (Phaseolus vulgaris); lima bean (Phaseolus lunatus); lentil (Lens culinaris or Lens esculenta); lupin (Lupinus albus, Lupinus angustifolius, or Lupinus luteus); moth bean (Vigna aconitifolia); mung bean (Vigna radiate); pea (Pisum sativum); pigeon pea (Cajanus cajan); sesame (Sesamum indicum); runner bean (Phaseolus coccineus); soybean (Glycine max); and wild vetch (Vicia sativa).

[0260] Subsequently, the inventors determined the physical and chemical composition of these legumes based on data provided by the United States Department of Agriculture (USDA). The following table summarizes the data for each legume:

[0261] Table 2: Proximates of raw legumes (per 100 grams, USDA 2022)

[0262]

[0263]

[0264] Then, the inventors evaluated the protein (i.e., vicilin), polyphenol, and lipid profiles of 19 candidate legumes and assigned a similarity to the cocoa score for each legume based on a comprehensive scoring system. In the case of characterizing the relative abundance and protein profile of the 7S fraction, the legumes were scored according to their relative similarity to cocoa. Other factors were also considered, such as polyphenol components rich in flavan-3-ols and proanthocyanidins, or high fat content. The following table summarizes the profiles and scores of the candidate legumes.

[0265] Table 3: Comparison of Key Cocoa Bean Quality Attributes among Bean Varieties

[0266]

[0267] As shown in the above table, among the selected candidate legumes, six legumes exhibited characteristics more similar to cocoa beans (i.e., legumes with a total score ≥ 4), including broad bean, white lupin, pea, moth bean, runner bean, and sesame.

[0268] Then these six legumes were fermented by a new fermentation process.

[0269] Example 2: Fermentation Process

[0270] Reference Figure 1 A, shows a method for preparing a fermentation inoculum for legume fermentation. It can be seen that yeast and / or bacterial colonies are transferred to a suitable separate growth medium. There are two options, where Option 1 involves combining yeast and bacteria into one inoculum, and Option 2 involves keeping the yeast and bacterial inocula separate.

[0271] Now two different embodiments of the fermentation protocol will be described, where the first embodiment uses a combined (yeast and bacteria - Option 1) inoculum throughout the fermentation process, and the second embodiment uses yeast first and then bacteria, inoculating and fermenting in sequence (Option 2).

[0272] As Figure 1As shown in Figure B, in one embodiment of the fermentation protocol, the selected beans or bean combination (as described in Example 1) is prepared for the fermentation step. This involves transferring the treated beans together with a synthetic slurry as the fermentation medium and a yeast and / or bacterial inoculum into a fermentation chamber (reactor). Then, combined microbial fermentation is allowed, for example, by culturing at 30 degrees Celsius (an ideal temperature for yeast cell growth and fermentation) for at least 48 hours. After that, once the first fermentation is completed, the synthetic slurry (fermentation medium) is discarded. Then, the beans are stirred and further cultured with the combined microorganisms at 42 degrees Celsius (an ideal temperature for bacterial cell growth and fermentation) for 72 hours. Once the second combined fermentation is completed, the beans are dried and then can be processed as described in Example 3 or 4.

[0273] As Figure 1 As shown in Figure C, in a second embodiment of the fermentation protocol, the selected beans or bean combination (as described in Example 1) is prepared for the fermentation step. This involves transferring the beans together with a synthetic slurry and a yeast-only inoculum into a fermentation chamber (reactor). Then, only yeast fermentation is allowed, for example, by culturing at 30 degrees Celsius for at least 48 hours. After that, once the first yeast fermentation is completed, the synthetic slurry is discarded. Then, a bacteria-only inoculum is added to the fermentation chamber / reactor, and then the beans are stirred and further cultured with the combined microorganisms (yeast and bacteria) at 42 degrees Celsius for 72 hours. Once the second combined fermentation is completed, the beans are dried and then can be processed as described in Example 3 or 4.

[0274] Reference Figure 4 , which shows photos of various broad beans dried after 3 hours of rehydration. (a) 0 hours, (b) 24 hours of culture, (c) 48 hours of culture, (d) 72 hours of culture, (e) 120 hours of culture, (f) beans dried after 120 hours of fermentation, (g) dried unfermented beans.

[0275] Example 3: Method for Making Chocolate Analogue Paste (e.g., for Ingredients and Chocolate Bars)

[0276] Reference Figure 2 , which shows an embodiment of a method for making a chocolate analogue paste.

