(19)
(11)EP 3 427 598 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
12.02.2020 Bulletin 2020/07

(21)Application number: 18170388.5

(22)Date of filing:  07.11.2012
(51)Int. Cl.: 
A23L 19/00  (2016.01)
A23L 5/43  (2016.01)
A23L 5/20  (2016.01)
A23P 10/30  (2016.01)

(54)

METHOD FOR PREPARING A COLORANT FROM A GENIPIN-RICH EXTRACT OF GENIPA AMERICANA

METHODE ZUR HERSTELLUGN EINES FABSTOFFS AUS EINEM GENIPIN REICHEN EXTRAKT VON GENIPA AMERICANA

MÉTHODE POUR LA PRODUCTION D'UN COLORANT À PARTIR D' UN EXTRAIT RICHE EN GÉNIPINE DU GENIPA AMERICANA


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 07.11.2011 US 201161556441 P

(43)Date of publication of application:
16.01.2019 Bulletin 2019/03

(62)Application number of the earlier application in accordance with Art. 76 EPC:
17175767.7 / 3238550
12790757.4 / 2775861

(73)Proprietor: Wild Flavors, Inc.
Erlanger, KY 41018 (US)

(72)Inventors:
  • Wu, Shaowen
    Erlanger, KY 41018 (US)
  • Horn, Gregory
    Erlanger, KY 41018 (US)

(74)Representative: dompatent von Kreisler Selting Werner - Partnerschaft von Patent- und Rechtsanwälten mbB 
Deichmannhaus am Dom Bahnhofsvorplatz 1
50667 Köln
50667 Köln (DE)


(56)References cited: : 
WO-A2-2005/105020
TW-B- 460 523
US-B1- 7 282 220
WO-A2-2011/122934
US-A1- 2009 246 343
  
  • MEENA R ET AL: "Development of a stable hydrogel network based on agar-kappa-carrageenan blend cross-linked with genipin", FOOD HYDROCOLLOIDS, ELSEVIER BV, NL, vol. 23, no. 2, 1 March 2009 (2009-03-01), pages 497-509, XP026128639, ISSN: 0268-005X, DOI: 10.1016/J.FOODHYD.2008.03.008 [retrieved on 2008-03-25]
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

Technical Field



[0001] This application is based on and claims priority from U.S. Provisional Application Serial No. 61/556,441, Wu and Horn, filed November 7, 2011.

[0002] This invention refers to to a method of preparing a colorant from Genipa americana. More specifically, the present invention relates to a method for producing novel genipin-rich materials from the fruit of Genipa americana; the genipin-rich material has a genipin content of at least 70% w/w of the solids which enables its use as raw material for colorant production.

Background



[0003] Today, synthetic chemicals, such as colorants or cross-linking reagents, tend to have decreasing acceptance in the food, cosmetic, animal feed and textile industries. For safety reasons, whether real or perceived, people tend to favor the use of natural or organic ingredients in food, cosmetic, textile, and biomaterial products.

[0004] Genipin is a colorless compound. It belongs to the iridoid group. It is very active chemically and reacts immediately when combined with compounds having primary amine groups, such as amino acids, collagen, chitosan, glucosamine-type compounds and various proteins and enzymes. When oxygen is present, the product may turn to blue, green, or black quickly. Genipin is an iridoid ester, therefore, it can be hydrolyzed to generate genipinic acid which also can react with different compounds to generate red and brown colorants. The colorants generated from genipin are heat and pH stable. Since genipin normally comes from plant materials, its Kosher characteristics provide great potential for use of genipin-derived colorants in bakery and canned food applications.

[0005] Large amount of iridoids, such as geniposide, gardenoside, genipin-1-b-gentiobioside, geniposidic acid and genipin, can be found in Gardenia jasminoides Ellis fruit. Geniposide, an iridoid glycoside, is a main component comprising 7-8% of fruit weight, while only trace amounts of genipin naturally exist in the Gardenia fruit. Historically, the iridoids in Gardenia fruit were extracted from the fruit with methanol or ethanol, and then separated as yellow pigment on active carbon, and treated with enzymes having beta-glucosidic activity or proteolytic activity to convert glycoside iridoids to aglycone form, genipin or genipinic acid. Then, genipin or genipinic acid can be reacted with other compounds to produce colorants (KR1020010096213A).

[0006] Genipin and other iridoid compounds, such as genipinic acid, genipin-gentiobioside, geniposide and geniposidic acid, are found also in the fruits and leaves of Genipa americana, also known as Genipap, or Huito, a tropical wild plant. Genipin is naturally present in the mature fruit, and its quantity is from 0 to 3.0% of fruit weight depending on the degree of ripeness. Genipin is stable in the plant cell even though it is not established where it is stored. Whenever the cell is broken, genipin will react spontaneously with the amino acids that naturally exist in the fruit pulp and turn color to blue or black in an air environment. In our previous patent application (US20090246343A1), we made coloring (blue) fruit juice by mixing Genipa americana fruit pulp with juices from other fruits or vegetables. The natural acid-stable blue juice has been used broadly in the food and beverage industries. Lopes et al. (US7927637B2) used unprocessed raw juice obtained from Genipa americana fruit pulp, and mixed with glycine or with glycine plus starch, to make blue colorants. Patrice Andre and co-worker (US20100196298A1) used coloring materials obtained from the plant extraction of Genipa americana, Gardenia jasminoides, Rothmannia, Adenorandia or Cremaspora, bound to a solid substrate, to make cosmetic compositions.

[0007] Genipin not only can generate colorants, but also can act as a good cross-linking agent. Campbell et al. discovered a new biodegradable plastic polymer involving genipin as cross-linking agent (EP2093256A2). Such plastics are useful in biological systems for wound repair, implants, stents, drug encapsulation and delivery, and other applications. Fujikawa et al. claimed (US4983524) the use of iridoid aglycone, genipin, as a cross-linking agent to produce immobilized enzyme on beads for food industry. Quijano, R and Tu, H (US 20080195230A1) disclosed the use of genipin to fix whole, natural tissues to reduce antigenicity and immunogenicity and prevent enzymatic degradation of the tissue when implanted in a host.

