Cocoa-free chocolate

Abstract

The present invention relates to non-cocoa or cocoa-free chocolate analogues and methods for making such analogues. The invention also extends to fermentation methods for producing cocoa / cocoa-free chocolate analogues, as well as foodstuffs and cosmetics containing such analogues.

Description

Detailed Description of the Invention

[0001] The present invention relates to non-cocoa or cocoa-free chocolate analogs and methods for making such analogs. The invention also extends to fermentation methods for producing cocoa / cocoa-free chocolate analogs, as well as ingredients, foodstuffs and cosmetics containing such analogs.

[0002] Cacao is the seed of the tropical cacao tree (Theobroma cacao), from which cocoa and chocolate are made, highly sought-after and relatively expensive ingredients used in a variety of foods. There are many well-documented health benefits associated with cocoa and products made from cocoa seeds, such as chocolate and cocoa. For example, research suggests that dark chocolate may reduce the risk of cardiovascular disease. Furthermore, unsweetened cocoa is relatively low in calories and contains essential minerals that support heart, bone, and immune system health.

[0003] The taste of cocoa is also enjoyed worldwide and is the basis for beverages and confectionery products with a variety of flavor profiles. For example, there are a wide variety of cocoa flavors depending on the type of cocoa bean and the region where it is grown, the amount of sugars present, the amount of free amino acids present, and the cocoa color pigments.

[0004] However, several side effects associated with cocoa use have been reported. These include triggering migraines in sensitive people, an increased risk of bleeding and bruising in people with bleeding disorders, worsening cardiac arrhythmias, disruption of 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 highly toxic to dogs, which is why chocolate should not be given to domestic dogs.

[0005] Chocolate is the fifth most carbon-intensive food worldwide. Governments, industry legislators, and consumers all demand lower carbon-intensive foods on the market. Furthermore, the future of the cocoa and chocolate industry is under threat due to growing concerns about climate change. Some models suggest that as early as 2030, 28% of all cocoa imports will be affected, leading to a significant reduction in cocoa supplies to the global market. Given that cocoa beans travel an average of approximately 3,000 miles before the resulting chocolate reaches local supermarket shelves, this translates into a significant increase in prices for consumers. Furthermore, approximately 1.56 million children, some as young as five, work on cocoa farms worldwide, exacerbating the situation by chocolate companies' reluctance to pay cocoa farmers higher prices for their beans. Combined with growing consumer demand, this means alternative options to cocoa-based chocolate are needed.

[0006] To overcome these challenges, artificial chocolate and cocoa flavors made from a variety of alternative synthetic compounds have been developed, but these chocolate analogs fall far short of mimicking the exact taste of cocoa and chocolate, exhibiting strong off-tastes and aromas, and some with poor safety profiles.

[0007] Therefore, there is a need to provide a chocolate and cocoa alternative that can be made from locally sourced natural products and that closely mimics (in terms of flavor, aroma, and texture) original chocolate made from cocoa beans, but does not exhibit the various associated drawbacks discussed above.

[0008] The inventors set out to develop a novel cocoa bean-free chocolate / cocoa analogue. After conducting an in-depth analysis of various locally produced products (i.e., legumes), the inventors identified several candidate legumes that could be used to provide a good replacement for cocoa in chocolate formulations, and developed and optimized a new method for making the alternative chocolate / cocoa analogue.

[0009] In one embodiment, the process involves a microbial fermentation treatment of legumes using yeast and / or bacteria to obtain a cocoa bean-free chocolate analogue having a taste and aroma similar to regular (cocoa bean) chocolate. In another embodiment, the process involves two separate treatments of legumes: in one treatment, the legumes are subjected to microbial fermentation, and in the other treatment, the legumes are treated in the absence of exogenously added microorganisms. A portion of the microbially fermented legumes is then blended with a portion of the non-microbially treated legumes to obtain a cocoa bean-free chocolate analogue, also having a taste and aroma similar to regular (cocoa bean) chocolate.

[0010] Thus, in a first aspect of the present invention, there is provided a cocoa bean-free chocolate analogue 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 a microbially fermented legume.

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

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

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

[0015] While cocoa bean substitutes such as cupuaçu (Theobroma grandiflorum) and carob (Ceratonia siliqua) are already known for their cocoa-like qualities, these are relatively underdeveloped crops with seasonal variations and the corresponding burden of international shipping costs. Advantageously, however, the inventors of the cocoa bean-free chocolate analogs of the first, second, or fourth aspects have identified several legume candidates that are low in cost, agriculturally well established, and grown relatively close to European manufacturing facilities. Furthermore, the chocolate analogs of the present invention closely resemble or mimic traditional chocolate made from cocoa beans in terms of flavor and aroma (i.e., notes and tones) and texture.

[0016] Legumes will be understood to be plants of the legume family (Fabaceae or Leguminosae), or the fruits or seeds of such plants. When dry particles are used, the seeds can also be called pulses. Legumes may be selected from kidney beans, soybeans, peas, chickpeas, peanuts, lentils, lupins, mesquite, carbo, tamarind, alfalfa, and clover.

[0017] Preferably, the fermented legume contains vicilin. Vicilin is a legumin-related globulin storage protein. Globulin fractions identified as vicilin globulins (VCGs; also known as 7S storage proteins) contain a common 66 kDa precursor, which, after proteolysis, is composed of three polypeptide subunits (47, 31, and 14.5 kD; Biehl et al., 1982; Spencer and Hodge, 1992; Voight, Biehl, and Wazir, 1993). More recently, it has been shown that the 14.5-15 kD band commonly observed by SDS-PAGE of cocoa proteins is a composite of three low-molecular-weight polypeptides of 14.5, 15, and 15.5 kDa in size (Kratzer et al., 2009). As is common with vicilins, the precursor can form a trimer. Warwicker and O'Connor (1995) constructed a homology model of a cocoa vicilin with a size of 138 kDa, which is close to the previously observed sizes of cocoa trimers of 144 and 150 kDa (Macdonald et al., 1991; Voight, Biehl, and Wazir, 1993, respectively). No 11S legumin has been identified in cocoa (Voight, Biehl, and Wazir, 1993).

[0018] During microbial or non-microbial fermentation, VCG is degraded by two important enzymes. First, aspartic endoprotease causes the release of hydrophobic oligopeptides, i.e., oligopeptides that terminate 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 bean fermentation; aspartic endopeptidase reaches a peak midway, and carboxypeptidase reaches a peak at the end of 144 h of fermentation (Amin, Jinap and Jamilah, 1997). As a result, the profile of cocoa VCG also changes during fermentation. For example, Kumari et al. (2016) observed five major protein bands, i.e., vicilin subunits (15, 31, and 47 kDa), albumin (21 kDa), and also a putative kinase superfamily protein at 43 kDa, after SDS-PAGE of unfermented cocoa protein. During the first 72 h of fermentation, the 15 kDa subunit was the main target of vicilin proteolysis, while 31 and 47 kDa began to degrade only after 96 h. However, this hydrolysis was rapid and complete, and by 120 h both the 31 and 47 kDa bands were completely degraded, while some of the 15 kDa band remained. In addition, aspartic endoprotease and carboxypeptidase have quite different pH optima (pH 3.5 vs 5.5 - 6, respectively), and during in vitro proteolysis of VCG isolates, "nutty" aroma precursors are dominant over a pH range of 4.8 - 5.6, while lower pH values (4.4 - 5.2) are associated with "cocoa" aroma precursors and more complete protein hydrolysis, accompanied by the release of a greater amount of free amino acids (Voigt et al., 2018). However, as mentioned by Biehl et al. (1985), low pH (<pH 5) during bean fermentation can be associated with a decrease in flavor and aroma, and thus "complete protein hydrolysis" is not desirable.

[0019] Preferably, the fermented legume contains convicilin, which is a legumin-related globulin storage protein.

[0020] Preferably, the fermented legume (which may be microbially fermented and / or non-microbially fermented) is selected from the group consisting of adzuki bean (phaseolus angularis); alfalfa (medicago sativa); bambara (vigna subterranean); black gram (vigna mungo); chickpea (cicer arietinum); cowpea (vigna unguiculata); broad bean (vicia faba); kidney bean (phaseolus vulgaris); lima bean (phaseolus lunatus); lentil (lens culinaris or Lens esculenta). esculenta); lupine (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 vetch (vicia sativa).

[0021] The inventors have determined the physicochemical composition of the legumes listed above based on data provided by the United States Department of Agriculture (USDA). Data for each of the legumes is summarized in Table 2 in the Examples. However, a summary of the protein profile and phenolic content of selected legume candidates is provided below.

[0022] Adzuki beans: Phaseolus angularis Approximately half of the proteins in adzuki beans (or red kidney beans) are globulins, and the majority of these (approximately 80%) are 7s vicilins. It is understood that catechins, quercetin glycosides, procyanidins, and protocatechuic acid are the major polyphenols found in adzuki beans.

[0023] Alfalfa: Medicago sativa Alfalfa contains three major storage proteins, consisting mainly of 7S-alfin, 30% of the total protein content, 11S-medicagin, and 20% of the total protein content. Regarding the phenolic profile of alfalfa seeds, the major flavonoids found in the aerial parts of the plant are glycosides of the flavones tricin and apigenin.

[0024] Bambara: Vigna Subterranean Bambara groundnuts (Bambarra pea) have a total protein content ranging from 18 to 27%, with vicilin proteins accounting for 46% of the total, and legumin and albumin accounting 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, are found in the bambara variety and are generally at higher levels in red and brown beans compared to browneye and blackeye types. Phenolics are concentrated in the husk.

[0025] Black Gram: Bigna Mungo Vicilin constitutes approximately 50% of the total soluble protein content in black gram (or mungo bean). The distribution of phenolic acids in black gram, consisting mainly of ferulic acid, has been shown to be concentrated in the seedlings and the seed coat. The distribution of total polyphenols and total anthocyanins ranged from 0.9 to 134.7 and 5.9 to 86.8 mg / 100 g, respectively, with the highest concentrations found in the seed coat.

[0026] Chickpea: Cicer arietinum The protein profile of chickpea contains 56% globulins, 18% glutelin, 12% albumin, and 2.8% prolamins. Intact 11s and 7s globulins were identified, and the vicilin subunit appeared to be similar to the 7S subunit observed in pea and soybean extracts. Over 96 polyphenols were identified in chickpea, with catechin (and related pentosides), sinapic acid hexoside, gallic acid, and kaempferol glycosides being the major contributors.

[0027] Cowpea: Vigna unguiculata Globulins represent 51-72% of total cowpea protein, the major component of which is the 7s vicilin-like β-vignin, which shares approximately 62% of its amino acid sequence with the subunits of β-conglycinin (derived from soybean). Analysis of four cowpea varieties showed that the total polyphenol (mainly flavonoids and phenolic acids) content ranged from 642 to 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 procyanidins.

[0028] Broad beans: Vicia faba Also known as broad beans, approximately 80% of the broad bean protein is globulin, consisting of both 11s and 7s. The broad bean protein profile is complex, containing both vicilins and convicilins. Broad beans have been reported as a good source of catechins, epicatechins, and procyanidins.

[0029] Kidney bean: Phaseolus vulgaris Phaseolus vulgaris is a highly diverse species encompassing several well-known bean varieties, including green beans, kidney beans, black beans, pinto beans, and white beans. They are typically rich in globular proteins. The 7S protein in kidney beans is called "phaseolin" and accounts for 50% of the total protein in green beans. Most kidney bean varieties contain similar phenolic acids, but there is diversity in their monomeric flavonoid profiles, which are partially responsible for their color. For example, black beans are rich in anthocyanidins, pinto beans are rich in kaempferol glycosides, red kidney beans are dominated by both quercetin and kaempferol glycosides, while white beans appear to have little to no detectable flavonoid content. Some cultivated species of Phaseolus vulgaris have significantly higher proanthocyanidin contents than other Phaseolus species, including Lima bean, Phaseolus polyanthus, Phaseolus tinctorius, and Phaseolus zebra.

