AU8695198A - Method for the conversion of xanthophylls in plant material - Google Patents

Description

WO 00/07458 PCT/US98/16393 5 Method For The Conversion Of Xanthophylls In Plant Material 10 Field of the Invention This invention relates to the in situ process for converting non free form xanthophylls to free xanthophylls in the biological material of the plant. The method more particularly relates to a method that would liberate xanthophylls by transesterification of acyl- xanthophylls in plant materials. 15 Background of the Invention Carotenoids are a group of red and yellow pigments contained in plants and fruits. Carotenoids include carotenes and hydroxylated carotenoids designated xanthophylls. Xanthophylls include lutein. zeaxanthin, 20 capsorubin, capsanthin. astaxanthin, and canthaxanthin. The animal feed industry and the food industry and the pharmaceutical industry have all indicated a strong interest in xanthophylls. The poultry industry receives a benefit in adding the xanthophylls to increase the yolk color of eggs. The pharmaceutical companies, have found that xanthophylls are useful 25 in certain tumor treatments and as an antioxidant. The food industry has found that consumers are looking for naturally occurring food colorants. Annatto, saffron and paprika are a few natural carotenoids that have traditionally been used for food coloring. The dramatic red and yellow coloration and the xanthophylls natural occurrence in edible plants such as green vegetables and fruit such as broccoli, green beans and peas and brussels sprouts, cabbage, kale, spinach, kiwi and honeydew have led 30 to many uses including a pigmentation additive for animal feeds. In certain vegetables the xanthophylls are in the free non esterilied form. However, the large quantities of chorophylls in green vegetables make concentration or extraction of the xanthophylls difficult. A number ofxanthophylls is also present in yellow colored fruits and vegetables such as mango, peaches, prunes, acorn squash and oranges. These contain less chorophylls but often the xanthophylls exists in the esterified form with fatty acids such as 35 myristic, lauric, and palmnitic acids. Unfortunately, to be metabolized in a feed additive, the xanthophylls ester must undergo conversion to the free xanthophylls. The free xanthophyll can then be metabolizable by the body.

WO 00/07458 PCT/US98/16393 5 Various plant materials contain xanthophylls; the desired xanthophylls will drive the selection of the material used. For example it is well know to use the petals of the marigold flower. Tagetes erecta for the extraction of the lutein xanthophylls. Marigolds are readily cultivated and have been used as a pigment source for poultry. Lutein occurs in the marigold flower diacylated with pahnitic and myristic acids in long fatty acid esters typically as diesters in the chromoplastids. 10 The animal feed industry has taken two different approaches to providing xanthophylls in animal feeds, particularly lutein to poultry feeds. The industry has used the dried marigold meal as a feed additive thus providing the lutein in the less useable acylated form. This form of the xanthophylls requires more consumption of the marigold meal to get the pigment desired. Alternatively, the industry has used a 15 number of processes, starting with the extraction of the xanthophylls from the plant material and the formation of oleoresins. The industry then goes further and processes the oleoresins to convert the xanthophylls from the acylated form to the free form by a number of differing processes including transesterfication for some oleoresin processing of paprika, though for lutein the process is primarily by saponification. The converted oleoresin requires less consumption by the animal to get the desired 20 xanthophylls. However. formation of oleoresins and the processing thereof by saponification is both time consuming and adds labor costs to the feed product. Saponification is the conversion of the fatty acid to into a soap by treating it with an alkali. The saponification number is the number of milligrams of potassium hydroxide required to saponify one gram 25 of the ester. After saponification the industry has often used solvents to crystallize the lutein from the oleoresin. This has made the xanthophylls more pure and available to the organism consuming the lutein but it has added time and labor to the process of supplying the xanthophylls to the feed mixture. Some of the following patents indicate the processes for recovering various compounds such as 30 xanthophylls from oleoresins. US patent 5,602,286 describes a process for recovering xanthophylls from corn gluten. The patent has the steps of: adding ethanol as an extraction step, filtering then stripping to form the crude xanthophyll and then using ethanol, KOH as the saponification step, then wash and filter, then purify to the refined xanthophylls. 35 Three Japanese references also show the use of similar oleoresin extractions. No.82,133,160 Japan 1982 shows a red pepper pigment production using a red pepper oleoresin, in either water or alcohol-mixtures, treated with KOH, NaOH, CaCO3, and then treated with acids such as HCI, H 2

SO

4 , H 3 PO4, HOAc, lactic and citrus acids. The pigment solutions are removed with organic solvents such as MeOH, Et OH PrOH and acetone.