[0008] However, pure genipin production historically involves many steps, including HPLC separation, and currently its cost is high. No description in the art teaches the preparation of a stable genipin-rich extract from Genipa americana plant, and its broad applications as natural colorants and cross-linking agent in foods, drugs, nutritional supplements, personal care products, cosmetics, animal feed, textiles, polymers, and in the biomaterial industries.

Summary



[0009] The present invention provides a method of preparing a colorant from Genipa americana comprising the steps:
  1. 1) preparing a genipin-rich extract from Genipa americana; and
  2. 2) use of said genepin-rich extract in colorant production;wherein the preparation of the genepin-rich extract comprises the following steps:
    1. a) washing and/or blanching the mature fruits of Genipin americana;
    2. b) peeling the fruits and cutting them into pieces;
    3. c) milling or blending the fruits with water or polar solvents in a weight ratio of 1:0.5-5.0;
    4. d) extracting the fruits with the water or polar solvents;
    5. e) separating the pulp, seeds and skin of the fruit by filtration and collecting the liquid filtrate;
    6. f) adjusting the pH of the filtrate to 3.8 to 4.0 using acids;
    7. g) concentrating the filtrate to produce a genepin-rich aqueous extract;
    8. h) extracting said aqueous extract with non-polar organic solvents through mixing in a ratio of about 1:1, wherein said non-polar organic solvents have a polarity index from 0 to 5.0 and water solubility of less than 30%;
    9. i) separating the organic solvent phase from the aqueous phase by settling or siphoning the top organic layer or by using a high speed centrifuge;
    10. j) evaporating the organic solvent to produce a genepin-rich extract as a yellow or off-white solid in which the genepin content is at least 70 % w/ w of the solids.


[0010] The invention is defined by the appended set of claims and any other aspects or embodiments set forth herein not falling within the scope of the claims are for information only.

[0011] The present disclosure further provides applications of the genipin-rich extracts in natural colorant production and forming polymers for use in a broad range of areas, such as in foodstuffs, drugs, nutritional supplements, personal care products, cosmetics, animal feed, textiles, biodegradable polymers, and biomaterials production. Examples of such uses are to be found in numerous patent documents, including US8283222, CN101899484, US20120189584, WO2012048188, CN102399370, CA2458821, DE602007013718, US20110082199, TW1334878, US20100183699.

[0012] As used herein, all ratios and proportions noted are "by weight" unless otherwise specified. In addition, all patents, patent applications and technical publications cited are incorporated herein by reference.

Brief Description of the Drawings



[0013] Fig. 1 is a flow chart representation of an embodiment of the process for making genipin-rich extract produced from Genipa americana fruit.

Detailed Description


Starting Materials



[0014] The starting materials used in this process are fruits or leaves from a plant of Genipa americana L. which is also known by numerous informal names: genipap, huito, jaguar, bilito, cafecillo denta, caruto, caruto rebalsero, confiture de singe, danipa, genipa, génipa, genipayer bitu, guaitil, guaricha, guayatil colorado, huitol, huitoc, huitu, irayol, jagua blanca, jagua amarilla, jagua colorado, jeipapeiro, juniper, maluco, mandipa, marmelade-box, nandipa, ñandipa genipapo, tapaculo, tapoeripa, taproepa totumillo, yagua, yanupa-i, yenipa-i, yenipapa bi, genipapo, huitoc, vito, chipara, guanapay, or other varieties such as jenipaporana, or jenipapo-bravo, etc. The fruit is optimal for harvest when mature in size, firm, and green to greenish brown in color.

[0015] Materials may be whole fruit, fruit pulp, fruit juice, fruit puree, fruit juice concentrate, dried powder from fruits or juice, water-insoluble part of fruit, and leaves from Genipa americana L.

Process for Genipin-Rich Extraction



[0016] In order to produce the genipin-rich extracts of the present invention, the mature fruits of Genipa americana are processed with water or polar solvent in about 1:0.5-5.0 (by weight) ratios depending on extraction efficiency, preferably from about 1:1-1.5 ratios. After washing and/or blanching, the fruit is peeled and cut into pieces; then milled or blended with water or polar solvents; extracted with or without heating, the pulp, seeds and skin are separated by filtration; and liquid is collected. Extraction may take place for up to about 1 hour, preferably about 15-30 min, at a temperature of about 15-40°C. The water or polar solvent extraction from fruit pulp of Genipa americana may be repeated 1-3 times. Then, the pH of polar solvent extracts is measured and adjusted to about 3.8-4.0 using acids. Concentration can be performed on a rotary evaporator with vacuum and temperature set at about 40-46 C. The solid content in the concentrate may be greater than 15% w/w, preferably about 40-70% w/w.

[0017] Next, the aqueous extract or the concentrated genipin-rich aqueous extract may be further extracted with non-polar organic solvents involving suitable mixing (by shaking or agitation) in a ratio of about 1:1. Those non-polar organic solvents have a polarity index from about 0 to 5.0 and water solubility less than about 30%. The organic solvent phase is then separated from aqueous phase by settling and siphoning the top organic layer or by using a high speed centrifuge. Non-polar solvent extraction may be repeated 2-3 times depending on the extraction efficiency. The genipin-rich extracts may subsequently be dried by evaporation and organic solvent can be recycled and reused for genipin extraction. The remaining yellow or off-white solid is a genipin-rich extract in which genipin content is at least about 70% w/w of the solids.

[0018] Genipin-rich extracts may be aqueous or polar solvent extracts obtained from the starting material sources described. Polar solvents include those with water solubility up to 100% and polarity index greater than about 5.0, such as water, acetic acid, methanol, ethanol, n-propanol, iso-propanol, dimethyl sulfoxide, dimethyl formamide, acetonitrile, acetone, dioxane tetrahydrofuran, etc., or acetic, citric, phosphate acid buffer solutions, or mixtures thereof in different ratios. This list is not intended to limit the solvent used, however considering food safety, water, ethanol, n-propanol, iso-propanol, methanol and acetic, citric and phosphate acid buffer solutions, are preferred for food uses of the extract.