[0030] Lima bean: Phaseolus lunatus Lima beans contain vicilin (phaseolin). Lima bean flour contains similar levels of p-coumaric acid and ferulic acid. Other phenolic acids and flavonoids are found in lima bean seed oil, including b-coumaric acid and its downstream metabolite, rosmarinic acid.

[0031] Lentil: Lens culinaris / Lens esculenta Globulins and albumins represent 42 and 11% of the total lentil protein content, respectively. However, the major native globulins in lentils, representing 34% of the total protein, were found to be legumin-type globulins. Lentils also contain a wide range of flavan-3-ol, procyanidin, and flavonol glycosides, particularly catechin glucoside, quercetin-diglucoside, and procyanidin dimers.

[0032] Lupine: Lupinus albus / Lupinus angustifolia / Lupinus luteus Lupin is a high-protein seed that also contains approximately 10% lipids. Blue lupin (Lupinus angustifolia or narrowleaf) and yellow lupin (Lupinus luteus) are typically grown as animal feed, while white lupin (Lupinus albus) is primarily used for human consumption. The globulin-to-albumin ratio in lupin species is approximately 9:1, and it is divided into α-conglutin (11s, legumin-like), β-conglutin (7s, vicilin-like), and two minor proteins called γ- and Δ-conglutin. Regarding the phenolic profile of lupin seeds, the total polyphenol content consisted of gallic acid equivalents / 100g in blue, yellow, and white lupins, as well as several phenylpropanoids (e.g., ferulic acid, catechol), benzoic acids (e.g., vanillin, vanillic acid), and flavones (e.g., apigenin-7-glucoside, apin). In addition, both lupin leaves and seeds are rich in isoflavones, mainly the aglycone genistein and several related glycosides and 2'-hydroxy forms.

[0033] Moss Bean: Vigna aconitifolia Globulins accounted for 63.9% of the total soluble moth bean protein, while glutelin, albumin, and prolamin accounted for 27.8, 5.1, and 3.2%, respectively. Total polyphenols in the 22 moth bean species ranged from 57±23 to 1034±12 mg GAE / 100 g, with catechin, tannic acid, and gallic acid as major contributors.

[0034] Mung beans: Vigna radiatae Mung beans (or green gram) contain both 7S and 11S globulins (3 and 8%, respectively), but their major storage protein is 8S vicilin. Mung beans also contain a diverse profile of flavonoid phenolic acids, with the glycosylated form of the flavone apigenin being the predominant form.

[0035] Pea: Pisum sativum Protein profiles of various pea species show that their globulin content accounts for 49.2–81.8% of total protein, with vicilin being the most abundant, accounting for 26.3–52.0% of the total globulin content. Legumin content ranges from 5.9–24.5% of total globulins, with convicilin accounting for only 3.9–8.3%. While hydroxybenzoic acids dominate the phenolic profile of pea cotyledons, polyphenols in the seed coat are primarily flavone (e.g., luteolin, apigenin) and flavonol (e.g., quercetin) glycosides, along with lower levels of flavanol-3-ol monomers and procyanidins. A variety of quercetin glycosides are found in pea leaves.

[0036] Pigeon pea: Cajanus cayan Intact pigeon pea contains vicilin. The phenolic profile of pigeon pea is made up of the glycosylated flavones orientin and vitexin, along with canjanol and cajanin.

[0037] Sesame: Sesamum indicum Approximately 67% of sesame protein is globulin, but 7s globulin accounts for only 5% of the total protein content. The major polyphenols in sesame are lignans, particularly sesamin, sesamolin, sesamol, and sesaminol. Flavonoids and phenolic acids, such as catechin and 1,2-dihydroxybenzoic acid, have also been reported in sesame seeds.

[0038] Scarlet runner bean: Phaseolus coccineus The major storage protein in runner beans is intact vicilin. On the other hand, caffeic acid and chlorogenic acid are the main phenolic compounds found in runner bean seed oil, while the seed coat is an excellent source of high molecular weight procyanidins.

[0039] Soybean: Glycine Max The vicilin storage protein in soybeans is known as β-conglycinin, which exists as a trimer of three glycosylated peptides. Over 70% of the protein content of soybeans is composed of 7S and 11s (glycinin) globulins, which are highly valued in the food industry for their nutritional value and gelling properties. Soybeans are also a rich source of the isoflavones genistein, daidzein, and glycitein, which exist primarily as malonic acid conjugates of the 7-O-glucosides genistin, daidzein, and glycitein.

[0040] Vicia sativa: Vicia sativa The protein content of vetch is approximately 50.8% globulin and 43.6% albumin. The globulin fraction is dominated by a legumin-like 10S and 6S storage protein called α-vicinin. Phenolic compounds such as kaempferol glycoside, myricetin, and naringenin have been reported in vetch roots and leaves. Furthermore, the total polyphenol content of vetch seeds is more than three times higher than that in soybeans.

[0041] Table 2 summarizes the physicochemical properties of candidate legumes based on data provided by the United States Department of Agriculture (USDA).

[0042] Advantageously, the inventors evaluated the protein, polyphenol, and lipid profiles of 19 candidate legumes and assigned each a similarity score to cocoa based on an aggregate scoring system. The relative abundance of 7S VCG and protein profile were characterized, while legumes were scored in terms of their relative similarity to cocoa. Additional factors, such as a polyphenol profile rich in flavan-3-ols and procyanidins, or a high lipid content, were also considered. Table 3 summarizes the profiles and scores obtained for each of the candidate legumes.

[0043] Through considerable effort by the inventors and based on their analysis, six of the legumes listed above are believed to exhibit characteristics sufficiently similar to cacao / cocoa beans (i.e., legumes having a combined score of ≧4 in Table 3), including broad beans, white lupine, peas, moth beans, scarlet beans, and sesame.

[0044] Thus, in a preferred embodiment, the legume to be fermented is selected from the group consisting of broad beans (Vicia faba), white lupine (Lupinus albus), pea (Pisum sativum), moth bean (Vigna aconitifolia), scarlet runner bean (Phaseolus cocchineus), and sesame (Sesamum indicum). Most preferably, the legume to be fermented is broad beans.

[0045] However, Example 7 illustrates how a variety of beans other than broad beans (e.g., lupin, green peas, moss, adzuki beans, and hemp seeds) can be used to develop cocoa-like analogs and are therefore preferred.

[0046] In one embodiment, the at least one microorganism used in the microbial fermentation may be selected from yeast and / or bacteria. In one embodiment, the microbial fermentation substantially comprises or exclusively comprises 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.

[0047] In another embodiment, the microbial fermentation comprises substantially or exclusively 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.

[0048] In yet another embodiment, the microbial fermentation comprises yeast and bacteria. Preferably, the ratio of yeast to bacterial cells is about 50:50. However, other ratios are contemplated, including 10:90, 20:80, 30:70, or 40:60 yeast cells to bacterial cells, or 10:90, 20:80, 30:70, or 40:60 yeast cells to bacterial cells.

[0049] In some embodiments, when yeast cells are used in microbial fermentation, only one species of yeast is used. Similarly, when bacterial cells are used in microbial fermentation, in some embodiments, only one species of bacteria is used. However, in other embodiments, microbial fermentation may involve the use of one or more yeast species and / or one or more bacterial species. For example, two, three, four, or more different species of yeast or bacteria may be used.

[0050] The bacteria may be gram-positive or gram-negative. Preferably, the bacterial family may be selected from the group of bacterial families consisting of Lactobacillales; and Acetobacter. Preferably, the bacterial species may be selected from the group of bacterial species consisting of Lactobacillus fermentum; Lactobacillus plantarum; and Acetobacter pasterianus. Example 6 illustrates the effect of these different microorganisms on the resulting chocolate analogue.

[0051] Preferably, the yeast genus may be selected from the group of yeast genera consisting of Pichia, Hanseniaspora, and Saccharomyces. Preferably, the yeast species may be selected from the group of yeast species consisting of Pichia kudriavzevii, Hanseniaspora opuntiae, and Saccharomyces cerevisiae.

[0052] Preferably, the microbial fermentation method comprises culturing at least one microorganism and a legume in a medium (referred to as "synthetic pulp" in the Examples and in FIG. 1). The medium is a solution of chemicals and nutrients used to ferment the legume. It will be understood that the legume, medium, and at least one microorganism collectively form a microbial fermentation mixture.

[0053] It will be appreciated that in one embodiment, the method comprises a microbial fermentation process of a legume, preferably broad beans, in a culture medium, as shown in Figures 1B and 1C. Two types of fermentation starters may be provided, one containing both yeast and bacteria (option 1), and the second containing either yeast only or bacteria only (option 2). Figure 1B illustrates an exemplary legume fermentation process using a fermentation starter according to option 1 to obtain a cocoa bean-free chocolate analog, and Figure 1C illustrates an exemplary legume fermentation process using a fermentation starter according to option 2 to obtain a cocoa bean-free chocolate analog.

[0054] However, in another embodiment, as shown in Figure ID, the method preferably comprises two separate treatments of legumes (preferably broad beans). The method preferably comprises a first treatment in which the legumes are microbially fermented, preferably in a culture medium (either Option 1 or Option 2), to obtain a microbially fermented legume, and a second treatment in which the legumes are separately non-microbially fermented, preferably in an incubation solution, in the absence of exogenously added microorganisms, to obtain a non-microbially treated legume. The method then preferably comprises combining a portion of the microbially fermented legume with a portion of the non-microbially fermented legume to obtain a cocoa bean-free chocolate analog.

[0055] The ratio of microbially fermented legumes to non-microbially fermented legumes is preferably about 10%:90% to 90%:10%, or about 50%:50%.

[0056] In one embodiment, the culture medium or incubation solution may comprise one or more sugars, preferably selected from the group consisting of sucrose, glucose, fructose, high viscosity carboxymethylcellulose, low viscosity carboxymethylcellulose, and / or pectin.

[0057] A suitable concentration of sucrose may be about 0.1% (w / v) to 10% (w / v), preferably about 0.5% (w / v) to 7% (w / v), more preferably about 1% (w / v) to 5% (w / v), and most preferably about 2% (w / v) to 3% (w / v). A glucose concentration of about 2.5% (w / v) is most preferred. A suitable glucose concentration may be about 0.1% (w / v) to 10% (w / v), preferably about 1% (w / v) to 8% (w / v), more preferably about 2% (w / v) to 6% (w / v), and most preferably about 3% (w / v) to 5% (w / v). A glucose concentration of about 4% (w / v) is most preferred. A suitable concentration of fructose may be about 0.1% (w / v) to 10% (w / v), preferably about 1% (w / v) to 8% (w / v), more preferably about 3% (w / v) to 7% (w / v), and most preferably about 4% (w / v) to 6% (w / v). About 5% (w / v) of fructose is most preferred. A suitable concentration of high-viscosity carboxymethylcellulose may be about 0.005% (w / v) to 5% (w / v), preferably about 0.005% (w / v) to 2% (w / v), more preferably about 0.005% (w / v) to 1% (w / v), and most preferably about 0.05% (w / v) to 0.5% (w / v). About 0.1% (w / v) of high-viscosity carboxymethylcellulose is most preferred. A suitable concentration of low-viscosity carboxymethylcellulose may be about 0.005% (w / v) to 5% (w / v), preferably about 0.005% (w / v) to 2% (w / v), more preferably about 0.005% (w / v) to 1% (w / v), and most preferably about 0.05% (w / v) to 1% (w / v). About 0.8% (w / v) low-viscosity carboxymethylcellulose is most preferred.

