CN1242761A

CN1242761A - Improved process for recovering xanthophylls from corn gluten meal

Info

Publication number: CN1242761A
Application number: CN 97181210
Authority: CN
Inventors: H·S·穆拉利达拉; T·L·科尔尼埃勒
Original assignee: Cargill Inc
Current assignee: Cargill Inc
Priority date: 1996-11-29
Filing date: 1997-11-24
Publication date: 2000-01-26

Abstract

Improved methods for the recovery of xanthophylls from corn gluten meal are provided. More specifically, methods for the recovery of xanthophylls from corn gluten whereby relatively dry corn gluten is extracted with an organic alcohol followed by a saponification reaction to convert xanthophyll esters to xanthophylls is described. Also described are methods for the recovery of xanthophylls from corn gluten meal whereby relatively dry corn gluten meal is extracted with a first solvent containing an antioxidant and the resulting extract is, optionally, purified. Using these methods, xanthophylls can be recovered from corn gluten meal in higher yields, at greater efficiencies, and in higher purity as to compared to prior art methods. Moreover, the xanthophyll products recovered in these methods are ideally suited for use in food products and in pharmaceuticals. These materials are especially suited for use in poultry feeds to provide the desired, healthy yellow color or hue in broiler and egg yolks.

Description

From the hominy chop powder, reclaim the improved method of xenthophylls

Invention field

The present invention relates to from the hominy chop powder, reclaim the xenthophylls improved method of (having another name called carotol).Use method of the present invention, can obviously improve yield, organic efficiency and the quality of the xenthophylls of recovery.

Background of invention

Xenthophylls is the oxidized carotinoid compounds that exists in green plants (as corn, mary bush etc.) and some animals productss (the most famous is yolk).Xenthophylls is xanthein, can be used for animal and human's food and medicine.Xenthophylls in the natural feed source often is used to poultry (especially chicken) and egg is painted.The example in these natural feed sources comprises Semen Maydis, hominy chop powder, mary bush powder and seaweed powder.These materials that contain xenthophylls in joining the chicken feed after, can give yellow color or the yellow hue of roaster with required health.

Use the natural food source to provide xenthophylls may cause the inconsistent of result, it is variant that this situation mainly shows as the color depth of roaster.In many cases, these natural feed sources are having very big difference aspect bioavailability, lutein content and the xenthophylls stability of xenthophylls.These problems often cause feed material composition test (that is the amount of xenthophylls) poor with the dependency between the biological property (that is the colour developing of broil food).

Existing trial of from vegetable matter (as corn and mary bush powder), extracting xenthophylls.For example, russian patent application No.1,819,619 provide a kind of method of extracting flavonoid and carotenoid from the mary bush petal.Petal is pulverized, extract each 40 minutes with ethanol 7 times at 70 ℃ then.Boil off after the ethanol, extract is dissolved in the Viscotrol C.This material from the flavonoid of about 30 weight % of initial mary bush petal (for example contains usually, patuletin, patuletrin, quercetagetin, Tayettin, Xanthaurine) and the carotenoid (for example, xenthophylls, rubsanthin, helenine, carotene and violet sulphur matter) of about 15 weight %.This material is used to medical usage.The present most of xenthophylls Industrial products that use are from the mary bush petal, and are consequently, more expensive relatively.

Recently, the U.S. Patent No. 5,254,673 (on October 19th, 1993) of Cook etc. invention provides a kind of the hominy chop powder has been handled with the proteic method of purifying corn.The pigment that comprises xenthophylls produces as by product.Corn gluten moistening or that slightly do is carried out the hydrolysis of enzyme starch, alkaline purification and alcohol washing, separate with alcoholic extraction or with impurity elimination flavor and the seitan that discolors then.Existing report, pigment is recovered in the alcohol washing step, and this alcohol washing step is made up of the washing of carrying out with " successive reflux type or the mode of washing with faints in each step " in batches.Quality and quantity about the by product pigment that reclaims does not then describe in detail.

We have used hominy chop powder (moisture that contains about 12 weight %) to repeat the method for Cook etc. now.The xenthophylls that reclaims with described method such as Cook is usually as rubber or batter thickness.The xenthophylls that reclaims with method of the present invention then is stronger crystallization of yellow color or flour.And the yield of the xenthophylls that reclaims with the method for Cook etc. and efficient is low than with method recovery of the present invention significantly.The xenthophylls that reclaims with the method for Cook etc. must be further purified (yield can reduce thus) significantly so that it can use in food.And be not further purified also and can in food, use with the xenthophylls that method of the present invention makes.

High yield and to reclaim xenthophylls expeditiously will be desirable from natural source (as the hominy chop powder).From natural source (as the hominy chop powder), obtain being crystal or powdered xenthophylls will be ideal.Obtaining xenthophylls with the purity that is adapted at using in the food from natural source (as the hominy chop powder) will be ideal equally.Method of the present invention provides such xenthophylls.

Brief summary of the invention

The present invention relates to from the hominy chop powder, reclaim the improved method of xenthophylls.

In (" method I "), the present invention relates to a kind of like this method that from the hominy chop powder, reclaims xenthophylls in one aspect, in the method, will extract with organic alcohol, carry out saponification reaction then, convert lutein ester to xenthophylls than the hominy chop powder of doing.Use this method of the present invention, can high yield, efficient and purity from the hominy chop powder, reclaim xenthophylls than the method for prior art.And the xenthophylls that reclaims with method of the present invention is crystallization, powdery solid, is suitable for very much food and medicine.

In second aspect (" method II "), the present invention relates to a kind of like this method that from the hominy chop powder, reclaims xenthophylls, in the method, the hominy chop powder that will contain less than the moisture of about 30 weight % extracts with the organic solvent (or mixed solution of organic solvent) that has added antioxidant, be present in resulting that the thick xenthophylls of soluble fractions collects in the extract, can be purified to improve final xenthophylls degree of purity of production.Different with method I of the present invention is that method II of the present invention does not use saponification reaction.In addition, compare with method I of the present invention, method II of the present invention has used antioxidant in the solvent (or solvent liquid) for preparing thick xenthophylls being used for from the hominy chop powder extracting xenthophylls, is preferably, and the temperature of using among the method II is higher than the temperature of using among the method I.Use method II of the present invention, also can reclaim xenthophylls than other high yield, efficient and purity of method that from the hominy chop powder, reclaims xenthophylls.The xenthophylls that reclaims with method II of the present invention is bisque or red mashed prod, and it can be used for food and medicine.Method II ratio method I of the present invention is better, because the yield of its pure xenthophylls is higher usually.

An object of the present invention is, a kind of method that reclaims xenthophylls from the hominy chop powder is provided, this method comprises:

(1) the hominy chop powder is dried to water content less than about 12 weight %;

(2) the hominy chop powder of doing is extracted with first kind of alcohol;

(3) collect to obtain thick xenthophylls from the extract that contains thick xenthophylls of step (2);

(4) with alkaliferous second kind of alcohol thick xenthophylls is handled;

(5) from the thick xenthophylls of handling, remove second kind of alcohol, reclaim refining xenthophylls;

(6) collect refining xenthophylls (method I).

Be preferably, will make with extra care xenthophylls and be further purified with technology such as adsorption chromatography or ion exchange chromatographies.