[0277] Method for making a chocolate analogue paste

[0278] 1. Bake the fermented and dried beans at a temperature of 180 - 280 degrees Celsius for about 5 - 20 minutes.

[0279] 2. The baked beans are cooled, then crushed in a CPL cracker and then air - classified in a CPL air classifier.

[0280] 3. Then the obtained granules are ground into a fine powder, fat (cocoa butter substitute) is added, weighed, and then heated to about 30 - 35 degrees Celsius.

[0281] 4. Then the heated powder - fat mixture (liquid) is refined to reduce the particle size and concentrated (i.e., gently mixed and heated) in ECGC65 for no more than 16 hours, depending on the batch size of the liquid being produced.

[0282] 5. After adding the relevant additives (such as milk, flavorings, sunflower lecithin, or sugar), the resulting chocolate - like liquid is further refined and concentrated as described above.

[0283] 6. Then the chocolate - like paste obtained through step 5 is tempered in an FBM tempering machine. The temperature and time of the tempering cycle mainly depend on the fat used in the formulation.

[0284] 7. Once tempered, the chocolate - like paste is molded and / or stored as required.

[0285] Suitable storage conditions include storing the product in a cool and dry place, at a temperature between 10 - 20 °C, under dry conditions, away from strong odors.

[0286] Example 4: Method for Making Chocolate Analogue Powder (e.g., for Beverages)

[0287] Reference Figure 3 , shows an exemplary embodiment of a method for manufacturing a chocolate - like powder.

[0288] Method for making chocolate - like powder:

[0289] 1. The fermented and dried beans are roasted at a temperature of 180 - 280 degrees Celsius for about 5 - 20 minutes.

[0290] 2. The roasted beans are cooled, then crushed in a CPL cracker and then air - classified in a CPL air classifier.

[0291] 3. Then the obtained granules are ground into a fine powder to produce a cocoa - powder - like product.

[0292] 4. Suitable storage conditions include storing the product in a cool and dry place, at a temperature between 10 - 20 °C, under dry conditions, away from strong odors.

[0294] It can be understood that the melting temperature, solubility, consistency, taste, etc. will vary according to the types of fat and sugar introduced into the chocolate - like formulation. However, ultimately, they will be very similar, if not identical, to actual cocoa - based or derived chocolate compounds.

[0295] Example 5: Development of Processed Broad Bean Flour without Adding Microorganisms

[0296] Examples 1-4 describe the use of microbial fermentation (yeast and / or bacteria) to process legumes (such as broad beans) to produce a cocoa-free chocolate analogue. However, on the basis of this work, the inventors subsequently determined an exemplary method for preparing a cocoa analogue powder that does not use added microorganisms, i.e., no microorganisms are added. In this way, a fourth embodiment of the legume processing protocol will now be described, which uses a microorganism-free culture solution.

[0297] 1. Surface sterilization of broad beans

[0298] Vicia fava was used.

[0299] · For each fermentation repeat experiment, weigh 125 grams of beans using an analytical balance and transfer them to a 500 mL reagent glass bottle.

[0300] · Prepare a legume sterilization solution (70% ethanol, 0.05% Triton X-100 (Catalog No.: T8787-250ML)) (prepare 200 mL for each fermentation repeat experiment).

[0301] · Add the sterilization solution to the beans and stir using a laboratory orbital shaker at 200 rpm and 30 °C for 1 hour.

[0302] The following steps are carried out under sterile conditions.

[0303] · Use a sterile 25 mL disposable polystyrene serological pipette to discard the sterilization solution.

[0304] · Subsequently, add 250 mL of sterile reverse osmosis water to the bottle, invert the bottle 10 times and discard the washing liquid to wash the beans. Repeat twice for a total of 3 washes.

[0305] 2. Rehydration of legumes

[0306] · Add sterile reverse osmosis water (250 mL) to the bottle and incubate the beans at 25 °C for 15 - 20 hours to rehydrate them.

[0307] 3. Cultivation

[0308] The following steps are carried out under sterile conditions.

[0309] · Discard the excess rehydration water and transfer the beans to a sterile SacO2 microbox (Catalog No.: TP1600+TPD1600#30WH).

[0310] · Add 200 mL of culture medium (Formulation 1). Using sugar (2.5% sucrose, 4% glucose, 5% fructose) + 1% citric acid is a simpler alternative but will reduce the flavor quality.