[0058] A suitable concentration of pectin may be about 0.01% (w / v) to 10% (w / v), preferably about 0.01% (w / v) to 5% (w / v), more preferably about 0.1% (w / v) to 3% (w / v), and most preferably about 0.5% (w / v) to 2% (w / v). About 1% (w / v) pectin is most preferred.

[0059] The culture medium or incubation solution may contain one or more salts. Preferably, the one or more salts may be selected from the group consisting of calcium lactate pentahydrate; magnesium sulfate heptahydrate; and manganese sulfate monohydrate. A suitable concentration of calcium lactate pentahydrate may be about 0.001% (w / v) to 2% (w / v), preferably about 0.001% (w / v) to 1% (w / v), more preferably about 0.01% (w / v) to 0.75% (w / v), and most preferably about 0.05% (w / v) to 0.5% (w / v). About 0.1% (w / v) of calcium lactate pentahydrate is most preferred. A suitable concentration of magnesium sulfate heptahydrate may be about 0.001% (w / v) to 1% (w / v), more preferably about 0.001% (w / v) to 0.5% (w / v), and most preferably about 0.01% (w / v) to 0.1% (w / v). Magnesium sulfate heptahydrate is most preferred at about 0.05% (w / v). A suitable concentration of manganese sulfate monohydrate may be about 0.001% (w / v) to 1% (w / v), more preferably about 0.001% (w / v) to 0.5% (w / v), and most preferably about 0.01% (w / v) to 0.1% (w / v). Manganese sulfate monohydrate is most preferred at about 0.02% (w / v).

[0060] The culture medium or incubation solution may contain one or more protein hydrolysates. Preferably, the one or more protein hydrolysates is peptone. A suitable concentration of peptone may be about 0.001% (w / v) to 7% (w / v), preferably about 0.001% (w / v) to 5% (w / v), more preferably about 0.01% (w / v) to 2% (w / v), and most preferably about 0.05% (w / v) to 1% (w / v). About 0.5% (w / v) of peptone is most preferred.

[0061] The culture medium or incubation solution may contain yeast extract. A suitable concentration of yeast extract may be about 0.001% (w / v) to 7% (w / v), preferably about 0.001% (w / v) to 5% (w / v), more preferably about 0.01% (w / v) to 2% (w / v), and most preferably about 0.05% (w / v) to 1% (w / v). About 0.5% (w / v) yeast extract is most preferred.

[0062] The culture medium or incubation solution may contain a surfactant. Preferably, the surfactant is Tween 80. A suitable concentration of Tween 80 may be about 0.001% (w / v) to 2% (w / v), preferably about 0.001% (w / v) to 1% (w / v), more preferably about 0.01% (w / v) to 0.75% (w / v), and most preferably about 0.05% (w / v) to 0.5% (w / v). About 0.1% (w / v) of Tween 80 is most preferred.

[0063] The culture medium or incubation solution may contain citric acid. A suitable concentration of citric acid may be about 0.0001% (w / v) to 6% (w / v), preferably about 0.005% (w / v) to 5% (w / v), more preferably about 0.05% (w / v) to 3% (w / v), and most preferably about 0.5% (w / v) to 2% (w / v). About 1% (w / v) citric acid is most preferred.

[0064] In a preferred embodiment, the culture medium or incubation solution comprises sucrose, glucose, fructose, citric acid, high viscosity carboxymethylcellulose, low viscosity carboxymethylcellulose, pectin, yeast extract, peptone, calcium lactate pentahydrate, Tween 80, magnesium sulfate heptahydrate, and manganese sulfate monohydrate.

[0065] In embodiments where microbial fermentation is carried out with either yeast or bacteria (but not both together), fermentation may be carried out in the following medium, to which the microorganisms and legumes (i.e., beans) are added and co-incubated:

[0066] Yeast and Lactobacillus - LB broth (Lennox) containing 5 g / L NaCl, 10 g / L tryptone, and 5 g / L yeast extract

[0067] Acetobacter - AA broth was prepared by mixing the following ingredients: 1% (w / v) D-glucose, 1.5% (w / v) bacteriological peptone, and 0.8% (w / v) yeast extract. After sterilization, 0.5% (v / v) ethanol and 0.3% (v / v) acetic acid were added to the mixture.

[0068] The culture medium or incubation solution is preferably prepared by mixing the reagents and adjusting the pH.

[0069] Preferably, the culture medium or incubation solution has an acidic pH. Preferably, the culture medium or incubation solution has a pH of 1 to 6, 2 to 5, or 3 to 4. In a preferred embodiment, the culture medium has a pH of about 3.6.

[0070] As explained in the Examples, the inventors have found that good chocolate aroma analogs are associated with fava bean flour processed at low pH. Referring to Figure 5, the pH optimum for chocolate aroma is shown to be between pH 4.8 and 5.1. Furthermore, Figure 6 shows that in chocolate analogs with low pH, chocolate aroma begins to develop 3-4 days after roasting and grinding, matures by day 7, and continues to improve for up to one month, remaining stable for at least four months thereafter.

[0071] Preferably, the chocolate analog has a pH of from about 4 to about 7, more preferably from about 4.3 to about 6.8, more preferably from about 4.4 to about 6.7.

[0072] Preferably, the chocolate analog has a pH of about 4.4 to about 6.3, more preferably about 4.7 to about 5.9, more preferably about 4.7 to about 5.5.

[0073] Preferably, the chocolate analog has a pH of about 4.4 to about 5.5, more preferably about 4.7 to about 5.2, and most preferably about 4.8 to about 5.1.

[0074] The medium or incubation solution may then preferably be autoclaved and then stored at 4°C.

[0075] In microbial fermentation, the legume is preferably contacted with at least one microorganism for one or more days at a temperature suitable to sustain the growth of the one or more microorganisms to allow the microbial fermentation to occur. Preferably, the microbial fermentation is carried out for at least 2, 3, 4, or 5 days.

[0076] In embodiments in which at least one microorganism is yeast, the microbial fermentation may be carried out 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 microbial fermentation mixture is incubated at a temperature of about 30° C., which is optimal for yeast cell growth.

[0077] However, in embodiments in which at least one microorganism is a bacterium, the microbial fermentation may be carried out at a temperature of 24° C. to 50° C., 26° C. to 48° C., 28° C. to 46° C., 30° C. to 44° C., or 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 microbial fermentation mixture is incubated at a temperature of about 42° C., which is optimal for bacterial cell growth.

[0078] It will be appreciated that when a combination of yeast and bacteria are cultured together, a suitable temperature may be used, for example, 24°C to 44°C, 26°C to 43°C, or 28°C to 42°C.

[0079] It will be understood that any of the temperatures described herein may be combined with any of the times described herein. Thus, for example, the microbial fermentation may include incubating the yeast and bacteria at 24°C to 44°C for at least one or two days. Alternatively, the microbial fermentation may include incubating the yeast at 21°C to 37°C for at least one or two days. Alternatively, the microbial fermentation may include incubating the bacteria at 24°C to 50°C for at least one or two days, etc.

[0080] As described in the Examples, the inventors have demonstrated two different embodiments of microbial fermentation of legumes to produce cocoa bean-free chocolate analogs, as shown in Figures 1B and 1C.

[0081] Thus, in one embodiment (shown in FIG. 1B), the microbial fermentation method may comprise simultaneously microbially fermenting a legume in the presence of yeast and bacteria. In this embodiment, preferably the yeast comprises Pichia kudriabzevi, Hanseniaspora opuntiae, and / or Saccharomyces cerevisiae, most preferably Pichia kudriabzevi, Hanseniaspora opuntiae, and Saccharomyces cerevisiae. Preferably, the bacteria comprise Lactobacillus fermentum, Lactobacillus plantarum, and / or Acetobacter pasteurianus, most preferably Lactobacillus fermentum, Lactobacillus plantarum, and Acetobacter pasteurianus. Preferably, the yeast and bacteria are mixed or pooled together, and preferably the concentration of each isolate is at least 1×10 7 or 1 x 10 8 cells / mL.

[0082] In a first embodiment (combined yeast and bacterial fermentation), the method comprises contacting the legume with yeast and bacterial cells for at least 24 hours at a temperature between 21° C. and 37° C. Preferably, the microbial fermentation mixture is incubated at a temperature between 21° C. and 37° C., 23° C. and 35° C., 24° C. and 34° C., 26° C. and 33° C., or 27° C. and 32° C. Preferably, the microbial fermentation mixture is incubated at a temperature of 30° C. Preferably, the microbial fermentation mixture is incubated for at least 36 or 48 hours.

[0083] Preferably, the method then includes discarding the medium after one or more days of initial microbial fermentation. Preferably, the method includes further incubating the legume and at least one microorganism at a temperature of 24°C to 50°C for one or more days. Preferably, the microbial fermentation mixture is incubated at a 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 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 of the temperatures described herein may be combined with any of the times described herein. At the end of the microbial fermentation process, the method preferably includes drying the legume. As described below, the dried legume can then be processed to produce a suitable substrate from which a food product can be prepared.

[0084] In a second embodiment (shown in FIG. 1C), the method may include first microbially fermenting the legume in the presence of yeast only, followed by subsequent microbial fermentation in the presence of bacteria (and yeast). In this embodiment, preferably the yeast comprises Pichia kudriabzevii, Hanseniaspora opuntiae, and / or Saccharomyces cerevisiae, most preferably Pichia kudriabzevii, Hanseniaspora opuntiae, and Saccharomyces cerevisiae. Preferably, the bacteria comprise Lactobacillus fermentum, Lactobacillus plantarum, and / or Acetobacter pasteurianus, most preferably Lactobacillus fermentum, Lactobacillus plantarum, and Acetobacter pasteurianus. Preferably, the yeast and bacteria are stored in separate pools, each at a concentration of at least 1×10 7 or 1 x 10 8 cells / mL.

[0085] In a second embodiment (initial yeast-only fermentation followed by yeast and bacterial fermentation), the method comprises contacting the legume with yeast cells for at least 24 hours at a temperature between 21° C. and 37° C. Preferably, the microbial fermentation mixture is incubated at a temperature between 21° C. and 37° C., 23° C. and 35° C., 24° C. and 34° C., 26° C. and 33° C., or 27° C. and 32° C. Preferably, the microbial fermentation mixture is incubated at a temperature of 30° C. Preferably, the microbial fermentation mixture is incubated for at least 36 or 48 hours.

[0086] Preferably, the method then includes discarding the medium after one or more days of initial microbial fermentation. Preferably, the method includes contacting the fermented legume with bacterial cells for at least 24 hours at a temperature of 24°C to 50°C. Preferably, the microbial fermentation mixture is incubated at a 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 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 of the temperatures described herein may be combined with any of the times described herein.

[0087] At the end of the microbial fermentation process, the method preferably includes a step of drying the legumes. As explained below, the dried legumes can then be processed to produce a suitable substrate from which a food product can be prepared.

[0088] In a third embodiment (not shown), the method may include first microbially fermenting the legume in the presence of bacteria only, followed by subsequent microbial fermentation in the presence of yeast (and bacteria). In this embodiment, preferably the yeast comprises Pichia kudriabzevi, Hanseniaspora opuntiae, and / or Saccharomyces cerevisiae, most preferably Pichia kudriabzevi, Hanseniaspora opuntiae, and Saccharomyces cerevisiae. Preferably, the bacteria comprise Lactobacillus fermentum, Lactobacillus plantarum, and / or Acetobacter pasteurianus, most preferably Lactobacillus fermentum, Lactobacillus plantarum, and Acetobacter pasteurianus. Preferably, the yeast and bacteria are kept separately in separate pools, each at a concentration of at least 1 x 10 7 or 1 x 10 8 cells / mL.