Another object of the present invention is, a kind of method that reclaims xenthophylls from the hominy chop powder is provided, and this method comprises:

(1) the hominy chop powder of water content less than about 12 weight % extracted with first kind of alcohol;

(2) collect to obtain thick xenthophylls from the extract that contains thick xenthophylls of step (1);

(3) with alkaliferous second kind of alcohol thick xenthophylls is handled;

(4) from the thick xenthophylls of handling, remove second kind of alcohol, reclaim refining xenthophylls;

(5) collect refining xenthophylls (method I).

Another purpose of the present invention is, a kind of method that reclaims xenthophylls from the hominy chop powder is provided, and this method comprises:

(3) with alkaliferous second kind of alcohol thick xenthophylls is handled, converted all lutein esters to xenthophylls;

(5) will make with extra care the xenthophylls purifying with chromatography or ion exchange technique;

(6) collect the refining xenthophylls (method I) that purifying is crossed.

A further object of the present invention is, a kind of method that reclaims xenthophylls from the hominy chop powder is provided, and this method comprises:

(1) the hominy chop powder of water content less than about 30 weight % extracted with the organic solvent or the organic solvent mixed solution that contain antioxidant, the amount of contained antioxidant is less than about 0.5 weight % of the xenthophylls total amount in the hominy chop powder, and organic solvent or organic solvent mixed solution are at least about 2: 1 with the ratio of hominy chop powder;

(2) collection is extract obtained, obtains xenthophylls (method II).

Be preferably, gained xenthophylls is carried out purifying with technology such as positive adsorption chromatographies.

Of the present invention also have a purpose to be, a kind of method that reclaims xenthophylls from the hominy chop powder is provided, and this method comprises:

(2) collection is extract obtained, obtains thick xenthophylls;

(3) thick xenthophylls is carried out purifying;

(4) collect pure xenthophylls (method II).

Of the present invention have a purpose to be again, and a kind of method that reclaims xenthophylls from the hominy chop powder is provided, and this method comprises:

(1) with hominy chop powder samples dried to water content less than about 30 weight %;

(2) add antioxidant in organic solvent or organic solvent mixed solution, the amount of the antioxidant of adding is less than about 0.5 weight % of the xenthophylls total amount in the hominy chop powder sample;

(3) hominy chop powder sample is extracted with organic solvent or organic solvent mixed solution, extracting method is to mix about 10 minutes to 90 minutes at about 10 ℃ to 80 ℃ with 1 part of hominy chop powder at least about 2 parts organic solvent or organic solvent mixed solution;

(4) collection is extract obtained, obtains thick xenthophylls;

(5) thick xenthophylls is carried out purifying;

(6) collect pure xenthophylls (method II).

By the following accompanying drawing of the present invention and the explanation of better embodiment, it is clear that these and other objects and advantages of the present invention will become.

The simple declaration of drawing

Fig. 1 is the functional diagram of the better embodiment of method I of the present invention.

Fig. 2 is the efficient of extracting xenthophylls from the hominy chop powder and the function relation figure that extracts the water content in the used ethanol of the inventive method I;

Fig. 3 is the efficient of extracting xenthophylls from the hominy chop powder and extraction functional relationship of temperature figure of the inventive method I;

Fig. 4 is the efficient of extracting xenthophylls from the hominy chop powder of the inventive method I and function relation figure extraction time of 25 ℃;

Fig. 5 and 5A are the functional diagrams of two better embodiment of method II of the present invention.

The description of preferred embodiments

The present invention is by reclaiming the improved method of lutein from the corn bran powder.

Method I of the present invention provides a kind of like this method that reclaims lutein from the corn bran powder, and in the method, the corn bran powder that will do extracts with Organic Alcohol, then carries out saponification, converts lutein ester to lutein. Method II of the present invention provides a kind of like this method that reclaims lutein from the corn bran powder, in the method, the corn bran powder that to do extracts with the organic solvent that contains antioxidant, also optionally with gained lutein product purification to obtain pure lutein product. When using the positive adsorption charomatography as the purification process in the optional purification step, the protein that the collection extract should be obtained in " thick lutein " product is removed, and makes it convert " refining lutein " product to. Then, can with gained " refining lutein " product purification, obtain pure lutein product.

Use method I of the present invention and method II, can high yield, efficient and purity from the corn bran powder, reclaim lutein than the method for prior art. And the lutein that reclaims with method I of the present invention is crystallization, powdery solid form, is bisque or red pasty state form with the lutein of method II recovery of the present invention, and they all are suitable for food and medicine very much.

In the practice, the source thing that contains lutein that is used for method I of the present invention and method II is the corn bran powder. The corn bran powder is the elementary accessory substance of industrial corn wet grinding. Corn bran powder used among method I of the present invention and the method II should be done. " more dried " word refers in method I of the present invention, the water content of corn bran powder is less than about 12 % by weight, be more preferably less than about 10 % by weight, in method II of the present invention, the water content of corn bran powder is less than about 30 % by weight, be preferably less than about 20 % by weight, be more preferably less than 12 % by weight. If in method I of the present invention and method II, use wet corn bran powder (namely, in method I of the present invention, the water content of corn bran powder surpasses about 12 % by weight, in method II of the present invention, the water content of corn bran powder surpasses about 30 % by weight, or add the water of significant quantity in the corn bran powder), the obviously decline of efficient of then extracting lutein with these two methods. And, resulting lutein can be as rubber thickness. Available common method well known by persons skilled in the art is dried to required water content with the corn bran powder. Usually, every pound in the dry corn bran powder of crossing contains about 100-200mg lutein.

Reagent and equipment used among method I of the present invention and the method II all are commercially available. The supplier of these reagent and equipment comprises Aldrich Chemical company (Michigan State Milwaukee city), Fisher Scientific company (Pennsylvania Pittsburgh city), Glitsh company (Texas Houston city), Grown Iron Works company (Minnesota State Minneapolis city), Ace Glass company (New Jersey Vineland city), Millipore company (Massachusetts Bedford city) and PCI Membrane Systems company (Whitchurch city, Britain Hampshire prefecture). Method I

In first key step of the inventive method I, the corn bran powder that will do extracts with the alcohol that lutein can be slightly soluble in wherein. Suitable alcohol comprises methyl alcohol, ethanol, isopropyl alcohol and butanols. Be preferably, in solvent extraction method, use ethanol (industrial or anhydrous) and/or methyl alcohol. Be preferably, the water content of used alcohol is more preferably less than about 10 % by weight less than about 20 % by weight; When the water content of alcohol solvent surpasses about 20 % by weight, extraction efficiency can descend (referring to Fig. 2). Usually, the corn bran powder is about 1: 2 with the ratio that extracts with alcoholic solvent. Usually, the amount of solvent for use must be enough to form slip. Usually, being extracted in environment temperature (that is, about 20-30 ℃) carries out with solvent extraction techniques commonly used. Should avoid making temperature to surpass about 40 ℃, otherwise extraction efficiency is decline (referring to Fig. 3) sharply. Particularly preferred extractive technique comprises continuous extraction, counter-flow extraction method and flows extraction method, centrifugal extraction method etc. The extract (or the extract that merges) that will contain first lutein filters to remove insoluble matter, is preferably then desolventizing. Can use the technology that desolventizes commonly used such as the way of distillation, vacuum distillation method, spray-on process, rotary evaporation method etc. Desolventizing (if the words of carrying out) needn't be complete, because also use solvent in saponification step. In fact, desolventizing optional (although being better) before saponification step. Common every pound of lutein extract that should the stage at method I contains about 800-1200mg lutein. This shows, compares with initial corn bran powder, and enrichment factor is about 7-8.