[0311] Formulation 1:

[0312] o 2.5% (w / v) sucrose

[0313] o 4% (w / v) glucose

[0314] o 5% (w / v) fructose

[0315] o 1% (w / v) citric acid

[0316] o 0.14% (w / v) high-viscosity carboxymethyl cellulose

[0317] o 0.77% (w / v) low-viscosity carboxymethyl cellulose

[0318] o 1.09% (w / v) pectin

[0319] o 0.5% (w / v) yeast extract

[0320] o 0.5% (w / v) peptone

[0321] o 0.1% (w / v) calcium lactate pentahydrate

[0322] o 0.1% (v / v) Tween 80

[0323] o 0.05% (w / v) magnesium sulfate heptahydrate

[0324] o 0.02% (w / v) manganese sulfate monohydrate

[0325] · Mix the beans, seal the box with a filter lid, and then incubate at 30 °C.

[0326] · After 48 hours, discard the medium and mix the beans.

[0327] The visible contamination at this stage is associated with the inhibition of the formation of ethanol and acetic acid (vinegary smell) during incubation at 42 °C, and produces bean powder with a more neutral pH and no chocolate aroma (see Table 4).

[0328] Table 4: There is a Significant Relationship among the pH Value of Bean Flour, the Vinegary Taste of Beans, the Microbial Contamination of the Culture Mixture and the Chocolate Aroma of Roasted Broad Bean Flour. Figure 5

[0329]

[0330]

[0331] · Then incubate the beans at 42 °C for another 72 hours. A chocolate aroma powder with a low pH will be produced by the vinegary incubation process and the beans that appear shiny and moist (see Table 4).

[0332] · Through cultivation at 30 °C, the pH value of the beans drops to 4.5. For beans that appear shiny and moist, during cultivation at 42 °C, the pH value of the beans remains at 4.5. For beans that do not appear shiny and moist, during cultivation at 42 °C, the pH value rises again to between 6.2 and 7.0.

[0333] 4. Drying

[0334] · Then the beans are placed in an oven and dried at 35 °C. The drying time is 7 days to enable the best aroma development of the roasted bean powder.

[0335] 5. Pre-roasting ripening of the beans

[0336] · To enable the best aroma of the roasted bean powder, before roasting, let the beans ripen at room temperature for 5 days.

[0337] · The dry beans can be stored for at least 10 days before roasting and grinding without any adverse effects on the change of powder aroma or powder pH value.

[0338] 6. Pre-roasting treatment to deepen the color

[0339] · Grind the dry beans, add water (1:1 w / w) to the powder, heat to 80 °C and hold for 60 minutes, and stir occasionally. Dry the paste at 35 °C for 7 days. A darker-colored powder is produced, whose darkness is equivalent to that of unground cocoa powder.

[0340] 7. Roasting and grinding

[0341] · Roast and grind the dried beans at 180 °C or 200 °C for 20 - 40 minutes to reach the predetermined color, (color judgment)

[0342] The internal standards established are used.

[0343] · Roasting causes the pH value of the powder made from dull beans to drop by about 1 pH unit. The pH value of shiny beans remains at a lower level, around 5.0.

[0344] · After roasting, the beans can be stored for at least 2 - 3 months before grinding without any adverse effects on the powder aroma or powder pH value.

[0345] 8. Roasting and ripening of the ground powder

[0346] · Detailed experiments show that the aroma of the roasted bean powder changes rapidly within the first 7 days after roasting and grinding. After 7 days, the aroma is stable for > 1 month. The pH value of the roasted powder is stable from immediately after grinding until up to 4 months.

[0347] · Powders with good chocolate aroma are associated with powders having a lower pH value (Table 1), see Figure 6 , showing that for chocolate aroma, the optimal pH value is 4.8 - 5.1. A bin is a mathematical term used to create a histogram from a continuous data set. In this graph, there are 163 processed broad bean powder samples with known pH values and whether they have chocolate aroma or not. The pH values of the samples range from 4 to 7. To create the histogram, pH value bins, i.e., narrow ranges of pH values, such as 4.8 - 5.1, are created. Within this pH range (bin), the number of powders with chocolate aroma having a pH value within this range is calculated. The percentage of samples with chocolate aroma in this pH value bin is calculated. This is repeated for all bins between 4 and 7, and then a graph is made based on these values. Thus, the proportion of samples with chocolate aroma is highest (about 60%) in samples with a pH value of 4.8 - 5.1. See Figure 7 , it can be seen that in powders with a lower pH value, chocolate aroma starts to develop on the 3rd - 4th day after roasting and grinding, starts to develop on the 7th day and continues to improve for up to 1 month, and then remains stable for up to 4 months.