[0089] In a third embodiment (initial yeast-only fermentation followed by bacterial and yeast fermentation), the method may include contacting the legume with the bacterial cells for at least 24 hours at a temperature between 24°C and 50°C. Preferably, the microbial fermentation mixture is incubated at a temperature between 26°C and 48°C, 28°C and 46°C, 30°C and 44°C, 32°C and 44°C, 34°C and 44°C, 36°C and 44°C, 38°C and 44°C, or 40°C and 44°C. Preferably, the microbial fermentation mixture is incubated at a temperature of 42°C. Preferably, the mixture is incubated for at least 2 or 3 days. Preferably, the microbial fermentation mixture is incubated for 3 days.

[0090] Preferably, the method then includes discarding the medium after one or more days of initial microbial fermentation. Preferably, the method includes contacting the microbially fermented legume with yeast cells for at least 24 hours at a temperature of 21°C to 37°C. Preferably, the microbial fermentation 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 microbial fermentation mixture is incubated at a temperature of 30°C. Preferably, the microbial fermentation mixture is incubated for at least 36 or 48 hours. It will be understood that any of the temperatures described herein may be combined with any of the times described herein. At the end of the microbial fermentation process, the method preferably includes drying the legume. As described below, the dried legume can then be processed to create a suitable substrate from which a food product can be prepared.

[0091] In embodiments where the method involves non-microbial fermentation, the method involves the use of a culture medium having a pH of 3-5 (preferably about 3.6), such that incubation in this medium tends to acidify the legume (preferably broad bean). Furthermore, if the legume (e.g., broad bean) is completely submerged when the legume begins to germinate, it will rapidly consume available oxygen for respiration. Under anaerobic conditions, fermentative metabolism begins with 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 decrease the pH of the legume (preferably broad bean).

[0092] Preferably, the non-microbial fermentation 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-microbial fermentation mixture is incubated at a temperature of 30°C. Preferably, the mixture is incubated for at least 1 or 2 days. Preferably, the non-microbial fermentation mixture is incubated for 2 days.

[0093] Preferably, the non-microbial fermentation 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-microbial fermentation mixture is incubated at a temperature of 42°C. Preferably, the mixture is incubated for at least 1 day, 2 days, or 3 days. Preferably, the non-microbial fermentation mixture is incubated for 3 days.

[0094] In a preferred embodiment, the method includes the step of drying the legumes after microbial or non-microbial fermentation. Preferably, the legumes are dried by heating in an oven. Preferably, the legumes are dried in an oven at a temperature of about 21°C to 37°C for one or more days. Preferably, the legumes are dried in an oven at a temperature of 23°C to 35°C, 25°C to 40°C, 28°C to 38°C, or 30°C to 36°C. During this time, the beans are mixed on the first, second, and third days of drying to minimize clumping of the beans. Preferably, the legumes are dried in an oven at a temperature of 35°C. Preferably, drying is carried out for 2, 3, 4, 5, 6, or 7 days. Preferably, the legumes are dried in an oven for 7 days.

[0095] In one embodiment, the cocoa bean-free chocolate analog containing microbially fermented and / or non-microbially fermented legumes may comprise a solid, liquid, or powder.

[0096] In one embodiment, the cocoa bean-free chocolate analog containing microbially fermented and / or non-microbially fermented legumes may comprise or be converted into a paste. Figure 2 shows an embodiment of how a paste can be prepared from fermented legumes.

[0097] Preferably, therefore, the method comprises roasting microbially fermented and / or non-microbially fermented legumes. Preferably, the method comprises crushing and sieving the roasted legumes to obtain a plurality of nibs. Preferably, the method comprises refining and conching the plurality of nibs to form a non-cocoa chocolate liquor. Preferably, the method comprises adding one or more additives to the non-cocoa chocolate liquor. It will be appreciated that when using real cocoa beans, grinding the roasted beans produces a viscous liquid / paste called "liquor." This is because cocoa beans contain approximately 40% lipids (called "cocoa butter"). However, fava beans are very low in lipids. Therefore, roasting and grinding the processed fava beans of the present invention produces a powder (not a liquid). To make "liquor" from fava bean powder, lipids with properties similar to cocoa butter (e.g., shea butter) may be added.

[0098] Preferably, the method includes repeating the previous step in a non-cocoa chocolate liquor, thereby producing a non-cocoa chocolate analog paste.

[0099] Preferably, the method includes the step of tempering the non-cocoa chocolate analog paste. Preferably, the method includes the step of shaping the tempered non-cocoa chocolate analog paste.

[0100] In one embodiment, roasting the microbially fermented 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 less than 350°C, 330°C, or 310°C, more preferably less than 300°C, 290°C, or 280°C. Preferably, roasting the fermented legumes comprises heat-treating the legumes at 150-350°C, more preferably 180-280°C. Preferably, roasting is for at least 2, 3, or 4 minutes, more preferably at least 5, 6, or 7 minutes. Preferably, roasting is done at 180-280° C. for 5-20 minutes. It will be understood that any of the temperatures described herein may be combined with any of the times described herein.

[0101] In another embodiment, the crushing of the legumes is performed in a mechanical or electrical crusher. In another embodiment, the sorting of the legumes is performed in a mechanical or electrical sorter.

[0102] In one embodiment, the crushing and sorting of the legumes are performed simultaneously. In one embodiment, the crushing and sorting of the legumes are performed sequentially. In a preferred embodiment, crushing of the legumes is performed before sorting of the legumes.

[0103] In one embodiment, the plurality of nibs are preheated prior to the refining and conching steps. In another embodiment, the plurality of nibs are not preheated prior to the refining and conching steps.

[0104] In one embodiment, refining and conching the fermented legumes comprises heat treating the legumes in a conching machine.

[0105] In another embodiment, tempering the fermented legumes comprises heat treating the legumes in a tempering machine.

[0106] In yet another embodiment, the non-cocoa chocolate analog paste is stored at a temperature ranging from 5° C. to 25° C.; 7° C. to 23° C., or 7° C. to 23° C. Preferably, the non-cocoa chocolate analog paste is stored at a temperature ranging from 10° C. to 20° C.

[0107] In an alternative embodiment, however, the cocoa bean-free chocolate analog comprising microbially fermented and / or non-microbially fermented legumes may comprise a powder. Figure 3 shows an embodiment of how a powder can be prepared from fermented legumes. In an alternative embodiment, however, the cocoa bean-free chocolate analog comprising microbially fermented and / or non-microbially fermented legumes may comprise a liquor.

[0108] In an alternative embodiment, a cocoa bean-free chocolate analog comprising microbially fermented and / or non-microbially fermented broad beans is preferably combined with a cocoa bean-free chocolate analog comprising a non-microbially fermented non-broad bean legume.

[0109] In another alternative embodiment, however, a cocoa bean-free chocolate analog comprising microbially fermented and / or non-microbially fermented broad beans is preferably combined with a cocoa bean-free chocolate analog comprising a microbially fermented non-broad bean legume.

[0110] The non-fava legume may be any of those described herein.

[0111] Preferably, the method includes roasting microbially fermented or non-microbially fermented legumes. Preferably, the method includes crushing and screening the legumes to form a plurality of nibs. Preferably, the method includes refining and conching the plurality of nibs to form a non-cocoa chocolate liquor. Preferably, the method includes pressing the non-cocoa chocolate liquor to form a non-cocoa chocolate powder cake. Preferably, the method includes grinding the non-cocoa chocolate powder cake, thereby producing a non-cocoa chocolate analog powder.

[0112] In one embodiment, roasting the microbially fermented or non-microbially fermented legumes comprises preheating the legumes to at least 150°C for at least 1 minute. Preferably, roasting the fermented legumes comprises preheating the legumes to 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 preheating the legumes to less than 350°C, 330°C, or 310°C, more preferably less than 300°C, 290°C, or 280°C. Preferably, roasting the fermented legumes comprises preheating the legumes to between 150 and 350°C, more preferably between 180 and 280°C. Preferably, roasting is for at least 2, 3, or 4 minutes, more preferably at least 5, 6, or 7 minutes. Preferably, roasting is done at 180-280° C. for 5-20 minutes. It will be understood that any of the temperatures described herein may be combined with any of the times described herein.

[0113] In another embodiment, the crushing of the legumes is performed in a mechanical or electrical crusher. In another embodiment, the sorting of the legumes is performed in a mechanical or electrical sorter.

[0114] In one embodiment, the crushing and sorting of the legumes are performed simultaneously. In one embodiment, the crushing and sorting of the legumes are performed sequentially. In a preferred embodiment, the crushing of the legumes is performed before the sorting of the legumes.

[0115] In one embodiment, the plurality of nibs are preheated prior to the refining and conching steps. In another embodiment, the plurality of nibs are not preheated prior to the refining and conching steps.

[0116] In one embodiment, grinding the non-cocoa chocolate powder cake comprises first coarsely grinding the non-cocoa chocolate powder cake and then finely grinding it.

[0117] In yet another embodiment, the non-cocoa chocolate analog powder is stored at a temperature ranging from 5° C. to 25° C.; 7° C. to 23° C., or 7° C. to 23° C. Preferably, the non-cocoa chocolate analog paste is stored at a temperature ranging from 10 to 20° C.

[0118] The inventors have shown that the cocoa bean-free chocolate analogs described herein can be used to prepare a variety of food products, solids, or liquids.

[0119] 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.

[0120] Thus, in a sixth aspect, there is provided a foodstuff or beverage comprising a cocoa bean-free chocolate analogue according to the first, second or fourth aspect, or an ingredient of the fifth aspect.

[0121] The cocoa bean-free chocolate analog may be in the form of a powder, liquor, liquid, solid, or paste.

[0122] A dry powder can be obtained from roasted and crushed broad beans. A lipid (e.g., shea butter) can be added to this dry powder to create a solid that liquefies when heated to 31-38°C (89-91°F). This liquid corresponds to the liquid obtained when cocoa beans are ground, but with cocoa, no lipid addition is required.

[0123] The food product is preferably a confectionery product. For example, the food product may be a block bar, a molded figure, a button, a truffle, a spread, a coated fruit, nut, and other inclusions, a bakery filling, a coating for products such as donuts or muffins, yogurt, ice cream, or other desserts. The beverage may be a hot or cold beverage.

[0124] In a seventh aspect, there is provided a cosmetic product comprising a cocoa bean-free chocolate analogue according to the first, second or fourth aspect, or an ingredient of the fifth aspect.

[0125] The cosmetic product may be a skin care product, such as a moisturizer, exfoliator, lip balm, eye shadow, soap, body cream or butter, or a hair mask.

[0126] All of the features described in this specification (including the accompanying claims, abstract, and drawings), and / or all steps of the disclosed methods or processes, may be combined with any of the above aspects in any combination, except combinations in which at least some of the features and / or steps are inconsistent with each other.