WO 00/07458 PCT/US98/16393 5 Patent No. 82.180,663 (1982), shows paprika, food coloring agents that are extracted as an oleoresmn. The oleoresin is heated with basic alkali metal compounds such as KOH. NaOH, K:Co 3 , NaCo 3 . and sodium alcoholate, and mixed one or more hydroxides and carbonates or alkali earth metals such as Ca (OH) 2 . The precipitates were extracted with organic solvents and yielded an odorless oleoresin pigment. Patent No. 83,173,164 shows paprika pigments can be prepared by treating paprika oleoresin with alkali at 10 temperatures below 500 C in the presence of halogen ions, sulfates. bicarbonate, carbonate, phosphate, and aliphatic carboxy ions, and then treated with an organic solvent and finally extracted with acetone. The US patent 5,382,714 describes a method of producing substantially pure lutein. In this patent the starting material was marigold petals. The process of saponification of the petals is briefly described in 15 column 5 example 1. The flower petals were tested for herbicides and pesticides and then the xanthophylls containing material was subjected to saponification with aqueous potassium hydroxide. This was accomplished by continuous mixing under heat (65-70 degrees C) of food grade potassium hydroxide 45%. This accomplished conversion of 98% of lutein into a form that was free of fatty acids and present as a yellow oil. This material could then be used as a feed or food additive. 20 The present invention provides a method of in situ conversion of the xanthophylls into the free form by liberation of the xanthophylls by transesterification. Thus the present invention avoids the need for the formation of the oleoresin. This oleoresin conversion requires an organic solvent extraction of the material from the plant material. Hexane is often used. The present invention provides in situ free form 25 xanthophylls by transesterification of in situ material thus eliminating the need for an oleoresin or saponification of the material. The present invention allows the marigold meal to be subjected to transesterification and then used without the extraction of the xanthophylls from the meal. Summary Of The Invention 30 It is evident that there is a need to convert acylated xanthophylls in plant material to the free form. The objective of this invention is to fulfill that need. Another object of my invention is to provide a marigold meal that has a high free lutein content. 35 Yet another object of my invention is to provide a marigold meal as a feed additive that provides additional pigment to eggs when compared to the same marigold meal as a feed additive that has not undergone the treatment of the present invention.