[0348] 9. Flavor

[0349] · See Example 6: Adding Microorganisms Produces Alternative Flavors of Broad Bean Flour , showing the flavor characteristics of a slurry (powder + shea butter 1:1 w / w) made from processed, roasted, and ground broad bean powder, including genuine cocoa powder for comparison. Compared with the genuine cocoa powder, the roasted broad bean powder scores high for each of the tested flavor elements and has excellent flavor characteristics.

[0350] Figure 1

[0351] The inventors found that adding different microorganisms leads to the development of alternative flavors in broad bean powder. In this way, a fifth embodiment of the bean processing protocol will now be described, which uses the fermentation inoculum described in Option 1 (i.e., Table 5 Selective Agar for Microbial Community Protocol 1 in B).

[0352] 1. Surface sterilization of broad beans

[0353] Broad beans (Vicia Fava) were used.

[0354] · For each fermentation replication experiment, 125 grams of beans were weighed using an analytical balance and transferred to a 500 - milliliter reagent glass bottle.

[0355] · A bean sterilization solution (70% ethanol, 0.05% Triton X - 100 (Catalog No.: T8787 - 250ML)) was prepared (200 mL was prepared for each fermentation replication experiment).

[0356] · Add the sterilization solution to the beans and stir for 30 minutes at 200 rpm and 25 °C using a laboratory orbital shaker.

[0357] The following steps are carried out under aseptic conditions.

[0358] · Discard the sterilization solution using a sterile 25 mL disposable polystyrene serological pipette.

[0359] · Subsequently, add 250 mL of sterile reverse osmosis water to the bottle, invert the bottle 10 times and discard the wash liquid to wash the beans. Repeat twice for a total of 3 washes.

[0360] 2. Rehydration of beans

[0361] · Add sterile reverse osmosis water (250 mL) to the bottle and incubate the beans at 25 °C for 15 - 20 hours to rehydrate them.

[0362] 3. Cultivation

[0363] The following steps are carried out under aseptic conditions.

[0364] · Discard the excess rehydration water and transfer the beans to a sterile SacO2 microbox (Catalog No.: TP1600 + TPD1600#30WH).

[0365] · Add 200 mL of culture medium (the formulation depends on the added microorganism - see below). The microorganisms used are Pichia kudriavzevii, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, Lactobacillus plantarum, and Acetobacter pasteurianus. Prepare the beans and inoculate them with one of the types of microorganisms used respectively.

[0366] · The fermentation inoculum is prepared as follows;

[0367] 1. Prepare selective agar and pour it into sterile 9 cm Petri dishes (20 mL each) and let it cool and solidify.

[0368] Acetic acid (AA) agar: 1% D - glucose, 1.5% bacteriological peptone, 0.8% yeast extract, 0.5% ethanol, 0.3% acetic acid, and 2% agar. The mixture is autoclaved. Note that ethanol and acetic acid should be added after autoclaving (the final pH of the medium should be 4.5).

[0369] De Man, Rogosa, and Sharpe (MRS) agar: MRS agar (Catalog No.: 69964-500G) was prepared according to the manufacturer's instructions. Briefly, 61.15 g of MRS agar was added to 800 mL of RO water containing 1 mL of Tween 80 (Catalog No.: P8074). The suspension was boiled to completely dissolve the medium, the volume was adjusted to 1000 mL, and then sterilized by autoclaving.

[0370] Yeast Peptone Glucose (YPG) agar: 1% yeast extract, 2% peptone, 2% glucose. The pH was adjusted to 5.6, 2% agar was added and autoclaved.

[0371] Microorganisms

[0372] Selective Agar Pichia kudriavzevii YPG Hanseniaspora opuntiae YPG Saccharomyces cerevisiae YPG Lactobacillus fermentum MRS Lactobacillus plantarum MRS Acetobacter pasteurianus AA Figure 8

[0373] 2. Inoculate the stock of each cell culture onto its respective selective agar plate (Table 1) and incubate at 28 °C for 72 hours.

[0374] 3. For each isolate, 15 mL tubes containing 3 mL of Lennox broth were prepared, except for Acetobacter pasteurianus, and one 15 mL tube containing 3 mL of AA broth (1% D-glucose, 1.5% bacteriological peptone, 0.8% yeast extract. Autoclaved and added 0.5%

[0375] ethanol and 0.3% acetic acid) was prepared.