[0019] Genipin-rich extracts may alternatively be organic solvent extracts obtained from aqueous or polar solvent extracts described above. Non-polar organic solvents include those with less than about 30% of water solubility and a polarity index from 0 to about 5.0, such as ethyl acetate, butyl acetate, n-butanol, diethyl ether, hexane, 2-butanone, chloroform, 1,2-dichloroethane, benzene, xylene, methyl-t-butyl ether, toluene, carbon tetrachloride, trichloroethylene, cyclohexane, pentane, and heptane, or mixtures thereof in different ratios. This list is not intended to limit solvent used, however considering food safety, ethyl acetate, butyl acetate, and n-butanol are preferred.

[0020] Methods used for solid-liquid separation can be, for example, regular filtration, centrifugation, press filtration, and membrane cartridge filtration. The liquid-liquid separation can be done, for example, by using high speed centrifuge, or by settling and siphoning the required liquid layer.

[0021] Acids used for pH adjustment can be any organic or inorganic acids, selected to be suitable for the intended end use.

[0022] Concentration can be performed by any method known in the art, for example, by evaporation using a rotary vacuum evaporator, a flash evaporator, an osmosis filtration device, or an ultra-filtration device with a suitable membrane.

Properties of Genipin-Rich Extract



[0023] Genipin-rich extract is an off-white powder or crystals. Some batches may have a slightly yellowish or greenish tint if purity is relatively low. The genipin content can be up to about 97% (w/w) depending on the extraction method. No geniposide, geniposidic acid, or other iridoid compounds were found in genipin-rich extract when analyzed by HPLC. The remainder of the composition of the genipin-rich extract is mainly moisture, fat, and also small amounts of acids and nitrogen-containing compounds, with the balance being carbohydrates, as noted in the following table.

Specification of Genipin Rich Material



[0024] 
Genipin 30-97%
Fatty Acids <1.0%
Fat <5.0%
Protein <1.0%
Organic Acids <0.5%


[0025] HPLC methods can be used to determine genipin content and perform other iridoid analysis.

[0026] Genipin content of the off-white genipin-rich powder is stable when stored at refrigerated temperatures. Genipin level decreased by less than about 5% after 3 months of storage. However, when mixing genipin-rich extract with compounds having a primary amine group, genipin will quickly react and generate colorants and/or polymers.

[0027] High genipin content extract readily dissolves in alcohol, alcohol-water mixtures, or hot water. Its solubility in cold water is limited.

Colorant Production



[0028] The present invention also provides a method of manufacturing a blue colorant by using the genipin-rich extract reaction and mixing with water and amino acids, for example (but not limited to), lysine, histidine, arginine, glutamine, asparagine, methionine, glycine, glutamic acids, tyrosine, valine, alanine, serine, leucine, taurine, carnitine, ornithine and citrulline, in the presence of oxygen. Heating can be used to accelerate the reaction speed. The molar ratios of genipin-rich extract to amino acids are from about 1:0.5 to about 1:10. The blue shades generated are variable among deep blue, violet-blue, bright-blue, and greenish-blue depending on the amino acid used. The blue colorant generated from genipin-rich extract is a heat- and acid-stable pigment.

[0029] Similar blue colorants also can be generated by reaction of the genipin-rich extract with other extracts, fruit and vegetable juices, plant and animal materials, including dairy and egg products, which contain amino acids, polypeptides, proteins, and compounds with one or more primary amine groups. The blue color also can be generated by reaction of the genipin-rich extract with collagen, gelatin, chitosan, enzymes, and microbes. The colorant produced can be further concentrated or deposited on clay or other carriers and used in foods, cosmetic (toothpaste, makeup, hair dye, etc), and textile (clothes) applications.

[0030] The present invention provides a method of manufacturing a red colorant by using the genipin-rich extract. The genipin-rich extract is hydrolyzed to remove a methyl group and convert the genipin to genipinic acid which further reacts with amine-containing compounds, with extra organic acid present and under anaerobic conditions, to generate red pigment. Heating can be used to accelerate red pigment formation. The red solution can be further purified on an ion exchange column and the eluted material can be concentrated on a rotary vacuum evaporator. The genipa red is a heat-stable pigment, and is also stable at a high pH.

[0031] Genipin-rich extract can be dispersed or dissolved in about 5-50% ethanol, preferably about 5-15% ethanol, and about 0.5-1.5 N sodium hydroxide or potassium hydroxide solution, to a concentration of extract of about 5-10 % w/w. Hydrolysis can be performed with or without heating for from about 30 min to 24 hours, preferably about 2-10 hours under room temperature (∼20°C).

[0032] Hydrolyzed genipin-rich extract can be neutralized in pH by using acids, preferably organic acids, such as acetic acid, formic acid, lactic acid, citric acid, tartaric acid, adipic acid, oxalic acid, succinic acid, fumaric acid, and malic acid, preferably acetic acid and/or tartaric acid. The pH of hydrolyzed solution can be adjusted to the about 3.5-5.0 range.

[0033] The acidified solution is then heated to about 65-85°C, and preferably about 70-75°C, for about 5-120 minutes, preferably about 10-20 minutes. The insoluble brown by-products are removed since they will not contribute to red color formation. Precipitate separation can be accomplished by any of the filtration or centrifugation methods known in the art, for example, using regular filtration, centrifuge, press filtration, or tangential flow filtration or membrane cartridge filtration methods. Formation of the insoluble brown by-products can be further enhanced by the addition of calcium carbonate to adjust the pH to about 5-7.5 and mixing for about 5-30 minutes. Precipitation separation can be accomplished by the methods described above.