WO 00/07458 PCT/US98/16393 S Another additional objective of my invention is to provide plant material that contains substantially a greater percentage of the free form of xanthophylls then contained in the original plant material prior to liberation. Broadly, then the present invention includes an improved plant material made from a natural plant 10 material. This plant material contains at least some non free form xanthophylls. comprising plant materials containing in situ, less of said non free form xanthophylls, and more of free form xanthophylls wherein the free form of xanthophylls in situ in the improved plant material has increased beyond the amount in the natural plant material. 15 The invention can have a number of different plant materials including flowers and the petals of flowers. A flower of particular usefulness is Tagetes erecta. This type of flower is useful if the desired xanthophylls are lutein. The bulk of the xanthophylls in marigolds exists in nature as non free form xanthophylls, as fatty acid esters. The present invention is adapted to convert the nonfree xanthophylls into the free form of xanthophylls. When the marigold flowers are employed, this conversion by 20 transesterification produces a nonacvlated lutein. The present invention can improve plant material so that it contains at least 5% more free form xanthophllys than does the natural plant material in situ. Another embodiment of the present invention is an improved animal feed composition comprising: vitamin and minerals, along with a source of carbohydrates selected from the group consisting of 25 soybean, peanuts, corn, alfalfa, wheat, barley and, improved plant material containing in situ more free form xanthophylls then the natural in situ amount of xanthophylls in the plant material that the improved plant material was formed from wherein the xanthophylls are more bioavailable to an animal fed the animal feed. 30 This feed can contain improved plant material that is flowers. If the desired xanthophyll is lutein then said flowers are marigolds. If the desired xanthophyll is capsanthin then paprika can be used. This animal feed is designed for the nutritional requirements of poultry, wherein the animal products such as eggs and meat can have the xanthophyll act as a pigmenter. 35 The present invention is believed particularly useful for chickens. Certain xanthophylls could also be useful in other animal feeds to color the meat or other animal byproducts. A feed for poultry is preferably characterized by having animal feed evidencing the bioavailability of the 40 free form xanthophylls by having increased pigmentation from the consumption of said free form WO 00/07458 PCT/US98/16393 5 xanthophylls. especially when the fed xanthophylls are lutein. The present invention encompasses a product and also the method of forming the free form xanthophylls. Thus broadly, the present invention covers a method of improved plant material made from a natural plant material containing at least some non free form xanthophylls by the following steps 10 of: treating said natural plant material in situ with a solvent; adding a base capable of transesterification of non free form xanthophylls to the free form xanthophylls; neutralizing the reaction wherein forming the improved plant material having more free form xanthophylls then the natural plant material. The method can also include the step of drying the improved plant materials to remove any solvent. 15 The method of the present invention includes using plant material such as flowers. If the desired product is lutein, then flowers such as Tagetes erecta can be used The method uses a solvent in the reaction. The solvent can be broadly an alcohol and more preferred an alkanol or alkenol. If an alcohol is used the alcohol preferably has one to four carbons. 20 The alcohol is selected from the group consisting of methanol, ethanol. isopropyl alcohol, butanol and the like. The method of the present invention uses a base. The base can be selected from the group consisting of, potassium hydroxide, potassium sorbate. NaOMe, animal liver lipase, yeast lipase, NaOEt, KOIVle, KOEt, NaCO 3 . K:CO. and the like. 25 The method also includes the step of stopping the reaction by neutralizing the reaction with a Lewis acid. The preferred Lewis acid is phosphoric acid. However, the Lewis acid can be selected from the group consisting of HCL, NI-H4CL, sulfuric acid, acetic acid, ALCL 3 and the like. 30 The method does not require an extractor such as petroleum ether or hexane or a number of other known extractors to remove the xanthophyll from the plant material. The method can have plant material such as flowers, roots or fruit but preferably without chlorophyll in 35 the product. Brief Description of the Drawings WO 00/07458 PCT/US98/16393 5 FIGURE 1- chroniatogram of marigold meal. FIGURE 2- chromatogram of paprika. 10 FIGURE 3-shows the chromatogram of conversion to free form of capsanthin from treated paprika. FIGURE 4-shows the chromatogram of conversion to free form of lutein from treated marigold meal. FIGURE 5-shows the Roche fan scale over days for the egg yolk color of the present invention at two 15 mg/Kg levels and of the commercial product. FIGURE 6- shows the mg/Kg of xanthophylls in the egg yolks over days of the three treatments. 20 Detailed Description of the Invention. This invention relates to the in situ process for converting non free fornim xanthophylls to free xanthophylls in the biological material of the plant. The method more particularly relates to a method that would liberate xanthophylls by transesterification of acyl-xanthophylls in plant materials. And improved plant 25 material having more free form xanthophyll then the original plant material from which it was made. Thus the present invention provides a method of liberating xanthophylls in situ in plant or vegetable or fruit material. A xanthophyll in the free form, such as lutein, zeaxanthin or capsanthin is formed from the xanthophyll diester in the plant material. The plant material should have relatively concentrated 30 amounts of the desired xanthophyll in the non free form that is usually and preferably the form of a fatty acid ester. Marigolds are an excellent source of lutein in the form of diesters. Also presently known in nature are the wolfberry fruit (Lycium barbarum) an excellent source of zeaxanthin diesters, and the pepper plant (Capsicum annuum) an excellent source capsanthin as diesters. Other plants and fruits and vegetables having high concentrations of desired xanthophylls can also be utilized. 35 The term non free form xanthophyll- refers to an xanthophyll that is in a form that can be transesterified to the free form of the xanthophyll.

WO 00/07458 PCT/US98/16393 5 The term free form xanthophyll- refers to an xanthophyll that is not an in an esterified form. The term bioavailability- refers to the extent to which the xanthophyll is available to the body of the organism consuming it. The term base -refers potassium hydroxide, potassium sorbate, NaOMe, animal liver lipase, NaOEt, 10 KOMe, KOEt, Na 2