[0376] 4. Select a single colony from the agar plate for each isolate and transfer it to 3 mL of the appropriate growth medium, and incubate in an orbital shaker at 28 °C,

[0377] 200 rpm for 72 hours.

[0378] 5. Harvest the cells by centrifuging at 4000 x rpm for 8 minutes at 4 °C.

[0379] 6. Discard the supernatant and add 5 mL of sterile 10 mM MgCl2 to the tube.

[0380] 7. Invert the tube 10 times to resuspend the pelleted cells, and then centrifuge at 4000 x rpm for 8 minutes at 4 °C.

[0381] 8. Repeat steps 6 - 7 two more times for a total of three washes.

[0382] 9. Finally, resuspend the cells in 1 mL of sterile 10 mM MgCl2 and measure the optical density (OD600) of each isolate at 600 nm (1 OD600 = 1×109 (cells / mL) to determine the cell concentration.

[0383] 10. Prepare the fermentation inoculum by pooling individual isolates, each at a concentration of 1×10 8 cells / mL (dilute the cells with sterile 10 mM MgCl2 if necessary). For each fermentation, prepare 1 mL of inoculum and use it immediately, or store it at 4 °C for no more than 24 hours.

[0384] · Mix the beans with the inoculum, seal the box with a filter lid, and then incubate at 30 °C.

[0385] · After 72 hours, discard the medium and mix the beans.

[0386] 4. Drying

[0387] · Then place the beans in an oven and dry at 35 °C. The drying time is 7 days to obtain the best aroma development of the roasted bean powder.

[0388] 5. Roasting and grinding

[0389] · Roast and grind the dried beans at 180 °C or 200 °C for 20 - 40 minutes until they reach a predetermined color (color judgment)

[0390] using in - house developed standards.

[0391] 6. Flavor

[0392] · Refer to Figure 1 , which shows the flavor profile of a slurry (bean powder, sugar, and shea butter) made from processed, roasted, and ground broad bean powder,

[0393] including using broad beans (NK0034) treated with our non - microbial process as a comparison. The flavor was evaluated by 5 independent individuals.

[0394] In addition, refer to Example 7: Use of Other Beans D, where another embodiment was developed that involves subjecting beans (such as broad beans) to two separate treatments and then mixing their products together to produce a cocoa - bean - free cocoa - powder analogue. First, treat the beans with a microorganism (Option 1 or Option 2) to produce microbially fermented beans. Additionally, treat the beans without any microorganism to produce non - microbially treated beans. Subsequently, mix a certain proportion of microbially fermented beans with a certain proportion of non - microbially treated beans to obtain a cocoa - bean - free cocoa - powder analogue.

[0395] Thus, studies have shown that the flavors produced by the treatment of broad beans with various microorganisms are different from the non-microbial process described in Example 5. Mixing the beans of the two types of processes, namely microbial fermentation and non-microbial fermentation, produces a unique flavor combination.

[0396] Figure 10

[0397] The inventors have found that legumes other than broad beans can also be used to develop cocoa-like powders. According to the legume processing protocol described in Example 5, namely the fourth protocol using a microbial-free culture solution, lupins, green peas, moth beans, adzuki beans, and hemp seeds are processed according to the same procedure as broad beans. The flavor of the pulp (powder mixed with shea butter at a ratio of 1:1) made from the processed seeds and beans is as Figure 10 shown.

[0398] Thus, studies have shown that all the tested legumes and seeds have different flavor profiles from each other and from broad beans. In addition, studies have shown that hemp seeds and lupins are superior to broad beans in terms of chocolate flavor. The inventors have also concluded that powders made from various processed legumes can be mixed with broad bean powder to produce a better flavor (for example, referring to Example 8: Manufacture of NuKoKo - like Cocoa Powder Using Broad Bean Flour and Additives. Various additives can be added to the broad bean flour made from , mixing hemp seeds with broad beans will introduce new sour, dried fruit, and nutty flavors to the broad bean powder).

[0399] microbially fermented or non - microbially fermented broad beans, which are roasted and ground, and can be mixed or not mixed as needed. These flavor - modifying ingredients are added in the range of 0.1 - 2.0% (w / w). They include but are not limited to beetroot powder, apple powder and malted barley. Example 9: Generating Cocoa - free Chocolate Analogue Using "NuKoKo" Powder The broad beans described in Example 5 and / or Example 6

[0400] Results of the Processing

[0401] The inventors have determined an exemplary method for preparing a cocoa-free chocolate analogue using a powder of the cocoa-free chocolate analogue of the present invention (i.e., "Nukoko" powder), which is Figure 9 Summary .

