the present disclosure provides a method of producing stable genipin - rich extracts from the genipa americana plant . the process involves aqueous or polar solvent extraction and organic non - polar solvent extraction . the resulting powder form of genipin - rich extract is off - white in color and has genipin content up to about 97 %. the present disclosure further provides applications of the genipin - rich extracts in the production of natural heat - stable colorants and as a new cross - linking agent for use in a broad range of areas , such as foodstuffs , drugs , nutritional supplements , personal care products , cosmetics , animal feed , textiles , biodegradable polymers , and biomaterial production . 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 . 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 . 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 . 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 . alternatively , the mature fruits of genipa americana are processed with organic solvents directly in a ratio of about 1 : 1 - 2 . after washing and / or blanching , the fruit is peeled and cut into pieces , then milled or blended , extracted with non - polar organic solvent directly , with or without heating , the pulp , seeds and skin are separated and the solvent extract is collected . extraction may take place for up to about 1 hour , preferably about 15 - 30 min at temperature of about 18 - 25 ° c . the organic solvent extraction may be repeated 2 - 3 times depending on the extraction efficiency . the genipin - rich extracts may subsequently be dried using an evaporator and condenser , and organic solvent can be recycled and reused . the resulting off - white or yellow solids is genipin - rich extract that contains genipin at greater than about 40 % w / w of solid . 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 . 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 . genipin - rich extracts may alternatively be organic solvent extracts obtained from the starting material sources described . organic solvents include those with polarity index less than about 6 . 0 , and mixtures thereof in different ratios . the solvent or solvent mixture with low solubility in water is preferred in order to obtained colorless genipin - rich extracts . 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 . acids used for ph adjustment can be any organic or inorganic acids , selected to be suitable for the intended end use . 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 . 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 . hplc methods can be used to determine genipin content and perform other iridoid analysis . 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 . high genipin content extract readily dissolves in alcohol , alcohol - water mixtures , or hot water . its solubility in cold water is limited . 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 . 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 . 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 . 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 .). 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 . 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 . 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 . 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 . 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 . 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 . the present invention 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 . the present invention 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 . 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 . following examples are provided for purpose of further illustrating the present disclosure , but should in no sense be taken as limiting . 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 ; 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 ; c ) the filtrate was adjusted to ph & lt ; 4 . 0 using citric acid , and concentrated to 25 - 55 % ( w / w ) of solid content on a rotovapor ; 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 ; 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 . a ) huito fruits , 462 g , were peeled and blended with di water 537 g to produce puree ; 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 ; c ) the combined aqueous extract was adjusted to ph & lt ; 4 . 0 using citric acid and concentrated to 24 . 92 % ( w / w ) of solid content via a rotovapor ; 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 ; 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 . 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 . 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 . three grams of genipin - rich extract made by the method in example 2 was dissolved in 23 . 5 g of 10 % ethanol solution . 3 . 78 g 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 . 20 g ) was added , genipin red color was developed by heating to 84 ° c . for 2 hours . the color was analyzed by taking 0 . 50 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 . one and a half grams of genipin - rich extract made by the method in example 2 was dissolved in 11 . 8 g of 10 % ethanol solution . 1 . 97 g 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 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 . 60 g ), taurine ( 0 . 60 g ), and magnesium chloride ( 0 . 4486 g ) was added , a dark brown - black color was developed by heating to 84 ° c . for 2 hours . the color was analyzed by taking 1 . 0 g of genipin 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 . 0 . 108 g of genipin - rich extract , made by the method in example 2 , was dispersed in 8 . 6 g of 8 % ethanol aqueous solution in a test tube . solids were completely dissolved after heating in a ˜ 50 ° c . water bath . then , 1 . 0 ml aliquots of the above genipin solution was put into test tubes separately and l - threonine 0 . 012 g , l - isoleucine 0 . 014 g , or l - histidine 0 . 016 g , respectively , were added . all test tubes were heated in an 80 ° c . water bath for 2 hours . the color was analyzed by taking 0 . 30 g of genipin color produced 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 . 0 . 37 g of genipin - rich extract , made by the method in example 2 , was dispersed in 34 . 63 g of 10 % ethanol aqueous solution in a beaker . alanine 0 . 7 g , and xylose , 1 . 4 g , were added and dissolved . sample heated at 92 ° c . for 1 hour . after cooling , the color was analyzed by taking 1 . 50 g of the color solution and diluting to 30 . 0 g 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 .
a method of preparing genipin - rich materials from the fruit of genipa americana fruit for their use as a cross - linking agent and as a raw material to produce colors is disclosed . the genipin - rich materials can be used in a broad range of applications including personal care , cosmetics , dietary supplements , packaging , textiles , beverages , foodstuffs , drugs , and animal feeds .