CO

3 , KCO 3 and such other non-nucleophilic and non-strong kinetically deprotonating material which does not cause kinetic deprotonation in the alpha-position of the carbonyls and highly conjugated double bond systems, for example LDA or BuLi would be excluded from the definition of base because these chemicals cause deprotonation in the alpha-position of the carbonyls and highly conjugated double bond systems. 15 The term Ratio of Base to plant material (Meal) by Weight- refers to the amount of based used in the reaction compared to the weight of the plant material. This amount is determined by the pH of the reaction and the desired reaction times. The preferred pH is between 11-14: lower pH can be used but increases the reaction time. 20 The term solvent- refers to a chemical in which the transesterification of the xanthophyll can be carried out. the chemical preferably has a hydroxyl group. The solvent is preferably an alcohol with 1-4 carbons More preferably the solvent is selected to have a boiling point that allows the reaction temperature of the transesterification to be kept at 75-85 degrees C, 25 such as MeOH. Plant material -refers to plants containing xanthophylls in the non free form of the xanthophyll. The contemplated plant sources contain xanthophylls in the esterified form as a mono- or di-C, 2 -Cl 8 long chain fatty acids such as lauric, myristic, oleic, linolenic and palmitic acids. Marigolds are an excellent 30 source of lutein in the form of diesters presently known in nature, the woliberry fruit (Lycium barbarum) is an excellent source of zeaxanthin diesters, and the pepper plant (Capsicum annuum) has capsanthin in the form of diesters. Other plants and fruits and vegetables having high concentrations of desired xanthophylls can be employed. 35 It is well known in the art to extract carotenoids from plants. It is equally well know in the art that marigold meal and certain other plant materials can be feed to animals as a feed additive to cause pigmentation of the animal products such as eggs or meat. Marigold meal has been supplied to pigment chicken eggs for a long period of time. Lutein has been extracted from marigold for the pigmentation properties. What the prior art has taught and suggested is that there are two ways to supply lutein. One is 40 as the naturally occurring plant material with the lutein in the non free form. Alternatively, extract the WO 00/07458 PCT/US98/16393 5 lutein by forming an oleoresin and saponify the oleoresin and supply the saponified material having the free form of the xanthophyll. The present invention provides a new and better alternative. The present invention is method of supplying an increased amount of free formed xanthophylls, in situ, in the plant material without having to form the oleoresin. 10 The prior art teaches that organic extractors have been used to extract carotenoids from plant material. Such extractors include hexane, acetone, petroleum ether, methanol, ethyl acetate, diethyl either, heptanes, chloroform, and tetrahydrofuran. These extractors result in what is called an oleoresin which contains diesters. The prior art then teaches the use of a saponification reaction that cleaves the fatty acids from the xanthophyll diesters. There are a number of known methods for the saponification. These 15 produce free form xanthophylls along with soaps of the fatty acids. The soaps are made with alkali solutions such as potassium hydroxide and sodium hydroxide in an aqueous solution. Even though similar chemicals may be used in the saponification process clearly the present invention does not require the organic extraction step nor the saponification step to provide an improved plant material. 20 The liberation of xanthophylls in situ plant material proceeds by the following chemical equation: 25 H3 C~ C'n CH 3 H3 0 H, CH3 CH3 N CC 30 HO v b C3 N3 " Clearly the diacyl-xanthophylls are converted by transesterification into free xanthophylls in situ (in the crude plant material). This process avoids the cost and processes and potentially hazardous chemicals associated with the production of an oleoresin. Additionally the plant material can act as the carrier 35 material increasing the efficiently of the process. Thus the steps of the present method include placing the plant material containing the nonfree form of xanthophylls in the solvent and base. Ratio of base to plant material (Meal) by weight is the amount of based used in the reaction compared to the weight of the plant material. This amount is determined by the WO 00/07458 PCT/US98/16393 5 pH of the reaction and the desired reaction times. The preferred pH is between 11-14: lower pH incmtases the reaction time. For approximately every 32 grams of xanthophyll activity in the raw plant material approximately 770g of base such as KOH was placed in 11 liters of the solvent. The solution containing the plant material was kept at a pH of approximately 13, between 11-14 being acceptable levels with 13 being the preferred 10 level. The reacting solution was stirred until complete conversion was observed. The run time was approximately 10 hours at 690 C and was monitored by HPLC. The reaction solution wias neutralized to a pH of 7 with phosphoric acid. Any number of neutralizing acids and agents could be employed. But phosphoric acid is preferred. The solvent, preferably an alcohol 15 and more preferably MeOH was remove with 16 hours of distillation at 690 C. The temperature of distillation can be higher or lower as long as it is above the boiling point of MeOH and the distillation is done in a commercially reasonable time. To the plant residue material other carriers can be added such as almond shall meal, silicates and the 20 like. The residue is dried after the distillation of the solvent either at room temperature at one ATM or by vacuum drying in an oven at a temperature <500 C. The method of drying is a question of drying time and is not critical. The plant residue material that now has in situ free form xanthophylls therein is mixed and a fine powder with a xanthophyll activity of 10-14 g/kg is achieved. The final product is preferably stored at room temperature under nitrogen. 25 The following bases can also be used in the present invention. Their selection can be based on the economics of the process, the speed of the process and the acceptability of trace amounts of the components. The present invention a plant material having free form xanthophylls, can be marigold meal that has been converted. The converted marigold meal was fed to poultry along with a control (the 30 same marigold meal from the same batch of plant materials) to the same type and age of egg laying hens. The data indicates that there was increased use of the xanthophylls in the converted meal. In other words there was more pigmentation of the yolks of the egg when the same amount of converted plant material was consumed compared to the non converted plant material. Thus the process is providing the xanthophylls in a more bioavailable form to the animal consuming the product. Generally, this test was 35 run with marigold meal that was processed by the steps as listed below to provide in situ free form xanthophylls. Experimeniit 1 WO 00/07458 PCT/US98/16393 5 Liberation of lutein by means of transesterification Step I Marigold meal (2 kg with a total xanthophyll activity of 16 g/kg) in MeOH/KOH (11 L/770 g; pH= 13) was stirred until complete conversion was observed (appr. 10 hours) at 690 C. The reaction turnover was monitored by HPLC. See Figure 1 and Figure 3. 10 Step 2 The reaction mixture was neutralized with phosphoric acid (pH=7) and MVeOH was removed by distillation at 690 C (distillation time = appr. 16 hours). Ste 3 The residue was then dried in a well ventilated room at room temperature and at 1 ATM or by 15 means of vacuum drying in an oven (temperature <500 C). Step 4 After mixing and blending, a fine powder was obtained with a total xanthophyll activity of 10 to 14 g/kg (depending on the duration of the reaction and drying process). The final product is stored at room temperature under nitrogen. 20 The process described above can use a number of different bases that include but are not limited to potassium hydroxide, potassium sorbate, NaOMe, animal liver lipase, NaOEt, KOMe, KOEt, Na 2 CO3,