In second key step of method I of the present invention, lutein extract and pure alkali are reacted in saponification, lutein ester being converted to free lutein, thereby improve the rate of recovery and the organic efficiency of lutein. It is conventional saponification that this step transforms. Although can use various pure alkali, usually preferred alcohol alkali. Usually, in whole process, preferably use with the first extraction step in used identical alcohol. Suitable alkali comprises the alkali hydroxide metal, such as lithium hydroxide, NaOH and potassium hydroxide. Potassium hydroxide-ethanol solution is most preferred saponification thing. Saponification was preferably carried out by the lutein extract is refluxed in pure alkali in about 1-2 hour. After washing, filter and desolventize with other alcohol (being preferably ethanol), purification and recover lutein.

The refining lutein that is obtained by method I of the present invention is flavous crystal powder. The powder of common every pound of extraction contains about 1600-2400mg lutein. This shows, compares with initial corn bran powder, and enrichment factor is about 10-15. Refining lutein can be used for various food materials, animal feed and medicine, perhaps, can and be preferably, and they are further purified. Can use purification technique commonly used. Particularly preferred purification technique comprises adsorption charomatography and ion-exchange chromatography. Use solvent liquid (being preferably hexane, acetone, toluene and ethanol), lutein can be adsorbed on silica gel and/or the siliceous earth column. Material by chromatographic column can be added to suitable going as in the bran powder of animal feed, also it can be given up; But preferably with solvent recovery and recycling. With another kind of solvent liquid (being preferably hexane, acetone and methyl alcohol) pure lutein is eluted from chromatographic column. Suitable chromatogram adsorbent comprises silica gel, magnesia, aluminium oxide, diatomite etc. Perhaps, available alcohols solvent is adsorbed on lutein on the ion exchange column, then carries out wash-out with the alcohols solvent that contains acetic acid. After common technology (such as distillation, vacuum distillation, rotary evaporation etc.) desolventizing, the yellow crystals powder common every pound be expected to contain about 10,000-20,000mg lutein. This shows, compares with initial corn bran powder, and enrichment factor is about 50-150. The lutein that this purifying can be crossed is used for various food (such as margarine), animal feed (such as poultry feed) and medicine as colouring agent or additive.

See now Fig. 1, shown in Figure 1 is a preferred approach implementing the inventive method I. In extraction unit 14, extract corn bran powder 10 with ethanol 12, then in unit 16, filter. Corn bran powder 10 must be done (that is, water content is preferably less than about 10 % by weight less than about 12 % by weight). Be preferably, extract 14 or condition near environment temperature (that is, about 25-35 ℃) under carry out. After extraction 14 and the filtration 16, collect the filtrate 24 and the merging that contain lutein. The solid of inherent filtration unit 16 and dry in unit 18 of can collecting obtains bran powder 20. Bran powder 20 can be used for animal feed etc. Optionally, the ethanol of self-desiccation unit 18 is circulated to extraction unit 14 or saponification unit 32 in the future.

Then in unit 26, remove the ethanol 28 in the filtrate 24 of merging. Desolventizing 26 can carry out with means (such as the way of distillation, vacuum distillation method, rotary evaporation method, falling-film evaporation etc.) commonly used. Optionally, the ethanol 28 of removing can be circulated in extraction unit 14 or the saponification unit 32. Remove after the ethanol, obtain thick lutein 30. Then in saponification unit 32, process thick lutein 30 with pure alkali 34 (being preferably the KOH ethanolic solution). The lutein ester that saponification 32 may be present in the thick lutein 28 converts free lutein to, thereby can improve overall recovery and the organic efficiency of lutein. After ethanol washing and filter 23 6, purification and recover lutein 38. Optionally, the ethanol 40 of removing can be circulated to extraction unit 14 or saponification unit 32 from washing and filtration step 36.

Refining lutein 38 can this form use, and also can be further purified. Usually, preferably will make with extra care lutein 38 is further purified. Therefore, usually preferably will make with extra care lutein 38 with the solvent that for example contains ethanol is further purified in chromatogram or cation exchange column 42. Lutein is adsorbed on the column material, then uses the second solvent elution. Then, remove the solvent in the lutein that elutes, obtain pure lutein 46. The material 48 that other can be adsorbed by chromatographic column (for example protein, starch and aliphatic acid) adds in the bran powder 30 as animal feed and goes, and perhaps can give up. Pure lutein product 46 is flavous crystal powders, can mix easily in the food (especially bird feed product).

The embodiment that provides method I of the present invention below is illustrating, but these embodiment are not the qualifications to method I of the present invention.In these embodiments, lutein content records with official method 970.64 (Associationof Official Analytical Chemists1990 the 15th edition).Embodiment 1

Present embodiment illustrates enforcement of the present invention shown in Figure 1.The hominy chop powder (100g, water content is less than 12 weight %) that drying is crossed and Denatured alcohol (200g contains the water of 0.4 weight %) mix in flask.The ratio of solvent/bran powder is about 2: 1.The hominy chop powder contains about 160mg xenthophylls for every pound.The vigorous stirring slurry mixture makes it keep suspended state about 30 minutes at about 35 ℃.Then this mixture is filtered, collect filtrate.Collect the dried bran powder of about 95g, it can be used as animal-feed.After with rotatory evaporator the ethanol in the filtrate being removed,, collect the 5g solid matter as extract.This solid matter (that is thick xenthophylls) that contains xenthophylls contains about 1150mg xenthophylls for every pound.This is equivalent to be recovered to about 58mg xenthophylls from every pound of initial hominy chop powder, and the rate of recovery is about 36%.

Then thick xenthophylls (5g) and about 5gKOH ethanolic soln (about 40gKOH is in about 100ml ethanol) were refluxed about 1 hour.Then with ethanol (50ml) washing reaction mixture and filtration.Remove ethanol in the filtrate with rotatory evaporator, obtain about 4g solid matter (that is, refining xenthophylls), this solid extract contains 2100mg xenthophylls for every pound approximately.This is equivalent to be recovered to about 76mg xenthophylls from every pound of initial hominy chop powder, and the rate of recovery is about 47%.

Should refining xenthophylls product be flavous crystal powder.It can be incorporated in food raw material and the animal-feed easily.Embodiment 2

Basically repeat the method for embodiment 1, different is that the water content in the ethanol used in extracting is for the first time changed; The amount of hominy chop powder initial substance is about 100g.Obtain following result.