[0034] The clear solution described above can be adjusted to pH about 4.0-4.6 with organic acid or salt, preferably acetic acid and/or sodium acetate. Red color is generated after mixing with compounds having primary amine groups and heating to about 70-95°C, preferably about 80-85° C, for about 2 hours, or about 90-95°C, for about 1 hour. The compounds with primary amine groups can be selected from amino acids, such as alanine, arginine, lysine, aspartic acid, glutamic acid, glycine, histidine, valine, leucine, and serine. This listing is not intended to limit useful compounds with primary amine groups.

[0035] The present invention, in addition, provides a method of manufacturing green colorant by using the genipin-rich extract. Genipin-rich extract can react with certain amino acids, such as isoleucine, threonine, cysteine, and tryptophan, to generate green color. It also can react with primary amine-containing compounds and mix with carotenoids, annatto, and turmeric pigments to generate green color. Since genipa green can provide a heat resistant green shade, it is very useful in the food industry because of heat instability of the natural green pigment, chlorophyll.

[0036] Similar green colorants also can be generated by reaction of the genipin-rich extract with other extracts, fruit and vegetable juice, plant and animal materials, including dairy and egg products, which are rich in amino acids, for example, isoleucine, threonine, cysteine, and tryptophan. The green color also can be generated by reaction of the genipin-rich extract with collagen, gelatin, chitosan, enzymes and microbes and mixing with carotenoids, annatto, and turmeric pigments to generate green color. The colorant produced can be further concentrated or deposited on clay or other carriers and used in foods, cosmetic (toothpaste, makeup, hair dye, etc), and textile (clothes) applications.

[0037] The present invention, in addition, provides a means to generate other colorants, like purple, red, yellow, orange, brown, and black by using the genipin-rich extract. Purples and reds can be created by mixing genipin-rich extract with carmine, beet juice, and anthcyanin colorants and solution with amine-containing compounds. Oranges and yellows can be created by reacting genipin-rich extract with pentoses, such as xylose, ribose, or vitamin C. Black color shade can be generated when alkaline earth metals (e.g. magnesium or calcium), or metals with multi-valences are involved during genipin reaction with compounds containing a primary amine group.

Cross-linking Agent



[0038] The present disclosure provides new materials which contain up to about 97% of genipin content. Genipin-rich extract can react with any compound that contains a primary amine group, such as collagen, gelatin, chitosan, glucosamine, and various enzymes and proteins, to form new texture materials used in, for example, the food, cosmetic, biomaterial, and polymer industries.

[0039] The present disclosure also provides a cross-linking agent obtained from the natural plant, Genipa americana, which can replace synthesized chemicals, such as glutaraldehyde, formaldehyde, glyoxal, malonaldehyde, succinaldehyde, epoxy compounds, etc. Genipin-rich extract used as a cross-linking agent has a much lower toxicity than those commonly used synthetic cross-linking reagents.

[0040] Genipin-rich extract can work as a natural cross-linking agent for encapsulation production in the food and pharmaceutical industries. Encapsulated materials can be nutritional ingredients, such as omega-3 oil, vitamin A, conjugated linolenic oil, or medicines. Gelatin, collagen, whey proteins, casein, chitosan, soy proteins, and other plant or animal proteins, are good starting materials in conjunction with the genipin-rich extract to use to make an encapsulation shell, film or micro-membrane. The products have good thermal and mechanical stability, as well as their biocompatible property.

[0041] Following examples are provided for purpose of further illustrating the present disclosure, but should in no sense be taken as limiting.

Example 1



[0042] A genipin-rich extract was generated based on the following process:
  1. a) Huito fruits were peeled to yield 866.0 g of peeled fruit. The peeled fruit was cut into pieces and blended with 1300 g of deionized (DI) water and pureed with a blender to produce a puree;
  2. b) the puree was allowed to stand for 15 min at room temperature; and the insoluble solid was separated from the aqueous extract by filtration. Then, 1212.4 g of DI water was added to the filtrate and mixed for 15 minutes followed by a second filtration step;
  3. c) the filtrate was adjusted to pH < 4.0 using citric acid, and concentrated to 25-55% (w/w) of solid content on a rotovapor;
  4. d) then, the concentrated aqueous extract was extracted using ethyl acetate in a 1:1 ratio. The colorless or slightly yellow clear ethyl acetate layer was collected. This liquid-liquid extraction was repeated 2 times;
  5. e) next, the combined ethyl acetate extract layers were evaporated on a rotovapor at a temperature of 40° C and vacuum of 35 mm Hg to remove the solvent. Condensed ethyl acetate can be reused for genipin extraction. After evaporating the ethyl acetate, an off-white or slightly yellow powder was obtained. The powder contained 74.4% w/w of genipin.

Example 2



[0043] A genipin-rich extract was generated based on following process:
  1. a) Huito fruits, 462 g, were peeled and blended with DI water 537 g to produce puree;
  2. b) the puree was allowed to stand for 15 min at room temperature, and the insoluble solid was separated from aqueous extract by filtration. Then, the same quantity of DI water was added to the solid part, and the extraction step was repeated 2 times;
  3. c) the combined aqueous extract was adjusted to pH < 4.0 using citric acid and concentrated to 24.92% (w/w) of solid content via a rotovapor;
  4. d) then, the concentrated aqueous extract was extracted using butyl acetate in 1:1 ratio. The colorless or slightly yellow clear butyl acetate layer was collected. This liquid-liquid extraction was repeated 2 times;
  5. e) next, the combined butyl acetate extract was evaporated on a rotovapor at a temperature of 55° C and vacuum of 35 mm Hg to remove the solvent. Condensed butyl acetate can be reused for genipin extraction. After evaporating butyl acetate, an off-white or slightly yellow powder was obtained. The powder contains 90.9% w/w of genipin.