K

2

CO

3 and such other non-nucleophilic and non-strong kinetically deprotonating material which does not cause deprotonation in the alpha-position of the carbonyls and highly conjugated double bond systems. 25 The preferred base is KOH due to its availability at an inexpensive price and its effectiveness in avoiding the issues of deprotonation. The amount to use of other bases is readily determined. As will be noted the process described in the following experiments does not involve the use of an aqueous solution. Instead the solvent is an alcohol solvent that further along in the process is removable by 30 distillation. MeOH is preferred as a solvent, but other types of alcohol, such as: isopropyl alcohol, ethanol and butanol and the like can be employed without undue experimentation by the ordinarily skilled person in the art. The solvent clearly is a chemical in which the transesterification of the xanthophyll can be carried out. The solvent preferably has a hydroxyl group and is not an aqueous solution. 35 The neutralizing agent is preferably phosphoric acid. But as a neutralizing acid, other Lewis acids, such as: HCL, Sulfuric acid, AIC13, NH 4 CL, Acetic acid and the like can be used. Plant material refers to plants containing xanthophylls in the non free form xanthophyll. The contemplated plant sources contain xanthophylls in the esterified form as a mono- or di-C 1 2 -C, 18 long 40 chain fatty acids such as lauric, myristic, oleic, linolenic and palmnitic acids. Xanthophylls are found in a WO 00/07458 PCT/US98/16393 5 number of differing plant materials. Marigold has lutein and paprika has capsanthin. This process works to liberate a number of nonfree form xanthophylls in plant materials. The experiment below is the reaction procedure for the liberation of capsanthin in paprika meal. 10 Experiment 2 Liberation of by means of transesterification of paprika to form free capsanthin 15 Paprika plant material having a determined total xanthophyll activity of per kg) is placed in MeOH (11 L, without ethoxyquin) and is stirred for 8 hours at 690 C and for 12 hours at rt. with KOH (770 g, pH=13). The reaction turnover is monitored by HPLC. The reaction mixture is neutralized with phosphoric acid (pH=7). MeOH (6 L) is removed by distillation at 690 C (distillation time = 16 hours at 20 690 C with a pause of 48 hours at rt.). The residue is then dried on standing at rt. at 1 atm, and is followed by vacuum drying in an oven (500 C, 100 Torr) for 2 hours. After mixing a fine powder is obtained with a total xanthophyll activity to be determined per kg (depending on the duration of the drying process and efficacy of the mixing). 25 Analysis: Can be performed with the following parameters: - Monitoring the transesterification: Chrompack chromsep 100*46 mm (L*ID) 15018 microspher C18 cat. no. 28076 30 flow 1 ml/min eluent: CH 2 Cl 2 / CH 3 CN 30 / 70 scanning at 450 mu - Measuring the total xanthophyll activity was performed by means of a spectrophotometer. 35 Experiment 3 Chromatography comparison of marigold meal with transesterified marigold meal to evaluate the free lutein WO 00/07458 PCT/US98/16393 The transesterification process described above in experiment one was employed on one of the marigold meal samples the other sample was not treated by the transesterification process. The two materials the plant material that had been treated and the untreated material were analyzed and the results are shown in Figure 1 and 3. Figure I shows the untreated marigold meal and Figure 3 shows the treated marigold 0 meal. Figure 3 as subjected to the following conditions: Column Pressure (PSI): 1866 Column Temperature (C): N/A Noise (microAU): 3e+O01 Drift (microAU/min): 3e+001. The untreated marigold meal had the following parameters and conditions: Column Pressure (PSI): 2391 Column Temperature (C): 5 N/A Noise (microAU): 4e+001 Drift (microAU/min): le+001 The graph in Figure 1 shows that the lutein of the marigold meals has a lot of ester activity in the 28-33 range and a lutein level in the 7-8 range. The transesterified marigold meal shows that the lutein peak is 0 still high and the esters in the 31-33 area no longer exist. The following data was gathered from the marigold meal and Figure 1. Component RT (min) Area Height Area % Peak Type 5 Unident0001 2.513 1110 194 0.05 Resolved Unident0002 4.220 4408 1331 0.19 Fused Unident0003 4.365 2763 670 0.12 Fused Unident0004 4.484 17210 2068 0.76 Fused CAPSANTHIN 6.602 8032 758 0.35 Fused 0 Unident0007 6.805 6003 891 0.26 Fused Unident0008 7.054 794979 90124 34.96 Fused LUTEIN K 1 7.862 85277 3973 3.75 Fused ZEAXANT'rHIN 8.110 123244 7773 5.42 Fused RUBIXANTHIN 8.797 15407 1288 0.68 Fused 5 CANTHAXANTHIN 9.900 0 0 0 NF CITRAXANTHIN 11.470 3141 223 0.14 Resolved B CRYPTOXANTHIN 12.626 8967 478 0.39 Resolved LYCOPENE 17.959 27086 689 1.19 Fused B CAROTENE 19.839 41511 2125 1.83 Fused 0 Unident0017 20.163 17368 1290 0.76 Fused WO 00/07458 PCT/US98/16393 5 Unident0018 21.777 12620 719 0.55 Fused Unident0019 22.138 6278 502 0.28 Fused Unident0020 28.596 16349 1191 0.72 Resolved Unident0021 30.268 92474 6473 4.07 Fused Unident0022 31.082 9061 802 0.40 Fused 0 Unident0023 31.445 2991 321 0.13 Fused Unident0024 31.816 228688 