[0127] For a better understanding of the present invention and to show how embodiments thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: FIG. [Brief explanation of the drawings]

[0128] [Figure 1]FIG. 1 shows a schematic diagram of legume fermentation according to two embodiments of the present invention. As shown in FIG. 1A, a fermentation seed is first prepared from a single colony of yeast or bacteria. The colony is grown in a selective medium, and the grown microbial cell preparation is pooled in vials and stored until the fermentation process. Two fermentation seed mixtures are prepared: one containing both yeast and bacteria (Option 1), and the second containing either yeast only or bacteria only (Option 2). FIG. 1B shows an exemplary legume fermentation process according to the present invention in which fermentation according to Option 1 is used, and FIG. 1C shows an exemplary legume fermentation according to the present invention in which a fermentation seed mixture according to Option 2 is used. FIG. 1D shows a further exemplary legume fermentation process according to the present invention in which two separate reactions, a microbial treatment and a non-microbial treatment of the legume, are performed. First, the legume is treated with a microorganism (either Option 1 or Option 2) to produce a microbially fermented legume, and then separately, the legume is treated in the absence of a microorganism to produce a non-microbially treated legume. The microbially fermented legume portion and the non-microbially treated legume portion are then blended or mixed together to obtain the cocoa bean-free chocolate analog. [Figure 2] FIG. 2 shows a schematic diagram of an embodiment of a method for making a chocolate analog paste. [Figure 3] FIG. 3 shows a schematic of an embodiment of a method for making a chocolate analog powder. [Figure 4] Figure 4 shows photographs of dried broad beans after soaking in water for 3 hours: (a) 0 hours, (b) 24 hours incubation, (c) 48 hours incubation, (d) 72 hours incubation, (e) 120 hours incubation, (f) dried beans after 120 hours of fermentation, and (g) dried unfermented beans. [Figure 5] Figure 5 shows a bar graph of the percent frequency of chocolate aroma per jar and therefore the optimum pH of roasted broad bean flour to produce the best chocolate aroma. [Figure 6] Figure 6 shows the evolution of the aroma profile over time after roasting and grinding the beans. [Figure 7] Figure 7 shows the flavour profile of the liquor (powder + shea butter 1:1 w / w) as scored by five independent judges. The fava bean flour is representative of powders made from 10 independent bean preparations. As can be seen, the fava bean flour of the present invention exhibits flavour characteristics very similar to supermarket (Tesco) cocoa powder and Peruvian cocoa powder. [Figure 8] Figure 8 shows the flavor profile of liquor (bean flour, sugar, and shea butter) made from processed roasted and milled fava bean flour, with fava beans (NK0034) processed using our non-microbial process included for comparison. Flavor was evaluated by five independent individuals. [Figure 9] Figure 9 shows the flavour profiles of a chocolate analogue according to the present invention (herein referred to as "NuKoKo choc") and authentic supermarket Tesco brand milk and dark chocolate (Mollys). The flavours were evaluated by five independent individuals. [Figure 10] Figure 10 shows the flavor profiles of liquors (bean flour, sugar, and shea butter) made from processed roasted and milled fava bean flour and from a selection of other beans and seeds, all processed using the non-microbial process of the present invention. (A) Hemp seeds, (B) Adzuki beans, (C) Moth beans, (D) Lupin beans, (E) Peas. Fava beans (NK0034) are shown for comparison (A, B, C, D, E). (F) is a comparison of all non-fava bean and seed groups. Flavor was evaluated by three independent individuals. [Example]

[0129] Fermentation is an essential step required for cocoa / cocoa beans to develop their unique and distinctive chocolate flavor and aroma. To develop a non-cocoa chocolate alternative, the inventors first identified various legumes that exhibit key characteristics they believe contribute distinctive viscosity, flavor, color, and aroma to traditional chocolate, and then selected a subset of legumes that met the required profile. The inventors also developed and highly optimized fermentation processes (both in the presence and absence of specific microorganisms, such as yeast and bacteria) applied to selected legumes (e.g., broad beans) that impart the same characteristics (in terms of taste and aroma) to cocoa analog powders made from these legumes as regular cocoa powder. The fermented legumes may then be processed to prepare chocolate analogs, which can then be formed into various food products.

[0130] In a first embodiment, the process involves a microbial fermentation process of legumes, such as broad beans, as shown in Figures 1B and 1C. Two fermentation starters are prepared, one containing both yeast and bacteria (Option 1), and the second containing either yeast only or bacteria only (Option 2). Figure 1B shows an exemplary legume fermentation process using a fermentation starter according to Option 1 to obtain a cocoa bean-free chocolate analog, and Figure 1C shows an exemplary legume fermentation process using a fermentation starter according to Option 2 to obtain a cocoa bean-free chocolate analog.

[0131] In a second embodiment, as shown in Figure ID, two separate treatments of legumes (e.g., broad beans) are performed. First, the legumes are treated with a microorganism (either option 1 or option 2) to produce a microbially fermented legume. Second, the legumes are separately treated in the absence of a microorganism to produce a non-microbially treated legume. A portion of the microbially fermented legume is then blended with a portion of the non-microbially treated legume to produce a cocoa bean-free cocoa powder analog.

[0132] Materials and Methods Selection of candidate legumes The inventors first present specific selection criteria for legumes to be considered for further testing, including crops that are low cost, agriculturally established, and grown relatively close to production facilities (particularly in Europe). From their extensive research, the inventors have identified the following characteristics as essential for candidate legumes to exhibit chocolate characteristics (taste and aroma) after fermentation. These characteristics include: 1) A sufficient content of 7S VCG (vicilin 7S class globulin) protein or its homologue. 2) A readily available source of carbohydrates to overcome the drawbacks associated with the reduction of sugar content during fermentation. 3) High content of flavan-3-ols and procyanidins to enhance chocolate-like flavor and color development.

[0133] For candidate legumes that met only the first characteristic, preliminary enrichment of the legumes with suitable food-grade sources of sugars and flavan-3-ols and procyanidins, such as apple juice or grape juice, was performed prior to fermentation.

[0134] Fermentation of legumes material Unless otherwise stated, chemicals and plastic consumables used in the fermentation of selected legumes were purchased from Sigma-Aldrich and Scientific Laboratory Supplies Ltd. All solutions were prepared using reverse osmosis (RO) water.

[0135] sterilization Solutions and consumables used in the present invention were heat sterilized by autoclaving at 121°C (130 kPa) for 15 minutes.

[0136] Step 1: Preparation of synthetic pulp (microbial fermentation medium) A synthetic pulp was prepared by mixing the following ingredients: 2.5% (w / v) sucrose (Cat. No.: S0389-1KG) 4% (w / v) glucose (Cat. No.: G7021-5KG) 5% (w / v) fructose (Cat. No.: F0127-1KG) 1% (w / v) citric acid (Cat. No.: C0759-1KG) 0.14% (w / v) high viscosity carboxymethylcellulose (Cat. No.: C5678-1KG) 0.77% (w / v) low viscosity carboxymethylcellulose (Cat. No.: C5013-1KG) 1.09% (w / v) pectin (Cat. No.: P9135-500G) 0.5% (w / v) yeast extract (Cat. No.: 70161-500G) 0.5% (w / v) peptone (Cat. No.: 91249-500G) 0.1% (w / v) calcium lactate pentahydrate (Cat. No.: C8356-250G) 0.1% (v / v) Tween 80 (Cat. No.: P4780-100ML) 0.05% (w / v) magnesium sulfate heptahydrate (Catalog number: M2773-1KG) 0.02% (w / v) manganese sulfate monohydrate (Cat. No.: M7899-500G)

[0137] The pH of the mixture of the above ingredients was then adjusted to a pH of 3.6 to create a medium suitable for yeast and bacteria so that the microorganisms could ferment the legumes. The mixture was then sterilized as above and then stored at 4°C.

[0138] Step 2: Preparing selective agar and broth (seed medium) The following agar media were used:

[0139] Acetic acid (AA) agar AA agar was prepared by mixing the following ingredients: 1% (w / v) D-glucose, 1.5% (w / v) bacteriological peptone, 0.8% (w / v) yeast extract, and 2% (w / v) agar. The mixture was then sterilized as described above. After sterilization, 0.5% (v / v) ethanol and 0.3% (v / v) acetic acid were added to the mixture, and the final pH of the mixture was adjusted to 4.5.

[0140] Acetic acid (AA) broth AA broth was prepared by mixing the following components: 1% (w / v) D-glucose, 1.5% (w / v) bacteriological peptone, and 0.8% (w / v) yeast extract. The mixture was then sterilized as described above. After sterilization, 0.5% (v / v) ethanol and 0.3% (v / v) acetic acid were added to the mixture.

[0141] De Man Rogosa Sharpe (MRS) agar: MRS agar (Cat. 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 (Cat. No. P8074). The suspension was then boiled until the medium was completely dissolved, and the total volume was adjusted to 1000 ml. The mixture was then sterilized as described above.

[0142] Yeast Peptone Glucose (YPG) Agar: YPG agar was prepared by mixing the following ingredients: 1% (w / v) yeast extract, 2% (w / v) peptone, and 2% (w / v) glucose. The pH of the mixture was adjusted to 5.6, and then 2% (w / v) agar was added. The solution was then sterilized as described above.

[0143] microorganisms The inventors identified six microorganisms (yeasts and bacteria) that showed optimal fermentation results with selected legumes. These included Pichia kudriabzevi, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, Lactobacillus plantarum, and Acetobacter pasteurianus. The inventors further observed that each of the microorganisms was selected on a specific agar medium, as summarized in the table below.

[0144] [Table 1]

[0145] Step 3: Preparation of fermentation starter To prepare the fermentation seed, the following steps were performed under sterile conditions: 1. Selective agar was prepared as above and poured into sterile 9 cm Petri dishes (20 ml each) and allowed to cool and harden. 2. Glycerol stocks of each microorganism were plated onto their respective selective agar plates (listed in Table 1) and incubated at 28°C for 72 hours. 3. For each isolate, a 15 mL tube containing 3 mL of Luria broth Lennox was prepared, except for Acetobacter pasteurianus, which required a 15 mL tube 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 tube after sterilization. 4. One colony from the agar plate for each isolate was selected and transferred to 3 ml of the respective medium and incubated at 28°C in a shaking incubator at 200 rpm for 72 hours. 5. Cells were harvested by centrifugation at 4,000 x rpm for 8 minutes at 4°C. 6. The supernatant was discarded and 5 ml of sterile 10 mM MgCl2 was added to the tube. 7. The tube was inverted 10 times to resuspend the precipitated cells, and then centrifuged at 4,000 x rpm at 4°C for 8 minutes. 8. Repeat steps 6-7 two more times for a total of three washes. 9. Finally, the cells were resuspended in 1 mL of sterile 10 mM MgCl2 and the optical density at 600 nm (OD600) was measured for each isolate (1 OD600 = 1 x 10 9 The cell concentration (cells / mL) was determined. 10. Once the microorganisms were purified, two separate fermentation seeds were prepared as follows.

[0146] Option 1 (yeast and bacteria combined) All purified yeast (Pichia kudriabzevii, Hanseniaspora opuntiae, and Saccharomyces cerevisiae) and bacterial (Lactobacillus fermentum, Lactobacillus plantarum, and Acetobacter pasteurianus) isolates were pooled together, with each isolate at a concentration of 1 × 10 8 cells / mL (cells were diluted using sterile 10 mM MgCl2 as needed).

[0147] Option 2 (yeast only or bacteria only) The yeast isolates (Pichia kudriabzevii, Hanseniaspora opuntiae, and Saccharomyces cerevisiae) were combined together, and the bacterial isolates (Lactobacillus fermentum, Lactobacillus plantarum, and Acetobacter pasteurianus) were combined together, with each group pooled into a separate pool, with each isolate at a concentration of 1 × 10 8 cells / mL (cells were diluted using sterile 10 mM MgCl2 as needed).

[0148] For each option, a 1 mL inoculum was prepared and stored at 4°C for no more than 24 hours.

[0149] Step 4: Legume Fermentation Two separate protocols were designed for fermenting legumes based on whether to use the fermentation starter of option 1 (Protocol 1 in Figure 1B) or option 2 (Protocol 2 in Figure 1C). The protocols include the following steps, which are performed under sterile conditions:

[0150] Sterilization and soaking of beans Selected beans (see below for selection criteria) are fermented as follows: For each fermentation replicate, weigh out 125 g of beans using an analytical balance and transfer to a 500 mL reagent glass bottle. The bean weight and solution volume can be increased or decreased as needed to obtain more or less material.