Example 3



[0044] The genipin-rich extract made by the method in Example 2 was tested for stability. Genipin-rich powder, 0.20 g each, was sealed in vials and stored at 4 °C. Samples were pulled at 0, 4 wks, 6 wks, and 12 wks, and genipin content was tested by using the HPLC method. Results are shown in Table 1.
Table 1. Stability of genipin-rich extract
Storage Time (wks)Genipin, % w/w
0 83.9
4 86.0
6 85.3
12 84.7


[0045] The genipin material was stable over the period of the test.

Example 4



[0046] Fifteen grams of genipin-rich extract made by the method in Example 1 was dissolved in 118.10 g of 10% ethanol solution. 16.89 g of 0.9 N solution of KOH was added to the above mixture and the genipin was hydrolyzed with stirring at room temperature (22° C) for 7.5 hours. The pH of the hydrolyzed solution was adjusted to less than 4.0 with tartaric acid. The solution was heated to 74 °C for 15 min and cooled. The precipitate was filtered out through #2 filter paper. Next, the pH of the filtrate was adjusted to ∼ 6.5 using calcium carbonate and the slurry was mixed for 10 minutes. The resulting precipitate was filtered off and the filtrate pH was adjusted to 4.0-4.5 by the addition of acetic acid. After alanine (4.50 g) was added, genipin red color was developed by heating to 82 °C for 2 hrs. The color was analyzed by taking 0.30 g of genipin red color and diluting to 30.0 g with DI water. L-, a-, and b-values were measured on a Hunter Lab spectrometer, and the absorption curve measured on a UV/VIS spectrometer. Results are shown in the following table.
ParametersValue
Hunter L-value 27.86
Hunter a-value 10.21
Hunter b-value 2.26
Wavelength (max,nm) 550.09
Absorption (max) 0.67314

Example 5



[0047] Three grams of genipin-rich extract made by the method in Example 2 was dissolved in 23.5g of 10% ethanol solution. 3.78g of 1.0N solution of KOH was added to the above mixture and the genipin was hydrolyzed with stirring at room temperature (22°C) for 8.0 hours. The pH of the hydrolyzed solution was adjusted to less than 4.0 with tartaric acid. The solution was heated to 74°C for 15 minutes and cooled. The precipate was removed by filtration on #2 filter paper. Next, the pH of the filtrate was adjusted to ∼7.0 using calcium carbonate and the slurry was mixed for 15 minutes. The resulting precipate was filtered off and the filtrate pH was adjusted to 4.0-4.5 by the addition of acetic acid. After alanine (1.20g) was added, genipin red color was developed by heating to 84°C for 2 hours. The color was analyzed by taking 0.50g of genipin red color and diluting to 30.0g with DI water. L-, a-, and b-values were measured on a Hunter Lab spectrometer, and the absorption curve measured on a UV/VIS spectrometer. Results are shown in the following table.
ParametersValue
Hunter L-value 27.42
Hunter a-value 9.46
Hunter b-value 2.32
Wavelength (max, nm) 549.97
Absorption (max) 0.94423

Example 6



[0048] One and a half grams of genipin-rich extract made by the method in Example 2 was dissolved in 11.8g of 10% ethanol solution. 1.97g of 1.0N solution of KOH was added to the above mixture and the genipin was hydrolyzed wih stirring at room temperature (22°C) for 8.0 hours. The pH of the hydrolyzed solution was adjusted to less than 4.0 with tartaric acid. The solution was heated to 74°C for 15 minutes and cooled. The precipitate was removed by filtration through #2 filter paper. Next, the pH of the filtrate was adjusted to ∼7.0 using calcium carbonate and the slurry was mixed for 15 minutes. The resulting precipitate was filtered off and the filtrate pH was adjusted to 4.0-4.5 by the addition of acetic acid. After alanine (0.60g), taurine (0.60g), and magnesium chloride (0.4486g) was added, a dark brown-black color was developed by heating to 84°C for 2 hours. The color was analyzed by taking 1.0g of genipin color and diluting to 30.0g with DI water. L-, a-, and b-values were measured on a Hunter Lab spectrometer, and the absorption curve measured on a UV/VIS spectrometer. Results are shown in the following table.
ParametersValue
Hunter L-value 24.73
Hunter a-value 0.29
Hunter b-value 0.85
Wavelength (max, nm) 542.83
Absorption (max) 0.0170
Wavelength (2nd peak, nm) 595.06
Absorption (2nd peak) 0.88795

Example 7



[0049] 0.108g of genipin-rich extract, made by the method in Example 2, was dispersed in 8.6g of 8% ethanol aqueous solution in a test tube. Solids were completely dissolved after heating in a ∼50°C water bath. Then, 1.0ml aliquots of the above genipin solution was put into test tubes separately and L-threonine 0.012g, L-isoleucine 0.014g, or L-histidine 0.016g, respectively, were added. All test tubes were heated in an 80°C water bath for 2 hours. The color was analyzed by taking 0.30g of genipin color produced and diluting to 30.0g with DI water. L-, a-, and b-values were measured on a Hunter Lab spectrometer, and the absorption curve measured on a UV/VIS spectrometer. Results are shown in the following table.
ParametersL-ThroenineL-IsoleucineL-Histidine
Dilution (g/ml) 0.50/100 0.30/100 0.10/100
Color Green Green Blue
Hunter L-value 79.12 87.41 71.13
Hunter a-value -5.91 -3.42 -7.82
Hunter b-value -2.42 -0.77 -17.46
Wavelenth (max, nm) 596.32 600.12 592.2
Absorption (max) 0.11914 0.06746 0.2537

Example 8



[0050] 0.37g of genipin-rich extract, made by the method in Example 2, was dispersed in 34.63g of 10% ethanol aqueous solution in a beaker. Alanine 0.7g, and xylose, 1.4g, were added and dissolved. Sample heated at 92°C for 1 hour. After cooling, the color was analyzed by taking 1.50g of the color solution and diluting to 30.0g with DI water. The color was observed to be orange-red in contrast to the red color observed when alanine was mixed alone with the genipin-rich extract. L-, a-, and b-values were measured on a Hunter Lab spectrometer, and the results are shown in the following table.
ParametersL-Alanine and Xylose
Color Orange-Red
Hunter L-value 25.12
Hunter a-value 2.48
Hunter b-value -0.22