16904 10.06 Fused Unident0025 32.225 13487 1254 0.59 Fused UnidentOO26 32.604 45440 2610 2.00 Fused Unident0027 32.847 10055 1418 0.44 Fused 5 Unident0028 33.184 408509 31639 17.96 Fused Unident0029 33.564 31417 2652 1.38 Fused Unident0030 33.940 79024 4915 3.48 Fused Uident0031 34.533 161146 8099 7.09 Fused Totals 2274045 0) 193374 100.00 WO 00/07458 PCT/US98/16393 Component RT (min) Area egt Area% Peak Type Unident0002 4.328 57885 4754 0.54 Resolved CAPSANTHIN 6.454 17237 901 0.16 Fused Unident0004 7.130 94538 8757 0.88 Fused LUTEIN K1 7.459 7571877 942410 70.17 Fused ZEAXANTHIN 7.959 1247667 54701 11.56 Fused RUBIXANTHIN 8.634 1533918 92877 14.21 Fused CANTHAXANTHIN 9.311 72773 5608 0.67 Fused CITRAXANTHIN 11.976 51048 1889 0.47 Fused B CRYPTOXANTHIN 12.475 9046 880 0.08 Fused Unident0011 13.231 87645 3194 0.81 Fused LYCOPEEN 16.350 0 0 0 NF B CAROTEEN 19.830 28395 1187 0.26 Resolved Unident0014 20.776 5077 454 0.05 Resolved Unident0015 31.145 2061 238 0.02 Resolved Unident0016 32.573 3773 477 0.03 Resolved Unident0017 33.801 8411 754 0.08 Resolved Totals 10791351 1119081 100.00 WO 00/07458 PCT/US98/16393 Experiment 4 Chromatography comparison of paprika with transesterified paprika to evaluate the free capsanthin ) The transesterification process described above in experiment 3 was employed on one of the paprika samples the other sample was not treated by the transesterification process. The two materials of the plant material: 1. that had been treated and 2. the untreated material were analyzed and the results are shown in Figure 2 and 4. Figure 2 shows the untreated paprika and Figure 4 shows the treated paprika. Figure 4 was subjected to the following conditions and gave the following data. Transesterified paprika Acquisition Log Column Pressure (PSI): 1799 Column Temperature (C): N/A S Noise (microAU): 5e+001 Drift (microAU/min): le+002 Component RT (min) Area Heiut Area % Peak Type Unident0002 3.350 5402 305 0.46 Fused S Unident0003 4.212 75241 6091 6.47 Fused Unident0004 5.939 167992 10409 14.44 Fused Unident0005 6.189 293468 17946 25.22 Fused CAPSANTHIN 6.455 89525 8942 7.69 Fused Unident0007 6.640 93922 9488 8.07 Fused Unident0008 7.028 163104 13380 14.02 Fused LUTEIN Kl1 7.936 4794 380 0.41 Fused EAXANTHIN 8.088 17197 921 1.48 Fused RUBIXANTHIN 8.550 0 0 0 NF CANTHAXANTHIN 9.900 0 0 0 NF i CITRAXANTHIN 11.143 7630 481 0.66 Resolvcd B CRYPTOXANTHIN 12.759 100344 5656 8.62 Resolvcd LYCOPENE 16.350 0 0 0 NF Unident0016 18.783 129299 6123 11.11 Fused B CAROTENE 19.418 15731 1033 1.35 Fused WO 00/07458 PCT/US98/16393 5 Totals 1163652 81155 Figure 2 was subjected to the following conditions and gave the following data 10 Non-transesterified paprika Acquisition Log Colunm Pressure (PSI): 1810 Colunn Temperaturz N/A Noise (microAU): 3e+001 I Drift (microAU/min 15 Component RT (min) Area H eigh Peak Type Unident0001 4.198 12917 1238 20 Unident0002 4.466 31213 2498 Unident0003 6.086 36925 2042 Unident0004 6.192 43531 3157 CAPSANTHIN 6.640 26476 1523 Unident0006 6.919 13869 2208 25 Unident0007 7.025 47360 4396 LUTEIN KI 7.900 0 0 ZEAXANTHIN 8.000 0 0 RUBIXANTHIN 8.550 0 0 CANTHAXANTHIN 9.900 0 0 30 CITRAXANTHIN 11.000 0 0 B CRYPTOXANTHIN 12.792 48661 2698 Unident0014 13.559 21569 873 1 Unident0015 15.054 74757 2979 5t LYCOPENE 16.096 46150 1463 35 Unident0017 16.647 37968 1994 Unident0018 17.747 28329 1296 1 Unident0019 18.362 21985 1169 I ' B CAROTENE 18.876 183451 8834 I 4. Unident0021 19.526 21641 1410 I 40 Unident0022 20.283 6424 585 0 WO 00/07458 PCT/US98/16393 5 Unident0023 23.517 45665 2343 3.11 Fused Unident0024 24.462 20179 669 1.37 Fused Unident0025 25.368 126787 5500 8.63 Fused Unident0026 26.136 66975 2185 4.56 Fused Unident0027 27.122 145853 6723 9.93 Fused 10 Unident0028 27.882 70164 2310 4.78 Fused Unident0029 28.833 100228 4103 6.82 Fused Unident0030 29.594 40246 1614 2.74 Fused Unident0031 30.509 32888 1624 2.24 Fused Unident0032 30.732 35873 2197 2.44 Fused 15 Unident0033 31.248 18710 891 1.27 Fused Unident0034 31.833 19004 1321 1.29 Fused Unident0035 32.239 17231 1315 1.17 Fused Unident0036 32.814 4583 399 0.31 Fused Unident0037 33.190 17009 1279 1.16 Fused 20 Unident0038 33.604 5286 525 0.36 Fused Totals 1468907 75361 100.00 Clearly the untreated paprika has a large number of esters in the 23-25 area that are not present in the treated material. In both plant materials whether or not treated, the capsanthin retains the high peak in 25 the 6-7 range. Experiment 5 Chicken testing of the pigmentation effect of marigold meal processed in situ by transesterification 30 Young Warren SEX-SAL-LINK hens were divided into three identical groups. Each group exists of 7 cages which contain each 3 hens. The pullets have access to feed and water at all times. The three groups were given the carrier feed (pigment free food) for 3 weeks as a control for the experiment. 35 Group A was fed ORO Glor a commercially available free form of lutein available form Kemin Industries, Inc. Des Moines, IA. Group B will be fed the new invention made according to the process of experiment one (7.5mg of lutein activity per Kg of feed). Group C will be fed the new invention made according to the process of experiment one (15.0mg of lutein 40 activity per Kg of feed).