[0151] 1. Prepare bean sterilization solution (70% ethanol, 0.05% Triton X-100 (Cat. No.: T8787-250ML)) (100 mL for each fermentation replicate). 2. Add the sterilizing solution to the beans and agitate using an orbital shaker at 200 rpm and 25°C for 30 minutes. The following steps are performed under sterile conditions. 3. Discard the sterilizing solution using a sterile 25 mL disposable polystyrene serological pipette. 4. The beans are then washed by adding 250 mL of sterile RO water to the jar, mixing by inverting the jar 10 times, and discarding the wash. 5. Repeat step 5 two more times for a total of three washes. 6. Add sterile RO water (250 mL) to the jar and rehydrate the beans by incubating them at 25°C for 3 hours. To speed up the germination process, the soaking time can be increased and the beans further rehydrated at either 4°C or 25°C. 7. Alternatively, suitable food-grade sources of sugars and flavan-3-ols / procyanidins (e.g., apple juice or grape juice) can be used to pre-fortify the beans prior to fermentation by adding these to the rehydration solution.

[0152] Protocol 1 (Combined Microbial Fermentation) 1. The beans were first soaked in water as described above. 2. Excess soaking water was discarded and the beans were transferred to a sterile SacO2 microbox (catalog number: TP1600+TPD1600 #30 WH). 3. 100 mL of synthetic pulp prepared as above was added to the beans in the microbox, followed by 1 mL of pooled fermentation starter containing both yeast and bacterial isolates (option 1 fermentation starter). 4. The beans, synthetic pulp, and fermentation starter were mixed, the fermentation box was closed with a filter lid, and incubated at 30°C. After 5.48 hours, the synthetic pulp was discarded and the beans were mixed. 6. The beans were then incubated at 42°C for an additional 72 hours for a total incubation of 120 hours (Figure 1B).

[0153] Protocol 2 (Sequential microbial fermentation: yeast followed by bacteria) 1. The beans were first soaked in water as described above. 2. Excess soaking water was discarded and the beans were transferred to a sterile SacO2 microbox (catalog number: TP1600+TPD1600 #30 WH). 3. 100 mL of synthetic pulp prepared as above was added to the beans in the microbox, followed by 1 mL of pooled fermentation starter containing only yeast isolate. 4. The beans, synthetic pulp, and fermentation starter were mixed, the fermentation box was closed with a filter lid, and incubated at 30°C. After 5.48 hours, the synthetic pulp was discarded and 1 mL of pooled fermentation inoculum containing only the bacterial isolate was added to the beans (option 2 fermentation inoculum), and the beans were then stirred. 6. The beans were then incubated at 42°C for an additional 72 hours (Figure 1C).

[0154] Step 5: Drying the legumes Once the fermentation process was complete, the beans were spread onto metal foil trays and placed in a 35°C oven for 7 days to reduce the moisture content of the beans to less than 7% to control the acidity of the beans. During this period, the beans were stirred on the first, second, and third days of drying to minimize clumping of the beans.

[0155] Method for neutralizing acidity, improving color, and producing mild flavor in chocolate analogues "Dutching" is accomplished by washing cocoa powder with a potassium carbonate solution. The solution neutralizes the powder's acidity to a pH of about 7, giving it a deep, dark brown color. Dutched powder tends to have a milder, more earthy flavor than natural powder.

[0156] Addition of lipid fractions to cocoa analogues In traditional chocolate made from cocoa beans, the lipid fraction of the cocoa beans is important for the physical properties of the chocolate, but does not change significantly during fermentation and is not thought to directly contribute to the flavor and aroma profile of the resulting chocolate. In light of the above, and given that legumes are generally lower in lipids compared to cocoa beans, lipid fractions (including standard cocoa butter substitutes and / or extenders) have been added to the cocoa analogues of the present invention to produce chocolate analogues. Examples of other lipids that can be used are lipids based on shea butter, palm and coconut, and rapeseed oil.

[0157] Adding sweeteners to chocolate analogues Beet or cane sugar, as well as coconut sugar, can be used. In addition, non-sugars can be used that provide more health benefits from other dietary fibers.

[0158] Adding flavorings to chocolate analogues Some brands of caramel or vanilla can be added to the base flavor. Any type of flavor such as orange, mint, etc. can be added to make the end consumer product.

[0159] result Example 1: Selection of candidate legumes The inventors have identified a number of candidate legumes that meet the preliminary criteria of being low-cost, agriculturally established, and grown relatively close to the manufacturing facility. These include adzuki bean (Phaseolus angularis), alfalfa (Medicago sativa), bambara (Vigna subterranean), black gram (Vigna mungo), chickpea (Cisser arietinum), cowpea (Vigna unguiculata), broad bean (Vicia faba), kidney bean (Phaseolus vulgaris), lima bean (Phaseolus lunatus), lentil (Lens culinalis or Lens esculenta), and peanut (Lens esculenta). These include lupine (Lupinus albus, Lupinus angustifolia, or Lupinus luteus), moth bean (Vigna aconitifolia), mung bean (Vigna radiatae), pea (Pisum sativum), pigeon pea (Cajanus cajan), sesame (Sesamum indicum), runner bean (Phaseolus coccineus), soybean (Glycine max), and vetch (Vicia sativa).

[0160] The inventors then identified their physicochemical composition based on data provided by the United States Department of Agriculture (USDA). The data for each of the legumes is summarized in the table below.

[0161] [Table 2]

[0162] The inventors then evaluated the protein (i.e., vicilin), polyphenol, and lipid profiles of the 19 candidate legumes and assigned each a similarity score to cocoa based on an aggregate scoring system. When characterizing the relative abundance of the 7S fraction and protein profile, legumes were scored in terms of their relative similarity to cocoa. Additional factors, such as a polyphenol profile rich in flavan-3-ols and procyanidins, or a high lipid content, were also taken into consideration. The table below summarizes the profiles and scores that the candidate legumes received.

[0163] [Table 3]

[0164] As can be seen in the table above, of the selected legume candidates, six legumes exhibited characteristics more similar to cocoa beans (i.e., legumes with a total score of ≧4), including broad beans, white lupine, peas, moth beans, scarlet beans, and sesame.

[0165] These six legumes were then fermented using a novel fermentation process.

[0166] Example 2: Fermentation Process Referring to Figure 1A, a method for preparing a fermentation inoculum for use in legume fermentation is shown. As shown, yeast and / or bacterial colonies are transferred to appropriate separate media. Two options are available: Option 1 requires combining both yeast and bacteria into one inoculum, while Option 2 requires keeping the yeast and bacterial inoculum separate.

[0167] Two different embodiments of the fermentation protocol will now be described, the first using a combined (yeast and bacteria - option 1) inoculum throughout the fermentation, and the second inoculating and fermenting yeast followed by bacteria sequentially (option 2).

[0168] As shown in Figure 1B, in one embodiment of the fermentation protocol, a selected legume or combination of legumes (as discussed in Example 1) is prepared for the fermentation step. This involves transferring the treated legumes to a fermentation box (reactor) along with a synthetic pulp, which serves as the fermentation medium, and a yeast and / or bacterial inoculum. Fermentation of the combined microorganisms is then carried out, e.g., incubation at 30°C (ideal for yeast cell growth and fermentation) for at least 48 hours, after which the synthetic pulp (fermentation medium) is discarded upon completion of the first fermentation. The legumes are then agitated and incubated with the combined microorganisms at 42°C (ideal for bacterial cell growth and fermentation) for an additional 72 hours. Upon completion of fermentation with the second combination, the legumes are dried and can then proceed as discussed in Examples 3 or 4.

[0169] As shown in Figure 1C, in a second embodiment of the fermentation protocol, a selected legume or combination of legumes (as discussed in Example 1) is prepared for the fermentation step. This requires the legumes to be transferred to a fermentation box (reactor) along with a synthetic pulp and a yeast-only inoculum. A yeast-only fermentation, e.g., incubation at 30°C for at least 48 hours, is then carried out, after which the synthetic pulp is discarded once the first yeast fermentation is complete. A bacteria-only inoculum is then added to the fermentation box / reactor, and the legumes are then agitated and incubated with the combined microorganisms (yeast and bacteria) at 42°C for an additional 72 hours. Once fermentation with the second combination is complete, the legumes are dried and can then proceed as discussed in 3 or 4.

[0170] Referring to Figure 4, various photographs of dried broad beans after 3 hours of rehydration are shown: (a) 0 hours, (b) 24 hours of incubation, (c) 48 hours of incubation, (d) 72 hours of incubation, (e) 120 hours of incubation, (f) dried beans after 120 hours of fermentation, and (g) dried unfermented beans.

[0171] Example 3: Method of Making Chocolate Analog Paste (e.g., for Toppings and Bars) Referring to Figure 2, an embodiment of a method for making a chocolate analog paste is shown.

[0172] How to make a similar chocolate paste: 1. The fermented and dried legumes were roasted in an oven at a temperature ranging from 180 to 280°C for approximately 5 to 20 minutes. 2. The roasted legumes were cooled and then crushed in a CPL crusher and then sorted in a CPL sorter. 3. The resulting nibs were then ground to a fine powder, lipid was added (cocoa butter substitute), weighed and then warmed to about 30-35°C. 4. The warmed powdered lipid mixture (liquor) was then refined to reduce particle size and conched (i.e. gently mixed and heated) in an ECGC65 for up to 16 hours depending on the batch size to obtain the liquor. 5. After adding any relevant additives (e.g., milk, flavorings, sunflower lecithin, or sugar), the resulting chocolate analog liquor was further refined and conched as above. 6. The chocolate analog paste resulting from step 5 was then tempered in an FBM tempering machine. The temperature and time of the tempering cycle will depend primarily on which lipid is used in the recipe. 7. Once tempered, the chocolate analog paste was molded and / or stored as desired.

[0173] Suitable storage conditions include storing the product in a cool, dry place at a temperature between 10 and 20°C, in dry conditions, and away from strong odors.

[0174] Example 4: Method of making a chocolate analog powder (e.g., for beverages) Referring to FIG. 3, an exemplary embodiment of a method for making a chocolate analog powder is shown.

[0175] How to make similar chocolate powder: 1. The fermented and dried legumes were roasted in an oven at a temperature ranging from 180 to 280°C for approximately 5 to 20 minutes. 2. The roasted legumes were cooled and then crushed in a CPL crusher and then sorted in a CPL sorter. 3. The resulting nibs were then ground to a fine powder to obtain a cocoa powder analog. 4. Proper storage conditions include storing the product in a cool, dry place at a temperature between 10-20°C, in dry conditions, and away from strong odors. It will be understood that melting point, solubility, viscosity, mouthfeel, etc. will vary depending on the types of lipids and sugars incorporated into the chocolate analog formulation, but ultimately they will all be very similar, if not identical, to those of the actual cocoa base or cocoa-derived chocolate compounds.

[0176] Example 5: Development of processed broad bean flour without added microorganisms Examples 1-4 described the use of microbial fermentation (yeast and / or bacteria) to process legumes (e.g., broad beans) to obtain a cocoa bean-free chocolate analog. However, following this work, the inventors identified an exemplary method for making a cocoa analog powder without the use of added microorganisms, i.e., in the absence of added microorganisms. Thus, a fourth embodiment of a legume processing protocol using a microorganism-free incubation solution will now be described.

[0177] 1. Surface sterilization of broad beans Vicia Fava (broad beans) was used. For each fermentation replicate, 125 g of beans were weighed out using an analytical balance and transferred to a 500 mL reagent glass bottle. A bean sterilization solution (70% ethanol, 0.05% Triton X-100 (Cat. No.: T8787-250ML)) was prepared (200 mL for each fermentation replicate). The sterilizing solution was added to the beans and agitated using a laboratory orbital shaker at 200 rpm and 30°C for 1 hour.

[0178] The following steps were performed under sterile conditions. The sterilizing solution was discarded using a sterile 25 mL disposable polystyrene serological pipette. The beans were then washed by adding 250 mL of sterile RO water to the jar, mixing by inverting the jar 10 times, and discarding the wash. This was repeated two more times for a total of three washes.