Claims

1. A method of preparing a colorant from Genipa americana comprising the steps:

1) preparing a genipin-rich extract from Genipa americana; and

2) use of said genepin-rich extract in colorant production;

wherein the preparation of the genepin-rich extract comprises the following steps:

a) washing and/or blanching the mature fruits of Genipin americana;

b) peeling the fruits and cutting them into pieces;

c) milling or blending the fruits with water or polar solvents in a weight ratio of 1:0.5-5.0;

d) extracting the fruits with the water or polar solvents;

e) separating the pulp, seeds and skin of the fruit by filtration and collecting the liquid filtrate;

f) adjusting the pH of the filtrate to 3.8 to 4.0 using acids;

g) concentrating the filtrate to produce a genepin-rich aqueous extract;

h) extracting said aqueous extract with non-polar organic solvents through mixing in a ratio of about 1:1, wherein said non-polar organic solvents have a polarity index from 0 to 5.0 and water solubility of less than 30%;

i) separating the organic solvent phase from the aqueous phase by settling or siphoning the top organic layer or by using a high speed centrifuge;

j) evaporating the organic solvent to produce a genepin-rich extract as a yellow or off-white solid in which the genepin content is at least 70 % w/w of the solids.


 
2. The method according to claim 1, wherein the non-polar organic solvent is selected from the group consisting of ethyl acetate, butyl acetate, n-butanol, diethyl ether, hexane, 2-butanone, chloroform, 1,2-dichloroethane, benzene, xylene, methyl-t-butyl ether, toluene, carbon tetrachloride, trichloroethylene, cyclohexane, pentane, heptanes or mixtures thereof.
 
3. The method according to either of claims 1 or 2, wherein the non-polar organic solvent is selected from the group consisting of ethyl acetate, butyl acetate, and n-butanol.
 
4. The method according to any of the preceding claims, wherein the polar solvent is a solvent having water solubility up to 100% and a polarity index greater than 5.0.
 
5. The method according to claim 4, wherein the polar solvent is selected from the group consisting of water, acetic acid, methanol, ethanol, n-propanol, iso-propanol, dimethyl sulfoxide, dimethyl formamide, acetonitrile, acetone, dioxane, tetrahydrofuran, and acetic, citric, phosphate acid buffer solutions, or mixtures thereof; preferably from the group consisting of water, ethanol, n-propanol, iso-propanol, methanol and acetic, citric and phosphate acid buffer solution.
 
6. The method according to any of the preceding claims, wherein a blue colorant is produced in step 2) by either

(i) mixing the genipin-rich extract with water and amino acids in a molar ratio of from 1:0.5 to 1:10 in the presence of oxygen, preferably wherein the amino acids are selected from the group consisting of lysine, histidine, arginine, glutamine, asparagine, methionine, glycine, glutamic acids, tyrosine, valine, alanine, serine, leucine, taurine, carnitine, ornithine and citrulline; or

(ii) reacting the genepin-rich extract with other extracts, fruit anding vegetable juices, plant and animal materials, including dairy and egg products, which contain amino acids, polypeptides, proteins, and compounds with one or more primary amine groups; or

(iii) reacting the genipin-rich extract with collagen, gelatin, chitosan, enzymes, and microbes.


 
7. The method according to any of claims 1 to 5, wherein a green colorant is produced in step 2) by either

(i) reacting the genepin-rich extract with amino acids, such as isoleucine, threonine, cysteine, and tryptophan; or

(ii) reacting the genepin-rich extract with primary amine-containing compounds and mixing with carotenoids, annatto, and turmeric pigments; or

(iii) reacting the genipin-rich extract with other extracts, fruit and vegetable juice, plant and animal materials, including dairy and egg products, which are rich in amino acids such as isoleucine, threonine, cysteine, and tryptophan; or

(iv) reacting the genipin-rich extract with collagen, gelatin, chitosan, enzymes and microbes and mixing with carotenoids, annatto, and turmeric pigments.


 
8. The method according to any of claims 1 to 5, wherein a black colorant is produced in step 2) by reacting the genepin-rich extract with compounds containing a primary amine group in the presence of alkaline earth metals or metals with multi-valences.
 
9. The method according to any of claims 1 to 5, wherein a purple or red colorant is produced in step 2) by mixing the genepin-rich extract with carmine, beet juice, and anthcyanin colorants and a solution with amine-containing compounds.
 
10. The method according to any of claims 1 to 5, wherein an orange or yellow colorant is produced in step 2) by reacting the genepin-rich extract with pentoses, or vitamin C, preferably wherein said pentoses are selected from xylose and ribose.
 


Ansprüche

1. Verfahren zur Herstellung eines Farbstoffs aus Genipa americana, umfassend die Schritte:

1) Bereitstellen eines genipinreichen Extrakts aus Genipa americana; und

2) Verwenden des genipinreichen Extrakts bei der Farbstoffproduktion;

wobei die Herstellung des genipinreichen Extrakts die folgenden Schritte umfasst:

a) Waschen und/oder Blanchieren der reifen Früchte von Genipa americana;

b) Schälen der Früchte und In-Stücke-Schneiden derselben;

c) Mahlen oder Mischen der Früchte mit Wasser oder polaren Lösungsmitteln in einem Gewichtsverhältnis von 1:0,5-5,0;

d) Extrahieren der Früchte mit dem Wasser oder den polaren Lösungsmitteln;

e) Abtrennen des Fruchtfleischs, der Samen und der Haut der Frucht durch Filtration und Auffangen des flüssigen Filtrats;

f) Einstellen des pH-Werts des Filtrats auf 3,8 bis 4,0 unter Verwendung von Säuren;

g) Einengen des Filtrats unter Bildung eines genipinreichen wässrigen Extrakts;

h) Extrahieren des wässrigen Extrakts mit unpolaren organischen Lösungsmitteln durch Mischen in einem Verhältnis von etwa 1:1, wobei die unpolaren organischen Lösungsmittel einen Polaritätsindex von 0 bis 5,0 und eine Wasserlöslichkeit von weniger als 30% aufweisen;

i) Trennen der organischen Lösungsmittelphase von der wässrigen Phase durch Absetzenlassen oder Absaugen der oberen organischen Schicht oder unter Verwendung einer Hochgeschwindigkeitszentrifuge;

j) Verdampfen des organischen Lösungsmittels unter Bildung eines genipinreichen Extrakts als gelben oder schmutzigweißen Feststoff, in dem der Genipingehalt wenigstens 70 Gew.-% der Feststoffe beträgt.