The ratio of ethanol/water	Extract (g)	Lutein content (mg/ pound extract)
The ratio of ethanol/water	Extract (g)	Lutein content (mg/ pound extract)	????100∶0	????2.9	????1300
????95∶5	????6.65	????758	????100∶0	????2.9	????1300
????95∶5	????6.65	????758	????90∶10	????6.79	????420
????85∶15	????12.53	????404	????90∶10	????6.79	????420
????85∶15	????12.53	????404	????80∶20	????14.24	????396
????75∶25	????17.8	????279	????80∶20	????14.24	????396

This data plot is shown among Fig. 2.As shown in Figure 2, when water content surpassed about 20 weight %, extraction efficiency descended.Be preferably, the water content of solvent should be remained on less than about 10 weight %.Embodiment 3

Repeat the method for embodiment 1, different is, the temperature of the hominy chop powder that drying is crossed changes between about 20 ℃ to 50 ℃.The results are shown in Figure 3.As shown in Figure 3, when temperature more than 40 ℃ the time, extraction efficiency obviously descends.Be preferably, should be maintained at about between 20 ℃ to 30 ℃ to obtain maximum extraction efficiency extracting temperature.Embodiment 4

Repeat the method for embodiment 1, different is, the extraction time of the hominy chop powder that drying is crossed changed between about 0.25-3 hour.The results are shown in Figure 4.As shown in Figure 4, when extraction time was 30 minutes, it is maximum that extraction efficiency reaches, and surpass about 2 hours when extraction time, and then extraction efficiency is on a declining curve.Therefore, they can not significantly improve total recovery or efficient usually though used extraction time can be grown (that is, about more than 2 hours).Embodiment 5

Present embodiment only is used for the purpose of comparison, institute is illustrational be Cook etc. in U.S. Patent No. 5,254, the method that from the hominy chop powder, reclaims xenthophylls described in 673.With the embodiment 1 of Cook etc. as guiding.With deionized water (510ml) and hominy chop powder (120g is with used identical among the embodiment 1) preparation slip.With this slip be heated to 80 ℃ and with 3N sodium hydroxide with pH regulator to about 6.4.Stir down and in slip, add α-Dian Fenmei (0.5ml).Continue digestion 1 hour, slip is cooled to 50 ℃.With centrifugal 10 minutes of slip, remove supernatant liquor with 1200 * g.In 80 ℃ of water of equal portions, carry out centrifugal with 1200 * g again solid suspension., the solid that removes destarching is handled pH regulator to 9 with 1M yellow soda ash and 1M sodium bicarbonate at 20 ℃.The slip of gained was heated 1 hour at 80 ℃, be cooled to 50 ℃ then.With centrifugal 20 minutes of slip, remove supernatant liquor with 1200 * g.Solid suspension in the water of equal portions, is neutralized with 3N hydrochloric acid.The water of using equal portions again with 1200 * g with the centrifugal secondary of solid.Then the ethanol (4 weight % water) of this solid with 2 deals was extracted 30 minutes and filtered in room temperature.Remove by rotary evaporation and to desolvate.The solid that reclaims is xanchromatic pasty state or rubber-like substance, and every pound contains 141mg xenthophylls approximately.Thus, the concentration basic identical (that is every pound of hominy chop powder 160mg xenthophylls) in the concentration of xenthophylls and the initial hominy chop powder in the extract; In other words, the bran powder of Cook etc. does not produce the spissated effect of the xenthophylls that makes in the extract.The total yield of xenthophylls is about 22%.Present embodiment is repeated, obtained essentially identical result.Because extract is lower than thickness and pigment content, therefore, this extract that contains xenthophylls can not be advantageously used in comprising the foodstuffs compositions of bird feed.Method II

Method II of the present invention uses organic solvent (or mixed solution of organic solvent) the extraction water content to contain antioxidant (to be preferably less than about 20 weight % less than about 30 weight %, be more preferably less than about 12 weight %, preferably less than 10 weight %) the general step of hominy chop powder, obtain " thick xenthophylls ", then, be preferably,, obtain pure xenthophylls " thick xenthophylls " purifying.

When " thick xenthophylls " that purifying is obtained by extract and when used purification process is positive adsorption chromatography (it is preferred purification process), should before this purification step, carry out from the extract that contains " slightly xenthophylls ", removing proteinic additional step.This removes proteinic step can adopt the precipitator method, membrane filtration or other same class methods well known by persons skilled in the art.Then can be, and pure xenthophylls product collection got up with " refining xenthophylls " purifying, the collection method that is adopted can with herein with regard to the method II that uses other method of purification and described mode is identical.This is removed proteinic step and should only adopt the positive adsorption chromatography to come " thick xenthophylls " time contained in the purified extract to carry out, and need not coming " thick xenthophylls " time contained in the purified extract to carry out with other method of purification.(1) extracts

Make hominy chop powder sample, if the water content of hominy chop powder greater than about 30 weight %, is preferably, to be dried to less than 20 weight % with regard to the described identical mode of method I of the present invention with the front, be more preferably less than about 12 weight %, preferably less than about 10 weight %.

Add antioxidant then in the mixed solution of organic solvent or organic solvent, its addition is preferably less than about 0.1 weight % less than about 0.5 weight % of the total xenthophylls in the hominy chop powder that is extracted.Total xenthophylls in the hominy chop powder that is extracted can record with foregoing official method 970.64 or following modification method.

Antioxidant can reduce the oxidative degradation of xenthophylls and can make and is extracted in higher temperature (between about 10-80 ℃, being preferably between about 50-70 ℃ preferably about 50 ℃ usually) and carries out.Add antioxidant and improve the extraction temperature and be proved to be and the xenthophylls yield in the final extract can be increased to 86%.

Be preferably, use feed grade or food grade antioxidants, as Santoflex (6-oxyethyl group-1,2-dihydro-2,2,4-trimethylquinoline), propyl gallate, butylated hydroxy anisole (BHA), Yoshinox BHT, Tert. Butyl Hydroquinone, tocopherol etc.

Identical with method I of the present invention, suitable solvent comprises methyl alcohol, ethanol, propyl alcohol, butanols and octanol.Preferred alcohol or ethanol-hexane mixed solution.In addition, identical with method I of the present invention, be preferably, the water content of solvent (with the every other reagent that uses among the inventive method II) is less than about 20 weight %, extraction efficiency is more preferably less than about 10 weight %, because when the water content of solvent surpassed about 20 weight %, can descend.Usually, the amount of solvent must be enough to form slip.

Then, (2: 1,3: 1,4: 1,5: 1,6: 1 etc.) extract the dry hominy chop powder of crossing to be at least about 2: 1 with the organic solvent that contains antioxidant (or organic solvent mixed solution) with the weight ratio of solvent and hominy chop powder, usually the method that is adopted is for example under vigorous stirring hominy chop powder and the solvent that contains antioxidant to be mixed between 10-80 ℃ about 10-90 minute.Usually, the amount of organic solvent should be enough to form slip, and therefore, organic solvent should be not less than about 2: 1 with the ratio of hominy chop powder.The hominy chop powder can and flow extraction method, continuous extraction, centrifugal extraction method and additive method well known by persons skilled in the art and extract with the countercurrent extraction method.In method II of the present invention, most preferred extracting method is the countercurrent extraction method.

With I in the method for the present invention should avoid temperature surpass about 40 ℃ different be that in the extraction step of method II of the present invention, used temperature is preferably in about more than 49 ℃.

Xenthophylls extract in this stage of the inventive method II (not carrying out purifying) contains about 500-2 for every pound usually, 000mg xenthophylls.This shows, compares with initial hominy chop powder, and enrichment factor is about 5-20.

Shown in the following examples 1, extract at 50 ℃ with the ratio use industrial ethyl alcohol of 3: 1 solvents with the hominy chop powder, the resulting dry extract of crossing contains 799mg xenthophylls (not purifying) for every pound.

Shown in the following examples 2, the extract that the drying that obtains with hexane/alcohol mixeding liquids of 20: 80 is crossed contains 1140mg xenthophylls (not purifying) for every pound.

Shown in the following examples 4, the purifying and the dry extract of crossing that obtain with hexane/alcohol mixeding liquids of 40: 60 contain 1410mg xenthophylls (not purifying) for every pound.