WO 00/07458 PCT/US98/16393 5 The experiment was run for 28 days. The feeding of the material requires 3 to 5 days before the effects on the yolk color are visible. Yolk color is uniform and consistent after approximately 21 days of feeding. ten (10) randomly chosen eggs laid by each group at day 0, 2, 4, 8, 16 and day 28 were broken and the egg yolk color was measured and the total amount of feed intake was determined on the 28th day. At days 0, 16 and day 28 the egg yolk of 3 randomly chosen eggs laid by each group had to be blended 0 and weighed, followed by extraction and analysis of the egg yolk from each group. The relationship between the egg yolk color and the mg xanthophylls/Kg of feed were calculated and the relationship between the amount of xanthophylls in the egg yolk and the xanthophylls fed. The result of this study are demonstrated in Figure 5, 6. Figure 5-shows the Roche fan scale over days for the egg yolk color of the present invention and of the conunercial product. Figure 6 shows the mg/Kg of xanthophylls 15 in the egg yolks over days of the three treatments. The results show that the plant material is providing more pigment and color at the 15 mg level then is the conunercial product. The 15mg level appears to be a more effective level then the 7.5 mg level. The lower level is providing less egg yolk color even though the egg appears to have more xanthophylls from the plant material in the egg yolk than the conunercial product. 20