 
2. Verfahren gemäß Anspruch 1, wobei das unpolare organische Lösungsmittel aus der Gruppe ausgewählt ist, die aus Ethylacetat, Butylacetat, n-Butanol, Diethylether, Hexan, 2-Butanon, Chloroform, 1,2-Dichlorethan, Benzol, Xylol, Methyl-t-butylether, Toluol, Tetrachlorkohlenstoff, Trichlorethylen, Cyclohexan, Pentan, Heptanen oder Mischungen davon besteht.
 
3. Verfahren gemäß einem der Ansprüche 1 oder 2, wobei das unpolare organische Lösungsmittel aus der Gruppe ausgewählt ist, die aus Ethylacetat, Butylacetat und n-Butanol besteht.
 
4. Verfahren gemäß einem der vorstehenden Ansprüche, wobei das polare Lösungsmittel ein Lösungsmittel ist, das eine Wasserlöslichkeit von bis zu 100% und einen Polaritätsindex von mehr als 5,0 aufweist.
 
5. Verfahren gemäß Anspruch 4, wobei das polare Lösungsmittel aus der Gruppe ausgewählt ist, die aus Wasser, Essigsäure, Methanol, Ethanol, n-Propanol, Isopropanol, Dimethylsulfoxid, Dimethylformamid, Acetonitril, Aceton, Dioxan, Tetrahydrofuran sowie Essig-, Zitronen-, Phosphorsäurepufferlösungen oder Gemischen davon besteht, vorzugsweise aus der Gruppe, die aus Wasser, Ethanol, n-Propanol, Isopropanol, Methanol sowie Essig-, Zitronen- und einer Phosphorsäurepufferlösung besteht.
 
6. Verfahren gemäß einem der vorstehenden Ansprüche, wobei in Schritt 2) ein blauer Farbstoff entsteht, und zwar durch entweder

(i) Mischen des genipinreichen Extrakts mit Wasser und Aminosäuren in einem Stoffmengenverhältnis von 1:0,5 bis 1:10 in Gegenwart von Sauerstoff, wobei vorzugsweise die Aminosäuren aus der Gruppe ausgewählt sind, die aus Lysin, Histidin, Arginin, Glutamin, Asparagin, Methionin, Glycin, Glutaminsäuren, Tyrosin, Valin, Alanin, Serin, Leucin, Taurin, Carnitin, Ornithin und Citrullin besteht; oder

(ii) Umsetzen des genipinreichen Extrakts mit anderen Extrakten, Frucht- und Gemüsesäften, pflanzlichen und tierischen Materialien einschließlich Milch- und Eiprodukten, die Aminosäuren, Polypeptide, Proteine und Verbindungen mit einer oder mehreren primären Amingruppen enthalten; oder

(iii) Umsetzen des genipinreichen Extrakts mit Collagen, Gelatine, Chitosan, Enzymen und Mikroben.


 
7. Verfahren gemäß einem der Ansprüche 1 bis 5, wobei in Schritt 2) ein grüner Farbstoff entsteht, und zwar durch entweder

(i) Umsetzen des genipinreichen Extrakts mit Aminosäuren wie Isoleucin, Threonin, Cystein und Tryptophan; oder

(ii) Umsetzen des genipinreichen Extrakts mit primäre Amingruppen enthaltenden Verbindungen und Mischen mit Carotinoiden, Annatto und Kurkumapigmenten; oder

(iii) Umsetzen des genipinreichen Extrakts mit anderen Extrakten, Frucht- und Gemüsesäften, pflanzlichen und tierischen Materialien einschließlich Milch- und Eiprodukten, die reich an Aminosäuren wie Isoleucin, Threonin, Cystein und Tryptophan sind; oder

(iv) Umsetzen des genipinreichen Extrakts mit Collagen, Gelatine, Chitosan, Enzymen und Mikroben und Mischen mit Carotinoiden, Annatto und Kurkumapigmenten.


 
8. Verfahren gemäß einem der Ansprüche 1 bis 5, wobei in Schritt 2) ein schwarzer Farbstoff entsteht, und zwar durch Umsetzen des genipinreichen Extrakts mit Verbindungen, die eine primäre Amingruppe enthalten, in Gegenwart von Erdalkalimetallen oder Metallen mit mehrfacher Wertigkeit.
 
9. Verfahren gemäß einem der Ansprüche 1 bis 5, wobei in Schritt 2) ein violetter oder roter Farbstoff entsteht, und zwar durch Mischen des genipinreichen Extrakts mit Karmin-, Rote-Bete-Saft- und Anthocyaninfarbstoffen und einer Lösung mit aminhaltigen Verbindungen.
 
10. Verfahren gemäß einem der Ansprüche 1 bis 5, wobei in Schritt 2) ein orangefarbener oder gelber Farbstoff entsteht, und zwar durch Umsetzen des genipinreichen Extrakts mit Pentosen oder Vitamin C, wobei vorzugsweise die Pentosen aus Xylose und Ribose ausgewählt sind.
 