Extracting an available step carries out, perhaps, can be same with the following examples 5, the extracting method that is adopted is, the extraction that is divided into 2 parts, 3 parts, 4 parts or many parts by a series of organic solvents (or organic solvent mixed solution) independent, that contain antioxidant is carried out.Under latter event, the hominy chop powder can extract respectively with each part in those parts solvent, and its mode is just carried out as such being extracted in only.After these independent extractions are carried out respectively, merge the extract that contains xenthophylls.Extracting method is preferably, is undertaken by a series of independent extractions, because this method is similar to countercurrent extraction, can obtain the yield of higher thick xenthophylls.

After mixing finishes, collection contains the extract (or by with the merging thing that is divided into the extract that a series of independent extraction that many parts solvent that contains antioxidant or solvent liquid carries out obtains) of xenthophylls, used method for example is, extract is filtered, from extract, remove insoluble substance, obtain " thick xenthophylls ".Perhaps, available centrifuging, hydraulic cyclone method or other similar approach well known by persons skilled in the art are carried out.Resulting extract contains " thick xenthophylls ", and it can this form be used for various foodstuff raw materials, animal-feed and medicine, also can and be preferably to be purified.

Shown in embodiment 6, the method for available for example rotary evaporation is the extract drying, and then, before purifying, the extract that drying is crossed extracts one or repeatedly again with identical or different organic solvents (or organic solvent mixed solution).(2) purifying (a) removes deproteinize

Removing proteinic operation from the extract that contains " thick xenthophylls " should be only carry out when the method for " thick xenthophylls " product that uses the positive adsorption chromatography to be obtained by extraction as purifying.The preferred method of this xenthophylls product that to be purifying obtained by extraction.Spendable other purification process (as distributing (anti-phase) adsorption chromatography, ion exchange chromatography, size exclusion chromatography and counter current chromatography) then do not need this step.

Protein accounts for the major portion that contains " thick xenthophylls " hominy chop powder extracts.Before carrying out purifying with normal phase chromatography, remove organic solvent with employing technology well known by persons skilled in the art (as distillation method, vacuum distillation method, spray method, rotary evaporation method etc.), extract is dissolved in the non-polar solvent (as hexane).Because protein is insoluble to hexane, it may block chromatographic column, thereby disturbs the xenthophylls purifying.

Can use the method for the multiple isolating protein that comprises the precipitator method and membrane filtration.

The method of precipitating proteins can be, adds reagent such as ammonium sulfate in the extract, and protein is forced out from solution.For example, add the solvent (as acetone, ethyl acetate etc.) that protein is insoluble to wherein and protein can be forced its precipitation of sening as an envoy to.It is optional that extract is concentrated (that is, removing the part organic solvent before with protein precipitation), but can do.Remove by the throw out of mainly forming that for example filters or additive method well known by persons skilled in the art (as centrifuging or hydraulic cyclone method) will form then by protein.The extract of the soluble fractions of gained contains " refining xenthophylls ".Can use common technology well known by persons skilled in the art (as distillation method, vacuum distillation method, spray method, rotary evaporation method etc.) with the filtrate drying.Yet, before purification step that the filtrate drying is optional.

Also available film is with other component separating in protein and the extract.This proteinic molecular weight ranges that records with gel electrophoresis is 20, and 000-25 is between 000 dalton.Can be used on the film market of the bigger component separating of composition that xenthophylls (molecular weight is 568.85) equimolecular quantity is lower and protein equimolecular quantity on sale.Manufacturer production weight shutoff values such as Millepore company and PCI Membrane Systems company are the film below 20,000.In such system, sent the film system with pump with organic solvent, protein remains is stayed in the concentrated solution, and xenthophylls and triglyceride level and lipid acid then will enter in the penetrating fluid.With habitual vaporizing extract process organic solvent is removed from penetrating fluid then, penetrating fluid is separated with the positive adsorption chromatography.(b) purifying

Then, can be with contained in the aforementioned extract " thick xenthophylls " (during the purification process purifying beyond using the positive adsorption chromatography " thick xenthophylls " product) or aforementioned " refining xenthophylls " product of obtaining after deproteinize step when " thick xenthophylls " product (in using positive adsorption chromatography purified extract contained) purifying that removes.

" refining xenthophylls " available positive adsorption chromatography purifying, and method known to those skilled in the art polarity purifying such as " thick xenthophylls " available allocation (anti-phase) adsorption chromatography, ion exchange chromatography, size exclusion chromatography, counter current chromatography.

The positive adsorption chromatography has description: K.Robards etc. in following document, Principles and Practiceof Mordern Chromatographic Methods, and Academic Press publishes (1995); R.P.W.Scott, Techniques and Practices of Chromatography, Marcel Dekker publishes, New York (1995); L.R.Snyder etc., Introduction to Mordern LiquidChromatography (the 2nd edition), John Wiley and Sons company publishes, New York (1979); And K.K.Unger, Packing and Stationary Phases in Chromatographic Techniques, Marcel Dekker company publishes, New York (1990).

Distribute (anti-phase) chromatography that description: El Rassi etc. are arranged in following document, " Reversed Phase andHydrophobic Interaction Chromatography of Peptides and Proteins ", in SeparationProcesses in Biotechnology, Marcel Dekker company publishes (1990); And K.K.Unger, Packing and Stationary Phases in Chromatographic Techniques, Marcel Dekker company publishes, New York (1990).

Ion exchange chromatography has description in following document: Charles D.Shuey, " Ion ExchangeProcesses ", in Separation Processes in Biotechnology, Marcel Dekker company publishes (1990); H.Small, Ion Chromatography, Plenum Press publishes (1989); K.K.Unger, Packing and Siationary Phases in Chromatographic Techniques, Marcel Dekker company publishes, New York (1990); With Phillip C.Wankat, " Large Scale Chromatography ", at Handbook of Separation Process Technology, John Wiley and Sons company publishes, NwYork (1987).

Size exclusion chromatography is at K.K.Unger, Packing and Stationary Phases inChromatographic Techniques, and Marcel Dekker company publishes, and New York has description in (1990).

Counter current chromatography is at Walter D.Conway etc., and Modern Countercurrent Chromatography, American Chemical Society publish, Wshington, and D.C. has description in (1995).

Above-mentioned full content about chromatographic document is all incorporated by reference herein.

For for the positive adsorption chromatography of preferred purification process, spendable suitable adsorbent (stationary phase) comprises silica gel, magnesium oxide, aluminum oxide, Mierocrystalline cellulose, sucrose, starch, calcium oxide, calcium phosphate, lime carbonate, salt of wormwood, yellow soda ash etc., also can use the mixture of these sorbent materials, promptly be a preferable sorbent material as the mixture of silica gel and aluminum oxide.

Successfully to be low to moderate 2: 1 and high sorbent material to 100: 1 has carried out purifying to the dry weight comparison " refining xenthophylls " of " refining xenthophylls ".Therefore, such sorbent material can be between about 2: 1 to 100: 1 to the dry weight ratio of " refining xenthophylls ".Be preferably, sorbent material is 2: 1 to the dry weight ratio of " refining xenthophylls ".

" refining xenthophylls " is adsorbed on the stationary phase.Optionally, can before being adsorbed to the post bed, they be added in the eluting solvent (mobile phase) that is dissolved in a small amount of.Mixed solution with organic solvent or two kinds or multiple organic solvent carries out wash-out to chromatographic column then, elutes from post with the xenthophylls product that purifying is crossed.

Can use equal solvent (that is, in whole elution process, using same solvent or solvent liquid) elution chromatography post, also can use gradient eluent or stepwise gradient elutriant (that is, solvent or solvent fluid component change) in elution process.