Claims (26)

1. An improved plant material made from a natural plant material containing at least some non free form xanthophylls, comprising: 10 plant materials containing in situ less of said non free form xanthophylls, and more of free form xanthophylls wherein the free form of xanthophylls in situ in the improved plant material has increased beyond the amount in the natural plant material.

2. Improved plant material of claim one wherein the plant material is flowers. 15

3. Improved plant material of claim one wherein the plant material is petals.

4. Improved plant material of claim one wherein the flowers are Tagetes erecta. 20

5. Improved plant material of claim one wherein the xanthophyll is lutein.

6. Improved plant material of claim one wherein the non free form of xanthophyll is the fatty acid esters. 25

7. Improved plant material of claim six wherein the free form of Xanthophylls is nonacylated lutein.

8. Improved plant material of claim one wherein the said improved plant material contains at least 5% more free form xanthophllys then does the natural plant material in situ. 30

9. An improved animal feed composition comprising: vitamin and minerals; a source of carbohydrates; and, improved plant material containing in situ more free form xanthophylls then the natural in situ amount of xanthophylls in the plant material that the improved plant material 35 was formed from wherein the xanthophylls are more bioavailable to an animal fed the animal feed.

10. A feed according to claim nine wherein said improved plant material is flowers. 40

11. A feed according to claim ten wherein said flowers are marigolds. WO 00/07458 PCT/US98/16393 5

12. A feed according to claim nine wherein said animal feed is designed for the nutritional requirements of poultry.

13. A feed according to claim 12 wherein said poultry are chickens. 10

14. A feed according to claim 12 wherein said poultry fed said animal feed evidence said bioavailability tby, having increased pigmentation from the consumption of said free form xanthophylls.

15. A feed according to claim 9 wherein said xanthophylls are lutein. 15

16 A method of improved plant material made from a natural plant material containing at least some non free form xanthophylls, comprising the steps of: treating said natural plant material in situ with a solvent; adding a base capable of transesterification of non free form xanthophylls to the free 20 form xanthophylls; neutralize the reaction wherein forming the improved plant material having more free form xanthophylls then the natural plant material.

17. The method of claim 16 including the step of drying the improved plant materials to remove any solvent. 25

18. The method of claim 16 wherein the plant material is flowers.

19. The method of claim 16 wherein the flowers are Tagetes erecta. 30

20. The method of claim 16 wherein the solvent is an alcohol.

21. The method of claim 20 wherein the alcohol has one to four carbons.

22. The method of claim 16 wherein the solvent is an alcohol. 35

23. The method of claim 16 wherein the alcohol is selected form the group consisting of methanol, ethanol, isopropyl alcohol.

24. The method of claim 16 wherein the base is selected from the group consisting of, Potassium 40 hydroxide, Potassium sorbate, NaOMe, animal liver lipase, NaOEt, KOMe, KOEt, Na 2 CO 3 , K 2 CO3. WO 00/07458 PCT/US98/16393

25. The method of claim 16 wherein the step of neutralizing includes the use of a Lewis acid.

26. The method of claim 25 wherein the Lewis acid is Phosphoric acid. 10 I The method of claim 25 wherein the Lewis Acid is selected from the group consisting of HCL, NH 4 CL, sulfuric acid, acetic acid, ALCL 3