[0402] 1. Prepare the NuKoKo chocolate analogue according to the following formula:

[0403] 21% 410g Nukoko powder

[0404] 34% 665g palm oil / sheanut oil

[0405] 24% 469g sucrose (pre-ground)

[0406] 14% 274g whole milk powder

[0407] 2% 39g beetroot powder

[0408] 2% 39g apple powder

[0409] 0.5% 10 g lecithin

[0410] 2% 39 g malt extract

[0411] 0.2% 3.5 g salt (pre-ground)

[0412] 0.25% 5 g chocolate malt barley

[0413] 2. Method: Melt the fat and pre-grind the granular ingredients (spice grinder). Add the semi-dry ingredients to the liquid fat and then place them in a pre-heated small stone mill. Balance the added dry ingredients.

[0414] Use a heating lamp in batches. Run for 8 hours.

[0415] 3. Appearance: It must be smooth, of uniform color, and shiny.

[0416] There should be no air bubbles, cracks, streaks, or cloudy appearance.

[0417] 4. Aroma: It should emit a strong chocolate flavor with fruity, earthy, or floral notes.

[0418] 5. Snapping sound: When broken, it should make a crisp, clean, sharp, and distinct snapping sound.

[0419] 6. Texture: It should melt in the mouth immediately. Its taste and smoothness should impress you instantly. It should feel like smooth velvet in the mouth. The taste should linger for a few minutes after eating the chocolate.

[0420] 7. Flavor: It should taste sweet, milky, caramel-like, and cocoa-like.

[0421] 8. Flavor profile

[0422] · Reference ​ , showing the flavor profiles of NuKoKo chocolate analog (Choc), the authentic Tesco brand milk chocolate, and dark chocolate (Mollys). Five independent individuals evaluated various chocolate flavor elements.

[0423] Therefore, a direct comparison is provided between NuKoKo Choc (a chocolate substitute made from broad bean flour according to the above formula and processed using the inventor's non-microbial method) and Tesco's milk chocolate and dark chocolate. NuKoKo chocolate scored very similarly to the authentic chocolate in all five test parameters, namely appearance, aroma, mouthfeel, texture, and flavor.

[0424] ​

[0425] As described in the embodiments, the inventors have developed an elaborate process for manufacturing a chocolate analogue that is not derived from raw cocoa beans and thus does not include any cocoa. Instead, the process involves using a carefully selected group of beans that meet the necessary criteria for replicating the appearance, taste, smell, and texture of traditional cocoa chocolate, but without the drawbacks of raw cocoa / cocoa. The non-cocoa chocolate analogue can be made into a paste or powder and can thus be used in a variety of foods, such as solid confectionery or cosmetics.

Claims

1. A method for producing a cocoa-free chocolate analogue, characterized in that, Comprising: Contacting the legumes with at least one microorganism under conditions suitable for fermentation of the legumes by the at least one microorganism to form a cocoa-free chocolate analogue comprising microbially fermented legumes.

2. The method according to claim 1, wherein: Among them, The method comprises a first treatment and a second treatment, In the first treatment, the legumes are microbially fermented to produce microbially fermented legumes, In the second treatment, in the absence of any exogenously added microorganisms, the legumes are non-microbially fermented alone to produce non-microbially treated legumes.

3. The method according to claim 2, wherein: Among them, The method comprises combining a proportion of the microbially fermented legumes and a proportion of the non-microbially fermented legumes to produce the cocoa-free chocolate analogue.

4. The method according to claim 3, wherein: Among them, The ratio of the microbially fermented legumes to the non-microbially fermented legumes is between about 10%:90% and 90%:10%, or about 50%:50%.

5. The method according to any one of the preceding claims, Characterized in that: Wherein, the fermented legumes are selected from the following legumes: adzuki bean (phaseolus angularis); alfalfa (medicagosativa); bambara (vigna subterranean); black bean (vigna mungo); chickpea (cicer arietinum); cowpea (vigna unguiculata); Broad bean (vicia fava); common bean (pHaseolus vulgaris); lima bean (phaseolus lunatus); lentil (lens culinaris or Lens esculenta); lupin (lupinusalbus, lupinus angustifolius, or lupinus luteus); moth bean (vigna aconitifolia); mung bean (vigna radiate); pea (pisum sativum); pigeon pea (cajanus cajan); sesame (sesamumindicum); runner bean (phaseolus coccineus); soybean (glycine max); and wild vetch (vicia sativa).