When using the equal solvent system, with organic solvent or organic solvent mixed solution elution chromatography post.According to the solvent that uses, non-polar solvent or solvent liquid (as hexane, hexanaphthene, heptane, sherwood oil etc.) can account for 100% to 50% of solvent or solvent liquid.But can adjust by bigger solvent or solvent liquid (as ethyl acetate, acetone, Virahol, ethanol, methyl alcohol, butanols etc.) with the polarity of 0-50%.May need the mobile phase of 5-200 column volume to come the elution chromatography post, appropriate vol can be determined by those skilled in the art.

The flow point that collection elutes from chromatographic column.The flow point that contains triglyceride level and lipid acid can be added back in the hominy chop powder that extracted and go.And lutein ester flow point and free lutein flow point can be merged, perhaps saponification merges with the free lutein flow point then.The free lutein flow point can be used as the poultry feed enriching substance, perhaps can be further purified.If collect the protein flow point, then also it can be added back in the hominy chop powder that extracted and go.

Perhaps, available gradient or stepwise gradient solvent systems elution chromatography post.After " refining xenthophylls " is adsorbed on stationary phase, with organic solvent or organic solvent mixed solution elution chromatography post.The component of mobile phase can be along with the time continuously or stepwise ground change, mobile phase polarity is increased.For example, initial mobile phase can be 100% to 50% non-polar solvent or solvent liquid (comprising hexane, hexanaphthene, heptane, sherwood oil equal solvent).Use bigger solvent or the solvent liquid (comprising ethyl acetate, acetone, Virahol, ethanol, methyl alcohol, butanols etc.) of polarity of 0-50% to adjust then.Increase the bigger solvent of polarity or the per-cent of solvent liquid with mode well known by persons skilled in the art with a given speed or with stepwise then.The solvent that can one or more polarity are bigger or the per-cent of solvent liquid are increased to and account for 0% or 100% of mobile phase, perhaps any numerical value therebetween.May need 5-200 mobile phase doubly to come the elution chromatography post, appropriate vol can be determined by those skilled in the art.

For example, available volume is adsorbed on " refining xenthophylls " mixture on the silicagel column than the hexane/acetone that is 97/3.With this solvent liquid triglyceride level, carotene and lutein ester are eluted from chromatographic column, but free " refining xenthophylls " still is adsorbed on the chromatographic column.The xenthophylls that the solvent liquid that available polarity is bigger (as the solvent liquid of hexane/acetone volume ratio between 94/6 to 0/100) is crossed purifying elutes from chromatographic column.

Available then aforesaid and/or well known by persons skilled in the art any method is then calculated the concentration of the pure xenthophylls in the exsiccant flow point with the improved method of aforementioned official method 970.64 or this method described below with pure free lutein flow point drying.

No matter which kind of purification process what use is, available chromatography is made further purifying, to improve final pure xenthophylls degree of purity of production.The employed chromatographic column of this additional chromatography can comprise positive sorbent material as previously described, anti-phase sorbent material is as bonding sorbent material octadecylsilane (C18), octyl group silane (C8), butyl silane (C4), cyano group propyl silane (CN), aminopropyl silane (NH2) etc.Can comprise water, methyl alcohol, acetonitrile, acetone, ethanol, Virahol, ethyl acetate, hexane etc. with the solvent that anti-phase sorbent material uses.Also but other chromatographys (as ion exchange chromatography, size exclusion chromatography, partition chromatography and counter current chromatography) are to improve the xenthophylls degree of purity of production.

The pure xenthophylls product that is obtained by method II of the present invention is orange or red mashed prod.It is about 5 that common every pound of pure product is expected to contain, 000-60,000mg xenthophylls.This shows, compares with the xenthophylls concentration (every pound of about 100-200mg xenthophylls of bran powder) in the initial hominy chop powder, and enrichment factor is up to about 600.Can use this pure xenthophylls product with regard to the described mode of method I of the present invention in the front.

See Fig. 5 now, Fig. 5 is the functional diagram of the better embodiment of method II of the present invention.

In extraction unit 214, use the ethanol 212 of the antioxidant Santoflex of the about 0.1 weight % that contains the xenthophylls total amount that is equivalent in the hominy chop powder to extract hominy chop powder 210.Collect extract obtainedly then, in filter unit 216, elimination insolubles 215 from this extract obtains filtrate 224.Hominy chop powder 210 should be done (that is, water content is preferably less than about 30 weight %, less than about 20 weight %, is more preferably, less than about 12 weight %, preferably less than about 10 weight %).Be preferably, extract 214 and carry out at about 50 ℃.

Thick xenthophylls in the filtrate 224 can directly use, and perhaps can be further purified.Usually, thick xenthophylls should be further purified 246.Usually should thick xenthophylls be further purified in chromatographic column with for example containing the alcoholic acid solvent, to obtain pure xenthophylls 250.Xenthophylls is adsorbed onto on the sorbent material, carries out wash-out with second solvent then, obtain pure xenthophylls 250.The material 248 that other can be adsorbed by chromatographic column (as protein, starch and lipid acid) adds in the bran powder as animal-feed and goes, and also it can be given up.Pure xenthophylls product 250 is orange or red mashed prod, can mix easily in the foodstuff products (especially poultry feed product).For example, it can be dissolved in solid or the liquid vehicle (as water), be spray dried to product and mix with method known to those skilled in the art.

Present 5A with the aid of pictures, Fig. 5 A are the functional diagrams of another better embodiment of method II of the present invention.

In extraction unit 114, use the ethanol 112 of the antioxidant Santoflex of the about 0.1 weight % that contains the xenthophylls total amount that is equivalent in the hominy chop powder to extract hominy chop powder 110.Collect then extract obtained, in filter unit 116, elimination insolubles 115 from this extract.Just as described in Fig. 5, hominy chop powder 110 should be done as the front.Be preferably, extract 114 and carry out at about 50 ℃.

In said extracted with after filtering, be preferably, then, in unit 126, the ethanol in the filtrate 124 128 is removed.Desolventize and to carry out with regard to the described mode of the inventive method I the front.Remove after the ethanol 128, contained the extract 130 of " thick xenthophylls ".

Thick xenthophylls can directly use, and perhaps can be further purified.Usually, thick xenthophylls 130 should be further purified.Therefore, should thick xenthophylls be further purified in chromatographic column with for example containing the alcoholic acid solvent usually.Xenthophylls is adsorbed onto on the sorbent material, carries out wash-out with second solvent then.Remove the solvent in the xenthophylls that elutes then, obtain pure xenthophylls.The material that other can be adsorbed by chromatographic column (as protein, starch and lipid acid) adds in the bran powder as animal-feed and goes, and also it can be given up.

Then, be preferably, hold in the unit 132 extract that will contain " thick xenthophylls " and be reduced to total solid concentration between about 10-40 weight % subtracting with rotatory evaporator or other similar devices.

Then, should be that 1/1 weight ratio joins second organic solvent (it is ethyl acetate 134 preferably) in the extract that contains " thick xenthophylls " with solvent/extract.Be preferably, under vigorous stirring, with extract 25 ℃ with 30 seconds of solvent.

In filter unit 136, remove the throw out of mainly forming 140 that forms in the superincumbent step then by protein.The soluble fractions of residual extract contains " refining xenthophylls " 138.