[0179] 2. Soak the beans in water Sterile RO water (250 mL) was added to the jars, and the beans were rehydrated by incubating at 25°C for 15–20 hours.

[0180] 3. Incubation The following steps were performed under sterile conditions. Excess soaking water was discarded and the beans were transferred to a sterile SacO2 microbox (catalog number: TP1600+TPD1600 #30 WH). 200 mL of incubation solution (recipe 1) was added. The use of sugars (2.5% sucrose, 4% glucose, 5% fructose) + 1% citric acid is a simpler alternative with some loss of flavor quality.

[0181] Recipe 1: 2.5% (w / v) sucrose 4% (w / v) glucose 5% (w / v) fructose 1% (w / v) citric acid 0.14% (w / v) high viscosity carboxymethylcellulose 0.77% (w / v) low viscosity carboxymethylcellulose 1.09% (w / v) pectin 0.5% (w / v) yeast extract 0.5% (w / v) peptone 0.1% (w / v) calcium lactate pentahydrate 0.1% (v / v) Tween 80 0.05% (w / v) magnesium sulfate heptahydrate 0.02% (w / v) manganese sulfate monohydrate

[0182] The beans were mixed, the box was closed with a filter lid and incubated at 30°C. After 48 hours, the incubation medium was discarded and the beans were mixed.

[0183] Apparent contamination at this stage involves the inhibition of ethanol and acetic acid formation upon incubation at 42°C (vinegar smell), resulting in a bean powder with a more neutral pH and no chocolate aroma (see Table 4).

[0184] [Table 4]

[0185] The beans were then incubated for an additional 72 hours at 42° C. The incubation, with the beans having a vinegar smell and appearing shiny and wet, results in a powder with a good chocolate aroma and a low pH (see Table 4).

[0186] The pH of the beans decreased throughout the 30°C incubation, reaching pH 4.5. For beans that appeared shiny and wet, the pH remained at pH 4.5 during the 42°C incubation. For beans that appeared dull and not wet, the pH rose again to pH 6.2-7.0 during the 42°C incubation.

[0187] 4. Drying The beans are then dried in a drying oven at 35°C. The drying time is 7 days for optimal aroma development in the roasted bean powder.

[0188] 5. Ageing the beans before roasting For optimal aroma development in the roasted bean powder, the beans are allowed to age at room temperature for 5 days before roasting. The dried beans can be stored for at least 10 days before roasting and grinding without adversely affecting the powder aroma or changing the powder pH.

[0189] 6. Pre-roasting treatment to develop a deeper color The dried beans are crushed and the powder is mixed with water (1:1 w / w) and heated to 80°C for 60 minutes with occasional stirring. The dried paste is dried at 35°C for 7 days. A deeper powder is obtained, with a color depth equivalent to that of unduched cocoa powder.

[0190] 7. Roasting and grinding The dried beans are roasted and ground at 180 or 200°C for 20 to 40 minutes until a predetermined color is reached using an in-house developed scale. Roasting lowered the pH of the powder made from dull beans by approximately 1 pH unit, while the pH of the glossy beans remained low at approximately pH 5.0. After roasting, the beans can be stored for at least 2-3 months before grinding without adversely affecting the powder aroma or changing the powder pH.

[0191] 8. Aging of roasted and ground powder Detailed experiments have shown that the aroma of roasted bean powder changes rapidly over the first 7 days after roasting and grinding. After 7 days, the aroma is stable for >1 month. The pH of the roasted powder remains stable for up to 4 months from immediately after grinding. Powders with good chocolate aroma are associated with powders with lower pH (Table 1), and Figure 5 shows that the optimal pH for chocolate aroma is pH 4.8 to 5.1. A bin is a mathematical term used to create a histogram from a continuous series of data. The plot shows 163 processed broad bean powder samples with known pH and whether they possess chocolate aroma. The pH of the samples ranged from 4 to 7. To create the histogram, pH bins were created with narrow ranges of pH values, for example, 4.8 to 5.1. Within that pH range (bin), the number of powders with a pH within that range and chocolate aroma was counted. The percentage of samples with chocolate aroma in that pH bin was calculated. This was repeated for all bins from 4 to 7, and the values ​​were then plotted. The results show that samples with a pH of 4.8 to 5.1 had the highest percentage of samples with chocolate aroma (approximately 60%). Referring to Figure 6, the low pH powder shows that chocolate aroma begins to develop 3-4 days after roasting and grinding, matures by day 7, and continues to improve for up to one month, after which it is stable for at least four months.

[0192] 9.Fragrance Referring to Figure 7, the flavor profile of a liquor made from processed roasted and ground fava bean flour (powder + shea butter 1:1 w / w) is shown, with real cocoa powder included for comparison. The flavor profile of the roasted fava bean flour scores favorably compared to the real cocoa powder for the various flavor components tested.

[0193] Example 6: Addition of microorganisms develops alternative flavors in broad bean flour The inventors have found that the addition of various microorganisms results in the development of alternative flavors in fava bean flour. Thus, a fifth embodiment of a legume processing protocol will now be described, using a fermentation starter as described in Option 1 (i.e., Protocol 1 in Figure 1B).

[0194] 1. Surface sterilization of broad beans Vicia Fava (broad beans) was used. For each fermentation replicate, 125 g of beans were weighed out using an analytical balance and transferred to a 500 mL reagent glass bottle. A bean sterilization solution (70% ethanol, 0.05% Triton X-100 (Cat. No.: T8787-250ML)) was prepared (200 mL for each fermentation replicate). The sterilizing solution was added to the beans and agitated using an orbital shaker at 200 rpm and 25°C for 30 minutes.

[0195] The following steps were performed under sterile conditions. The sterilizing solution was discarded using a sterile 25 mL disposable polystyrene serological pipette. The beans were then washed by adding 250 mL of sterile RO water to the jar, mixing by inverting the jar 10 times, and discarding the wash. This was repeated two more times for a total of three washes.

[0196] 2. Soak the beans in water Sterile RO water (250 mL) was added to the jars, and the beans were rehydrated by incubating at 25°C for 15–20 hours.

[0197] 3. Incubation The following steps were performed under sterile conditions. Excess soaking water was discarded and the beans were transferred to a sterile SacO2 microbox (catalog number: TP1600+TPD1600 #30 WH). 200 mL of incubation solution (recipe dependent on the microorganisms added - see below) was added. The microorganisms used were Pichia kudriabzevii, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, Lactobacillus plantarum, and Acetobacter pasteurianus. Individual beans were prepared and inoculated with one of the microorganisms used.

[0198] The fermentation seed was prepared as follows: 1. Selective agar was prepared and poured into sterile 9 cm Petri dishes (20 ml each) and allowed to cool and harden. 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 was sterilized by autoclaving. Note that the ethanol and acetic acid should be added after autoclaving (the final pH of the medium should be 4.5). De Man Rogosa Sharpe (MRS) Agar: MRS agar (Cat. 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 (Cat. No. P8074). The suspension was boiled until the medium was completely dissolved, and the total volume was adjusted to 1000 ml, then sterilized by autoclaving. Yeast peptone glucose (YPG) agar: 1% yeast extract, 2% peptone, 2% glucose. The pH was adjusted to 5.6, 2% agar was added, and the mixture was autoclaved.

[0199] [Table 5]

[0200] 2. Stocks of each cell culture were plated onto their respective selective agar plates (Table 1) and incubated at 28°C for 72 hours. 3. For each isolate, a 15 mL tube containing 3 mL of Luria broth Lennox was prepared, except for Acetobacter pasteurianus, which required a 15 mL tube containing 3 mL of AA broth (1% D-glucose, 1.5% bacteriological peptone, 0.8% yeast extract; autoclaved; added 0.5% ethanol and 0.3% acetic acid). 4. One colony from the agar plate for each isolate was selected and transferred to 3 ml of the respective medium and incubated at 28°C in a shaking incubator at 200 rpm for 72 hours. 5. Cells were harvested by centrifugation at 4,000 x rpm for 8 minutes at 4°C. 6. The supernatant was discarded and 5 ml of sterile 10 mM MgCl2 was added to the tube. 7. The tube was inverted 10 times to resuspend the cells, and centrifuged at 4,000 x rpm at 4°C for 8 minutes. 8. Repeat steps 6-7 two more times for a total of three washes. 9. Cells were resuspended in 1 mL of sterile 10 mM MgCl2 and the optical density at 600 nm (OD600) was measured for each isolate (1 OD600 = 1 x 10 9 The cell concentration (cells / mL) was determined. 10. Prepare a fermentation inoculum by pooling individual isolates, with a concentration of 1 x 10 for each isolate. 8 (Cells were diluted with sterile 10 mM MgCl2 if necessary.) For each fermentation, 1 mL of inoculum was prepared and used immediately or stored at 4°C for no more than 24 hours. The beans were mixed with the inoculum and the box was closed with a filter lid and then incubated at 30°C. After 72 hours, the incubation medium was discarded and the beans were mixed.

[0201] 4. Drying The beans are then dried in a drying oven at 35°C. The drying time is 7 days for optimal aroma development in the roasted bean powder.

[0202] 5. Roasting and grinding The dried beans are roasted and ground at 180 or 200°C for 20 to 40 minutes until a predetermined color is reached using an in-house developed scale.

[0203] 6. Flavor Referring to Figure 8, the flavor profile of liquor (bean flour, sugar, and shea butter) made from processed roasted and milled fava bean flour is shown, with fava beans (NK0034) processed using our non-microbial process included for comparison. The flavor was evaluated by five independent individuals.

[0204] Further, with reference to Figure ID, a further embodiment has been developed which involves two separate processings of legumes (e.g., broad beans), the products of which are then blended together to produce a cocoa bean-free cocoa powder analog. First, the legumes are treated with a microorganism (either Option 1 or Option 2) to produce a microbially fermented legume. Separately, the legumes are treated in the absence of a microorganism to produce a non-microbially treated legume. A portion of the microbially fermented legume is then blended with a portion of the non-microbially treated legume to produce a cocoa bean-free cocoa powder analog. A portion of the microbially fermented legume is then blended with a portion of the non-microbially treated legume to produce a cocoa bean-free cocoa powder analog.

[0205] Thus, it is shown that various microbial fava bean treatment processes result in different flavors than the non-microbial process described in Example 5, and that blending beans from both types of processes, i.e., microbial and non-microbial fermentation, results in unique flavor combinations.

[0206] Example 7: Use of other beans The inventors have found that beans other than fava beans can also be used to develop a cocoa-like powder, and have processed lupins, green peas, moss, adzuki beans, and hemp seeds following the same procedure as fava beans, following the fourth protocol, the legume processing protocol described in Example 5, which uses a microorganism-free incubation solution. The flavor of the liquor (powder mixed 1:1 with shea butter) produced from the processed seeds and beans is shown in Figure 10.

[0207] The results show that all beans and seeds tested are different from each other and have a different flavor profile than broad beans. Furthermore, hemp seeds and lupin beans are shown to compare favorably with broad beans in terms of chocolate flavor. The inventors also concluded that flours made from various processed beans can be blended with broad bean flour to create improved flavor profiles (e.g., see FIG. 10, blending hemp seeds with broad beans introduces new flavors of sourness, dried fruit, and nuts into the broad bean flour).

[0208] Example 8: Use of fava bean flour and additives to make NuKoKo cocoa-like powder. Various additives to modify the flavor of the flour can be added to broad bean flour made from either microbially fermented or non-microbially fermented beans, which are roasted and milled, blended or unblended. These flavor-modifying ingredients are added in the range of 0.1-2.0% (w / w). These include, but are not limited to, beetroot powder, apple powder, and malted barley.