Revendications

1. Procédé de préparation d'un colorant à partir de Genipa americana comprenant les étapes qui consistent :

1) à préparer un extrait riche en génipine à partir de Genipa americana ; et

2) à utiliser ledit extrait riche en génipine dans la production de colorant ;

dans lequel la préparation de l'extrait riche en génipine comprend les étapes suivantes qui consistent :

a) à laver et/ou à blanchir les fruits mûrs de Genipa americana ;

b) à éplucher les fruits et à les couper en morceaux ;

c) à broyer ou à mélanger les fruits avec de l'eau ou des solvants polaires selon un rapport pondéral de 1 : 0,5-5,0 ;

d) à extraire les fruits avec de l'eau ou des solvants polaires ;

e) à séparer la pulpe, les graines et la peau du fruit par filtration et à collecter le filtrat liquide ;

f) à ajuster le pH du filtrat à une valeur allant de 3,8 à 4,0 en utilisant des acides ;

g) à concentrer le filtrat pour produire un extrait aqueux riche en génipine ;

h) à extraire ledit extrait aqueux avec des solvants organiques non polaires par mélange selon un rapport d'environ 1:1, où lesdits solvants organiques non polaires ont un indice de polarité allant de 0 à 5,0 et une hydrosolubilité inférieure à 30% ;

i) à séparer la phase de solvant organique de la phase aqueuse par décantation ou siphonnage de la couche organique supérieure ou en utilisant une centrifugeuse à grande vitesse ;

j) à évaporer le solvant organique pour produire un extrait riche en génipine sous forme d'un solide jaune ou blanc cassé dans lequel la teneur en génipine est d'au moins 70% p/p des matières solides.


 
2. Procédé selon la revendication 1, dans lequel le solvant organique non polaire est choisi dans le groupe constitué par l'acétate d'éthyle, l'acétate de butyle, le n-butanol, l'éther diéthylique, l'hexane, la 2-butanone, le chloroforme, le 1,2-dichloroéthane, le benzène, le xylène, le méthyl-t-butyl éther, le toluène, le tétrachlorure de carbone, le trichloréthylène, le cyclohexane, le pentane, les heptanes ou des mélanges de ceux-ci.
 
3. Procédé selon l'une des revendications 1 et 2, dans lequel le solvant organique non polaire est choisi dans le groupe constitué par l'acétate d'éthyle, l'acétate de butyle et le n-butanol.
 
4. Procédé selon l'une des revendications précédentes, dans lequel le solvant polaire est un solvant ayant une hydrosolubilité allant jusqu'à 100% et un indice de polarité supérieur à 5,0.
 
5. Procédé selon la revendication 4, dans lequel le solvant polaire est choisi dans le groupe constitué par l'eau, l'acide acétique, le méthanol, l'éthanol, le n-propanol, l'isopropanol, le diméthylsulfoxyde, le diméthylformamide, l'acétonitrile, l'acétone, le dioxane, le tétrahydrofurane et des solutions tampons d'acide acétique, citrique, de phosphate, ou des mélanges de ceux-ci ; de préférence dans le groupe constitué par l'eau, l'éthanol, le n-propanol, l'isopropanol, le méthanol et une solution tampon d'acide acétique, citrique et de phosphate.
 
6. Procédé selon l'une des revendications précédentes, dans lequel un colorant bleu est produit à l'étape 2)

(i) en mélangeant l'extrait riche en génipine avec de l'eau et des acides aminés selon un rapport molaire allant de 1 : 0,5 à 1 : 10 en présence d'oxygène, de préférence où les acides aminés sont choisis dans le groupe constitué par la lysine, l'histidine, l'arginine, la glutamine, l'asparagine, la méthionine, la glycine, les acides glutamiques, la tyrosine, la valine, l'alanine, la sérine, la leucine, la taurine, la carnitine, l'ornithine et la citrulline ; ou

(ii) en faisant réagir l'extrait riche en génipine avec d'autres extraits, des jus de fruits et de légumes, des matières végétales et animales, y compris des produits laitiers et à base d'œufs, qui contiennent des acides aminés, des polypeptides, des protéines et des composés avec un ou plusieurs groupe(s) amine primaire ; ou

(iii) en faisant réagir l'extrait riche en génipine avec du collagène, de la gélatine, du chitosane, des enzymes et des microbes.


 
7. Procédé selon l'une des revendications 1 à 5, dans lequel un colorant vert est produit à l'étape 2)

(i) en faisant réagir l'extrait riche en génipine avec des acides aminés, tels que l'isoleucine, la thréonine, la cystéine et le tryptophane ; ou

(ii) en faisant réagir l'extrait riche en génipine avec des composés contenant une amine primaire et en mélangeant avec des pigments de turmérique, du rocou et des caroténoïdes ; ou

(iii) en faisant réagir l'extrait riche en génipine avec d'autres extraits, un jus de fruits et de légumes, des matières végétales et animales, y compris des produits laitiers et à base d'œufs, qui sont riches en acides aminés tels que l'isoleucine, la thréonine, la cystéine et le tryptophane ; ou

(iv) en faisant réagir l'extrait riche en génipine avec du collagène, de la gélatine, du chitosane, des enzymes et des microbes et en mélangeant avec des pigments de turmérique, du rocou et des caroténoïdes.


 
8. Procédé selon l'une des revendications 1 à 5, dans lequel un colorant noir est produit à l'étape 2) en faisant réagir l'extrait riche en génipine avec des composés contenant un groupe amine primaire en présence de métaux alcalino-terreux ou de métaux à valences multiples.
 
9. Procédé selon l'une des revendications 1 à 5, dans lequel un colorant pourpre ou rouge est produit à l'étape 2) en mélangeant l'extrait riche en génipine avec du carmin, du jus de betterave et des colorants à base d'anthocyanine et une solution avec des composés contenant une amine.
 
10. Procédé selon l'une des revendications 1 à 5, dans lequel un colorant orange ou jaune est produit à l'étape 2) en faisant réagir l'extrait riche en génipine avec des pentoses, ou la vitamine C, de préférence dans lequel lesdits pentoses sont choisis parmi le xylose et le ribose.
 




Drawing






REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description