Then, should in rotary evaporation unit 142, from the soluble fractions that contains the extract made from extra care xenthophylls 138, remove solvent ethyl acetate 144.

Silica gel/magnesium oxide with 1/1 is as sorbent material, with hexane as organic solvent, refining xenthophylls 138 is adsorbed on the chromatographic column, with ethanol as second solvent, pure refining xenthophylls 150 is eluted from chromatographic column, will make with extra care xenthophylls 138 by the positive adsorption chromatography thus and be further purified.The material 148 that other can be adsorbed by chromatographic column (as protein, starch, lipid acid and triglyceride level) adds in the animal-feed, also it can be given up.Pure refining xenthophylls product 150 is orange or red mashed prod, can mix easily in the foodstuff products (especially poultry feed product) with regard to the described mode of Fig. 5 the front.

Providing the following examples is to illustrate rather than limit method II of the present invention for example.The same with regard to the embodiment that method I of the present invention provides with the front, the lutein content in the different step described in the embodiment of the inventive method II all records with aforementioned official method 970.64.Yet, in the embodiment of method II of the present invention, with the dehydrated alcohol in VWR methylated spirits (Fisher Scientific company products catalogue VW0475-3 number) the replacement extraction agent.This prescription (below will describe) is to determine by comparative studies, so that equal result to be provided.

Prescription

SDI alcohol 100 volumes

Methyl iso-butyl ketone (MIBK) 1 volume

Ethyl acetate 1 volume

Varsol 1 volume

In addition, the hexane/acetone of the hexane/acetone replacement 96/4 with 97/3 can make top xenthophylls band (monohydroxy pigment) better separate as the elutriant of carotene because find 97/3 hexane/acetone.At last, before the optical density of the aliquots containig of the xenthophylls solution that produces reading, carry out this five equilibrium sample centrifugal.Have found that cleansing soln can improve the precision of analysis.Embodiment 1

Stir down, contain less than the water of 12 weight % and contain the hominy chop powder (100g) 30 minutes of 170mg xenthophylls at 50 ℃ of extraction using alcohols that contain the antioxidant Santoflex with 300g.Reclaim the hominy chop powder by vacuum filtration, extract again 2 times with same way as then.0.1% of xenthophylls amount in the hominy chop powder that the total amount of used Santoflex is equivalent to record with aforementioned official method 970.64.Three parts of extracts are merged, carry out drying by rotary evaporation.Xenthophylls concentration in the dry extract that records with aforementioned official method 970.64 is 799mg/ pound (not being further purified), compares with the amount of xenthophylls in the hominy chop powder of initial (initial), and concentration has improved about 5 times.Embodiment 2

Stir down, contain less than the water of 12 weight % and contain the hominy chop powder (100g) 30 minutes of 170mg xenthophylls at 60 ℃ of hexane/extraction using alcohols of 20/80 that contain the antioxidant Santoflex with 200g.Reclaim the hominy chop powder by vacuum filtration, extract again 2 times with same way as then.0.1% of xenthophylls amount in the hominy chop powder that the total amount of used Santoflex is equivalent to record with aforementioned official method 970.64.Three parts of extracts are merged, carry out drying by rotary evaporation.Xenthophylls concentration in the dry extract that records with aforementioned official method 970.64 is 1140mg/ pound (not being further purified), compares with the amount of xenthophylls in the hominy chop powder of initial (initial), and concentration has improved about 7 times.Embodiment 3

Stir down, contain the water of 20 weight % and contain the hominy chop powder (100g) 30 minutes of 170mg xenthophylls at 60 ℃ of extraction using alcohols that contain the antioxidant Santoflex with 300g.Reclaim the hominy chop powder by vacuum filtration, extract again 2 times with same way as then.0.1% of xenthophylls amount in the hominy chop powder that the total amount of used Santoflex is equivalent to record with aforementioned official method 970.64.Three parts of extracts are merged, carry out drying by rotary evaporation.Xenthophylls concentration in the dry extract that records with aforementioned official method 970.64 is 549mg/ pound (not being further purified), compares with the amount of xenthophylls in the hominy chop powder of initial (initial), and concentration has improved about 3 times.Embodiment 4

Stir down, contain less than the water of 12 weight % and contain the hominy chop powder (100g) 30 minutes of 170mg xenthophylls at 60 ℃ of hexane/extraction using alcohols of 40/60 that contain the antioxidant Santoflex with 200g.Reclaim the hominy chop powder by vacuum filtration, extract again 2 times with same way as then.0.1% of xenthophylls amount in the hominy chop powder that the total amount of used Santoflex is equivalent to record with aforementioned official method 970.64.Three parts of extracts are merged, carry out drying by rotary evaporation.Xenthophylls concentration in the dry extract that records with aforementioned official method 970.64 is 1410mg/ pound (not being further purified), compares with the amount of xenthophylls in the hominy chop powder of initial (initial), and concentration has improved about 8 times.Embodiment 5 extracts

100g is done hominy chop powder (containing the hominy chop powder less than 12 weight % water) to take by weighing in the extraction vessel.

Calculate the xenthophylls total amount of doing in the hominy chop powder (in g) with aforementioned official method 970.64 then.Because the xenthophylls concentration of hominy chop powder is every pound of 170mg, therefore, 100g hominy chop powder contains 37.4mg xenthophylls.The amount that joins the antioxidant Santoflex in the solvent for use (ethanol) is 0.1% of 37.4mg, i.e. 37.4 μ g.

In solvent/hominy chop powder is 3/1 extraction in batches, antioxidant is distributed in 3 parts of 300g 96% ethanol (0.4 weight % water).

Have in the extraction vessel of dried hominy chop powder in first part of 300g ethanol is poured into, extracted 30 minutes at 50 ℃ under the vigorous stirring.Reclaim extract by vacuum filtration.

The hominy chop powder is turned back in the extraction vessel.Second part of ethanol is poured over above the hominy chop powder, carries out similar 30 minutes of the second time at 50 ℃ and extract.By vacuum filtration extract is reclaimed again.

The hominy chop powder turned back in the extraction vessel goes, in a similar fashion with the 3rd and last a ethanol extract.Extract and under agitation carried out 30 minutes in 50 ℃.Reclaim last extract by vacuum filtration.

Above-mentioned three parts of extracts are merged.The heavy 713.4g of extract after the merging contains 16.81g total solids (solid content is 2.36 weight %).(the extract sample that 10.0g is merged carries out drying by rotary evaporation, records heavy 0.236g.According to this value, record the total solids in the extract of merging).Recording gained dry weight " thick xenthophylls " concentration with aforementioned official method 970.64 is that every pound of extract contains 799mg (before the purifying).Isolating protein

Remove part ethanol by rotary evaporation, like this, the weight of extract reduces to 156.23g from 713.74g.At this moment, the solid content of enriched material is 10.8 weight %.

Be that 2/1 ratio adds the ethyl acetate that contains 0.004% water with the weight ratio of solvent and enriched material in enriched material, that is, the ethyl acetate of 312.5g joined in the enriched material.So, form by the light yellow precipitate of mainly forming by protein.Then, by the vacuum filtration disgorging.

Remove from filtrate by rotary evaporation and to desolvate.Recording the dried concentration that leaches thing every pound contained " refining xenthophylls " with aforementioned official method 970.64 is 2150mg.The positive adsorption chromatography

At little glass column (1: 1 the silica gel of 12g 60 (Devisil633 level 60A type) of packing in the 30mm internal diameter * 60mm): magnesium oxide (the hyflo super cel that Fisher Scientific company produces).