6. The method according to any one of the above claims, wherein: Among them, The fermented legumes are selected from the following legumes: broad bean (vicia fava); white lupin (lupinus albus); pea (pisum sativum); moth bean (vigna aconitifolia); runner bean (phaseolus coccineus); and sesame (sesamumindicum).

7. The method according to any one of the above claims, wherein: Among them, The at least one microorganism for fermentation is selected from yeast and / or bacteria.

8. The method according to any one of the preceding claims, characterized in that: Among them, The fermentation substantially comprises yeast or consists only of yeast. Optionally, in the fermentation, at least 70%, 80% or 90% of the microorganisms comprise yeast cells.

9. The method according to any one of the preceding claims, characterized in that: Among them, The fermentation substantially comprises or consists only of bacteria. Optionally, in the fermentation, at least 70%, 80% or 90% of the microorganisms comprise bacterial cells.

10. The method according to any one of the preceding claims, characterized in that: Among them, The fermentation comprises yeast and bacteria. Preferably, the ratio of yeast to bacteria is about 50:50, 10:90, 20:80, 30:70, 40:60 of yeast cells to bacterial cells, or 10:90, 20:80, 30:70, 40:60 of bacterial cells to yeast cells.

11. The method according to any one of claims 4-7, characterized in that: Among them, The family of the bacteria is selected from the following bacterial families: Lactobacillaceae; and Acetobacteraceae.

12. The method according to any one of claims 4-8, characterized in that: Among them, The species of the bacteria is selected from the following bacterial species: Lactobacillus fermentum; Lactobacillus plantarum; and Acetobacter pasteurianus.

13. The method according to any one of claims 4-9, characterized in that: Among them, The genus of the yeast is selected from the following yeast genera: Pichia; Hanseniaspora; and Saccharomyces.

14. The method according to any one of claims 4-10, characterized in that: Among them, The species of the yeast is selected from the following yeast species: Pichia kudriavzevii; Hanseniaspora cactophila; and Saccharomyces cerevisiae.

15. The method according to any one of the preceding claims, Characterized in that: Wherein, the method comprises growing the at least one microorganism and the legumes in a growth medium. Optionally, the growth medium comprises: (i) One or more sugars selected from the following sugars: sucrose; glucose; fructose; high-viscosity carboxymethyl cellulose; low-viscosity carboxymethyl cellulose; and / or pectin; (ii) One or more salts selected from the following salts: calcium lactate pentahydrate; magnesium sulfate heptahydrate; And manganese sulfate monohydrate; (iii) One or more protein hydrolysates. Optionally, the protein hydrolysate is peptone; (iv) Yeast extract; (v) A surfactant, optionally it is Tween 80; and (vi) Citric acid.

16. The method according to any one of the preceding claims, characterized in that: Among them, The pH value of the growth medium is 2-5 or 3-4.

17. The method according to any one of the preceding claims, characterized in that: Among them, The fermentation comprises culturing yeast and bacteria at 24°C - 44°C for at least 1 day or 2 days.

18. The method according to any one of the preceding claims, characterized in that: Among them, The fermentation comprises culturing bacteria at 24°C - 50°C for at least 1 day or 2 days.

19. The method according to any one of the preceding claims, characterized in that: Among them, The method comprises co-fermenting the legumes in the presence of yeast and bacteria.

20. The method according to claim 16, wherein: Among them, the yeast includes Pichia kudriavzevii; Hanseniaspora opuntiae; and / or Saccharomyces cerevisiae, and the bacteria includes Lactobacillus fermentum, Lactobacillus plantarum, and / or Acetobacter pasteurianus.

21. The method according to claim 16 or 17, wherein: Among them, the method includes contacting the legumes with the yeast and the bacteria at a temperature of 21°C - 37°C for at least 24 hours, discarding the growth medium after an initial fermentation for one or more days, and then culturing the legumes with the at least one microorganism at a temperature of 24°C - 50°C for one or more days.

22. The method according to any one of claims 16 - 18, wherein: Among them, the fermentation mixture is cultured at a temperature of 34°C - 44°C for at least two or three days.

23. The method according to any one of claims 1 - 15, wherein: Among them, the method includes first fermenting the legumes in the presence of only yeast and then fermenting the legumes in the presence of bacteria.