[0209] Example 9: Use of "NuKoKo" powder to make a cocoa-free chocolate analogue. The inventors The broad bean processing process described in Example 5 and / or Example 6 The present invention has been described in detail in order to provide an exemplary method for making a cocoa-free chocolate analog using the resulting cocoa bean-free chocolate analog powder (i.e., "Nukoko" powder) of the present invention. 1. NuKoKo chocolate analog was prepared according to the following recipe: 21% 410g Nukoko powder 34% 665g Palm / Shea Butter 24% 469g cane sugar (pre-ground) 14% 274g whole milk powder 2% 39g beet root powder 2% 39g apple powder 0.5% 10g lecithin 2% 39g malt extract 0.2% 3.5g salt (pre-ground) 0.25% 5g of chocolate malted barley

[0210] 2. Method: Melt fat and pre-grind granular ingredients (spice grinder). Add semi-dry ingredients to liquid fat, then place in a preheated small millstone. Add remaining dry ingredients. Use the heat lamp in batches. Run for 8 hours.

[0211] 3. Appearance: It should be smooth, uniform in color, and shiny. It should not have any bubbles, cracks, streaks, or a cloudy appearance.

[0212] 4. Aroma: Should be a rich chocolate aroma with hints of fruity, earthy or floral notes.

[0213] 5. Breaking sound: There should be a clear, crisp, clean, sharp sound when broken.

[0214] 6. Texture: It should melt the moment it is put in the mouth. Its flavor and smoothness should be an immediate impression. It should feel smooth and velvety in the mouth. The flavor should linger for several minutes after the chocolate piece has been eaten.

[0215] 7. Flavor: Should taste sweet, milky, caramel, and cocoa.

[0216] 8. Flavor Profile Referring to Figure 9, the flavor profiles of NuKoKo chocolate analog (Choc), authentic Tesco brand milk chocolate, and dark chocolate (Mollys) are shown. The flavor components of the various chocolates were evaluated by five independent individuals.

[0217] Thus, a direct comparison is shown between NuKoKo Choc (a chocolate substitute made from broad bean flour processed using the inventors' non-microbial method according to the recipe above) and Tesco's milk and dark chocolates, with NuKoKo Choc scoring very close to the real chocolate in all five parameters tested: appearance, aroma, snap, texture and flavour.

[0218] summary As described in the Examples, the inventors have developed an elegant method for producing chocolate analogs that are not derived from cocoa beans and therefore do not contain cocoa. Instead, this method requires the use of a select group of legumes that meet the necessary criteria for replicating the appearance, taste, aroma, and mouthfeel of traditional cocoa-derived chocolate, but without the drawbacks of cocoa / cocoa. The non-cocoa chocolate analogs can be made into pastes or powders, allowing them to be used in a variety of food products, such as bar confectionery, or in cosmetics.

Claims

1. A method for producing a cocoa bean-free chocolate substitute, comprising the step of contacting a leguminous plant with at least one microorganism under conditions suitable for the fermentation of the leguminous plant by the at least one microorganism, thereby forming a cocoa bean-free chocolate substitute containing the microbially fermented leguminous plant.

2. The method according to claim 1, comprising: a first treatment of microbially fermenting the leguminous plant to obtain a microbially fermented leguminous plant; and a second treatment, separately therefrom, of non-microbially fermenting the leguminous plant in the absence of externally added microorganisms to obtain a non-microbially treated leguminous plant.

3. The method according to claim 2, comprising the step of combining a portion of the microbially fermented leguminous plant with a portion of the non-microbially fermented leguminous plant to obtain the cocoa bean-free chocolate analogue.

4. The method according to claim 3, wherein the ratio of microbially fermented leguminous plants to non-microbially fermented leguminous plants is approximately 10%:90% to 90%:10%, or approximately 50%:50%.

5. The fermented legumes mentioned above include: adzuki beans (Phaseolus angularis); alfalfa (Medicago sativa); bambara (Vigna subterranean); black gram (Vigna mung); chickpeas (Cicer arietinum); cowpeas (Vigna unguiculata); broad beans (Vicia faba); and kidney beans (Phaseolus vulgaris) Vulgaris); Green bean (Phaseolus lunatus); Lentil (Lens culinaris or Lens esculenta); Lupine (Lupinus albus, Lupine angustifolia, or Lupine luteus); Moss bean (Vigna aconitifolia); Green bean (Vigna radiate); Pea (Pisum sativum) The method according to any one of claims 1 to 4, selected from the group of leguminous plants consisting of sativum; pigeon pea (cajanus cajan); sesamum indicum; red bean (phaseolus coccineus); soybean (glycine max); and vetch (vicia sativa).

6. The method according to any one of claims 1 to 4, wherein the fermented leguminous plant is selected from the group consisting of broad bean (vicia faba); white lupine (lupinus albus); pea (pisum sativum); moss bean (vigna aconitifolia); red bean (phaseolus coccineus); and sesame (sesamum indicum).

7. The method according to any one of claims 1 to 4, wherein the at least one microorganism used in the fermentation is selected from yeast and / or bacteria.

8. The method according to any one of claims 1 to 4, wherein the fermentation substantially comprises yeast or comprises only yeast, and optionally, at least 70%, 80%, or 90% of the microorganisms in the fermentation comprises yeast cells.

9. The method according to any one of claims 1 to 4, wherein the fermentation substantially contains bacteria or contains only bacteria, and optionally, at least 70%, 80%, or 90% of the microorganisms in the fermentation contain bacterial cells.

10. The method according to any one of claims 1 to 4, wherein the fermentation comprises yeast and bacteria, preferably with a yeast-to-bacterial cell ratio of about 50:50, 10:90, 20:80, 30:70, 40:60, or a bacterial-to-yeast cell ratio of 10:90, 20:80, 30:70, 40:

60.

11. The method according to claim 4, wherein the bacterial family is selected from the group of bacteriaceae consisting of Lactobacillus and Acetobacter.

12. The method according to claim 4, wherein the bacterial species is selected from the group of bacterial species consisting of Lactobacillus fermentum; Lactobacillus plantarum; and Acetobacter pasterianus.

13. The method according to claim 4, wherein the genus of yeast is selected from the group of yeast genera consisting of Pichia, Hanseniaspora, and Saccharomyces.

14. The method according to claim 4, wherein the yeast species is selected from the group of yeast species consisting of Pichia kudriavzevii; Hanseniaspora opuntiae; and Saccharomyces cerevisiae.

15. The step includes culturing the at least one microorganism and the leguminous plant in a culture medium, optionally wherein the culture medium is (i) One or more sugars selected from the group consisting of sucrose; glucose; fructose; high viscosity carboxymethylcellulose; low viscosity carboxymethylcellulose; and / or pectin; (ii) One or more salts selected from the group consisting of calcium lactate pentahydrate; magnesium sulfate heptahydrate; and manganese sulfate monohydrate; (iii) One or more protein hydrolysates, optionally being peptones; (iv) Yeast extract; (v) Surfactants, optionally Tween 80; and / or (vi) Citric acid The method according to any one of claims 1 to 4, including the method described in any one of claims 1 to 4.

16. The method according to any one of claims 1 to 4, wherein the culture medium has a pH of 2 to 5 or 3 to 4.

17. The method according to any one of claims 1 to 4, wherein the fermentation comprises the step of incubating yeast and bacteria at 24°C to 44°C for at least one or two days.

18. The method according to any one of claims 1 to 4, wherein the fermentation comprises the step of incubating bacteria at 24°C to 50°C for at least one or two days.

19. The method according to any one of claims 1 to 4, comprising the step of simultaneously fermenting the leguminous plant in the presence of yeast and bacteria.

20. The method according to claim 16, wherein the yeast comprises Pichia kudriavzevii, Hanseniaspora opuntiae, and / or Saccharomyces cerevisiae, and the bacteria comprises Lactobacillus fermentum, Lactobacillus plantarum, and / or Acetobacter pasterianus.

21. The method according to claim 16, comprising the steps of: contacting the leguminous plant with yeast cells and bacterial cells at a temperature of 21°C to 37°C for at least 24 hours; discarding the culture medium one day or more days after the first fermentation; and then incubating the leguminous plant and at least one microorganism at a temperature of 24°C to 50°C for one day or more.

22. The method according to claim 16, wherein the fermentation mixture is incubated at a temperature of 34°C to 44°C for at least two or three days.

23. The method according to any one of claims 1 to 4, comprising the step of fermenting the leguminous plant first in the presence of yeast alone, and then in the presence of bacteria.

24. The method according to claim 20, wherein the yeast comprises Pichia kudriavzevii, Hanseniaspora opuntiae, and / or Saccharomyces cerevisiae, and the bacteria comprises Lactobacillus fermentum, Lactobacillus plantarum, and / or Acetobacter pasterianus.

25. The method according to claim 20, comprising the steps of: contacting the leguminous plant with yeast cells at a temperature of 21°C to 37°C for at least 24 hours; discarding the culture medium one day or more days after the first fermentation; and subsequently contacting the fermented leguminous plant with bacterial cells at a temperature of 24°C to 50°C for at least 24 hours.

26. The method according to claim 20, wherein the fermentation mixture is incubated at a temperature of 32°C to 44°C for at least two or three days.

27. The method according to any one of claims 2 to 4, wherein the method includes non-microbial fermentation, the method comprising the use of an incubation medium having a pH of 3 to 5, and the step of submerging the leguminous plants in water when the leguminous plants begin to germinate to consume the oxygen available for respiration, so that the fermentation metabolism starts with the oxidation of sugars and results in the production of ethanol, lactic acid, and optionally acetic acid.

28. The method according to claim 27, wherein the non-microbial fermentation 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.

29. The method according to claim 27, wherein the non-microbial fermentation is incubated for at least one or two days.

30. The method according to claim 27, wherein the non-microbial fermentation 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.

31. The method according to claim 30, wherein the non-microbial fermentation is incubated for at least one, two, or three days.

32. The aforementioned chocolate-like substance (i) Approximately 4 to 7, approximately 4.3 to 6.8, or approximately 4.4 to 6.7; (ii) Approximately 4.4 to 6.3, approximately 4.7 to 5.9, or approximately 4.7 to 5.5; or (iii) Approximately 4.4 to 5.5, 4.7 to 5.2, or 4.8 to 5.1 The method according to any one of claims 1 to 4, having a pH of [value].

33. The method according to any one of claims 1 to 4, comprising the step of drying the leguminous plants after fermentation, optionally drying the leguminous plants in an oven at a temperature of about 21°C to 37°C for one or more days.

34. The method according to any one of claims 1 to 4, wherein the cocoa bean-free chocolate analogue containing fermented leguminous plants comprises or is converted into a paste.

35. The method according to any one of claims 1 to 4, wherein the cocoa bean-free chocolate analogue containing fermented leguminous plants includes or is converted into a powder.

36. A cocoa bean-free chocolate analogue obtained or available by the method described in claim 1.

37. A cocoa bean-free chocolate substitute containing leguminous plants that have been fermented by microorganisms and / or non-microorganisms.

38. A cocoa bean-free chocolate analogue according to claim 37, as defined in any one of claims 1 to 4.

39. A product ingredient comprising the cocoa bean-free chocolate analogue described in claim 36.

40. A food product or beverage comprising a cocoa bean-free chocolate substitute as described in claim 36, or an ingredient as described in claim 39, wherein the cocoa bean-free chocolate substitute is optionally in the form of a powder, liquor, liquid, solid, or paste.

41. The food or beverage according to claim 40, wherein the food is a confectionery product, and optionally the food is a block bar, molded figure, button-shaped object, truffle, spread, coated fruit, nut, and other inclusions, bakery filling, coating for products such as donuts or muffins, yogurt, ice cream, or other dessert.

42. A cosmetic comprising a cocoa bean-free chocolate substitute as described in claim 36, or an ingredient as described in claim 39.

43. The cosmetic according to claim 42, which is a skincare product, moisturizer, exfoliator, lip balm, eyeshadow, soap, body cream or body butter, or hair mask.

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