24. The method according to claim 20, wherein: Among them, the yeast includes Pichia kudriavzevii; Hanseniaspora opuntiae; and / or Saccharomyces cerevisiae, and the bacteria includes Lactobacillus fermentum, Lactobacillus plantarum, and / or Acetobacter pasteurianus.

25. The method according to claim 20 or 21, wherein: Among them, the method includes contacting the legumes with the yeast and the bacteria at a temperature of 21°C - 37°C for at least 24 hours, discarding the growth medium after an initial fermentation for one or more days, and then contacting the legumes with the at least one microorganism at a temperature of 24°C - 50°C for at least 24 hours.

26. The method according to any one of claims 20 - 22, wherein: Among them, the fermentation mixture is cultured at a temperature of 32°C - 44°C for at least two or three days.

27. The method according to any one of claims 2 - 26, wherein: Among them, when the method includes non - microbial fermentation, the method further includes using a medium with a pH value between 3 and 5 and submerging the legumes when they start to germinate to consume the oxygen for respiration, thereby initiating the fermentation metabolism through the oxidation of sugars, resulting in the production of ethanol, lactic acid, and optionally, the production of acetic acid.

28. The method according to claim 27, wherein: Among them, the non - microbial fermentation is cultured at a first temperature, and the first temperature is 20°C - 38°C, 23°C - 37°C, 25°C - 35°C, 26°C - 34°C, 27°C - 33°C, 28°C - 32°C, or 29°C - 31°C.

29. The method according to claim 27 or 28, wherein: Among them, the non - microbial fermentation is cultured for at least one or two days.

30. The method according to any one of claims 27 - 29, wherein: Among them, The non-microbial fermentation mixture is cultured at a second temperature, which is 26°C - 48°C, 28°C - 46°C, 30°C - 44°C, 32°C - 44°C, 34°C - 44°C, 36°C - 44°C, 38°C - 44°C, or 40°C - 31°C.

31. The method according to claim 30, wherein: Among them, The non-microbial fermentation is cultured for at least one day, two days or three days.

32. The method according to any one of the above claims, wherein: Among them, The pH value of the chocolate analogue is: (i) between about 4 and about 7, between about 4.3 and about 6.8, or between about 4.4 and about 6.7; (ii) between about 4.4 and about 6.3, between about 4.7 and about 5.9, or between about 4.7 and about 5.5, (iii) between about 4.4 and about 5.5, between about 4.7 and about 5.2, or between about 4.8 and about 5.

1.

33. The method according to any one of the above claims, wherein: Among them, The method includes drying the beans after fermentation, optionally, wherein the beans are dried in an oven at a temperature of about 21°C - 37°C for one day or more.

34. The method according to any one of the above claims, wherein: Among them, The cocoa-bean-free chocolate analogue containing fermented beans contains a paste or is converted into a paste.

35. The method according to any one of the above claims, wherein: Among them, The cocoa-bean-free chocolate analogue containing fermented beans contains a powder or is converted into a powder.

36. A cocoa-free chocolate analogue, characterized in that, Obtained by the method according to any one of claims 1 - 35, or capable of being obtained by the method according to any one of claims 1 - 35.

37. A cocoa-free chocolate analogue, characterized in that, Containing microbially fermented / non-microbially fermented beans.

38. The cocoa-bean-free chocolate analogue according to claim 37, wherein: Among them, The analogue is as described in any one of claims 1 - 35.

39. A product ingredient, characterized in that, Comprising: The cocoa-bean-free chocolate analogue according to any one of claims 36 - 38.

40. A food or beverage, characterized in that, Comprising: The cocoa-bean-free chocolate analogue according to any one of claims 36 - 38 or the composition according to claim 39, optionally, wherein the cocoa-bean-free chocolate analogue is a powder, pulp, liquid, solid or paste.

41. The food or beverage according to claim 40, wherein: Among them, The food is a confectionery product, optionally, wherein the food is a bar, a molded shape, a bean, a truffle, a sauce, a coated fruit, nut or other inner layer, a baking filling, a product coating, such as a coating on a doughnut or muffin, yogurt, ice cream or other desserts.

42. A cosmetic, characterized in that, Comprising: The cocoa-bean-free chocolate analogue according to any one of claims 36 - 38 or the composition according to claim 39.

43. The cosmetic according to claim 42, wherein: Among them, The cosmetic is a skin care product, a moisturizer, an exfoliant, a lip balm, eye shadow, soap, a body cream or butter, or a hair mask.

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