To make with extra care xenthophylls (that is, the dried thing that leaches) and be dissolved in the 175g hexane (not moisture), be adsorbed on the chromatographic column then.

Carry out wash-out with 79g hexane/acetone (97: 3v/v contains 0.02 weight % water), use 100g acetone (containing 0.5 weight % water) to carry out wash-out then.

To collect from 3 parts of flow points that chromatographic column elutes: (1) hexane flow point; (2) 97: 3 hexane/acetone flow points; (3) acetone flow point.

Each flow point is carried out drying by rotary evaporation.The dry weight that records the hexane flow point is 3.53g, and the xenthophylls concentration of the every pound of solid flow point that records with aforementioned official method 970.64 is 205mg.The dry weight that records 97: 3 hexane/acetone flow points is 0.85g, and the xenthophylls concentration of the every pound of solid flow point that records with aforementioned official method 970.64 is 820mg.These two the main oil-containings of flow point, lipid acid, carotene and lutein esters can be added back to going as in the bran powder of animal-feed of recovery, also it can be given up.(that is, the acetone flow point) dry weight is 1.32g to last flow point, and the pure xenthophylls concentration of the every pound of solid flow point that records with aforementioned official method 970.64 is 7430mg, and the solid flow point is orange/red pasty state.

Thus, after aforementioned process was finished, the xenthophylls concentration in the hominy chop powder had improved about 44 times from every pound of 7430mg that every pound of 170mg of original corn bran powder brings up to the dry extract that purifying crosses.Embodiment 6

Stir down, contain less than the water of 12 weight % and contain the hominy chop powder (300g) 30 minutes of 170mg xenthophylls at 60 ℃ of extraction using alcohols that contain the antioxidant Santoflex with 900g.Reclaim the hominy chop powder by vacuum filtration, extract again 3 times with same way as then.0.3% of xenthophylls amount in the hominy chop powder that the total amount of used Santoflex is equivalent to record with aforementioned official method 970.64.Three parts of extracts are merged, carry out drying by rotary evaporation.Xenthophylls concentration in the dry extract that records with aforementioned official method 970.64 is 596mg/ pound (before the purifying).

(55.7g) is dissolved in the 100ml ethanol with a dry extract.In extract, add hexane (500ml) and acetone (100ml), solution is distributed with 10% aqueous sodium persulfate solution of 300ml in the 1L separatory funnel.Take out hexane layer, extract water layer again 2 times with the 500ml hexane.Three parts of hexanes are merged mutually and carry out drying by rotary evaporation.Recording xenthophylls concentration with aforementioned official method 970.64 is the 1716mg/ pound.

With 1: 1 silica gel of 78g (Devisil633 level 60A type): in the chromatographic column of magnesium oxide (the hyflo super cel that Fisher Scientific company produces) pack into 1 " (internal diameter) * 25 ".(dry weight 7.33g, xenthophylls concentration is the 1716mg/ pound, by the hexane phase composite of above-mentioned merging) is dissolved in the 246g hexane with the sample introduction material, is adsorbed onto on the chromatographic column then.Then carry out wash-out with hexane (69g), 97: 3 hexane/acetone (515g), 70: 15: 15 hexane/acetone/methyl alcohol (384g) and methyl alcohol (212g).Claim to such an extent that the dry weight of xenthophylls flow point is 0.2g, the xenthophylls concentration that records with aforementioned official method 970.64 is 30, and the 760mg/ pound promptly, is compared with the amount of xenthophylls in the hominy chop powder of initial (initial), and xenthophylls concentration has improved about 180 times.Embodiment 7

Stir down, contain the water of 15 weight % and contain the hominy chop powder (100g) 30 minutes of 170mg xenthophylls at 60 ℃ of extraction using alcohols that contain the antioxidant Santoflex with 300g.Reclaim the hominy chop powder by vacuum filtration, extract again 2 times with same way as then.0.1% of xenthophylls amount in the hominy chop powder that the total amount of used Santoflex is equivalent to record with aforementioned official method 970.64.Three parts of extracts are merged, and from extract, take out sample, carry out drying by rotary evaporation.Xenthophylls concentration in the dry extract that records with aforementioned official method 970.64 is 600mg/ pound (before the purifying).

Remove the solvent in the extract of merging by rotary evaporation, making total solid concentration is 41% (recording by the 10.0ml sample is carried out drying).In extract, add ethyl acetate (132g), mixture was stirred 1 or 2 minute with spatula.With mixture vacuum filtration, disgorging.With the filtrate drying, recording its xenthophylls concentration with aforementioned official method 970.64 is the 1835mg/ pound by rotary evaporation.

With 1: 1 silica gel of 9.4g (Devisil633 level 60A type): in the chromatographic column of magnesium oxide (the hyflo super cel that the Fisher Scientific company produces) 32mm that packs into (internal diameter) * 30mm.Sample introduction material (dry weight 4.7g, xenthophylls concentration is the 1835mg/ pound, is made up of the part that dissolves in ethyl acetate in the above-mentioned hominy chop powder ethanol extraction) is dissolved in 97: 3 hexane/ethyl acetate of 5g, is adsorbed onto on the chromatographic column then.Then carry out wash-out with hexane (40g), ethyl acetate (55g).Claim to such an extent that the dry weight of xenthophylls flow point is 0.9g, the xenthophylls concentration that records with aforementioned official method 970.64 is 8, and the 320mg/ pound promptly, is compared with the amount of xenthophylls in the hominy chop powder of initial (initial), and xenthophylls concentration has improved about 49 times.Embodiment 8

Stir down, contain less than the water of 12 weight % and contain the hominy chop powder (300g) 30 minutes of 170mg xenthophylls at 60 ℃ of extraction using alcohols that contain the antioxidant Santoflex with 900g.Reclaim the hominy chop powder by vacuum filtration, extract again 2 times with same way as then.0.1% of xenthophylls amount in the hominy chop powder that the total amount of used Santoflex is equivalent to record with aforementioned official method 970.64.Three parts of extracts are merged, and from extract, take out sample, carry out drying by rotary evaporation.Xenthophylls concentration in the dry extract that records with aforementioned official method 970.64 is 470mg/ pound (before the purifying).

Remove the solvent in the extract of merging by rotary evaporation, making its concentration is 54% (recording by the 10.0ml sample is carried out drying).In extract (100g), add ethyl acetate (400g), mixture was stirred 15 minutes with spatula.With mixture vacuum filtration, disgorging.With the filtrate drying, recording its xenthophylls concentration with aforementioned official method 970.64 is the 1518mg/ pound by rotary evaporation.

With 1: 1 silica gel of 16.5g (Devisil633 level 60A type): in the chromatographic column of magnesium oxide (the hyflo super cel that the Fisher Scientific company produces) 30mm that packs into (internal diameter) * 60mm.With a part of dry weight is that the dried thing that leaches of 8.35g is dissolved in the 70g hexane, is adsorbed onto on the chromatographic column then.Then carry out wash-out with hexane (90g), 97: 3 hexane/ethyl acetate (98g), ethyl acetate (142g) and ethanol (118g).Claim to such an extent that the dry weight of xenthophylls flow point is 2.32g, the xenthophylls concentration that records with aforementioned official method 970.64 is the 5150mg/ pound.

With 1: 1 silica gel of 80.0g (Devisil633 level 60A type): in the chromatographic column of magnesium oxide (the hyflo super cel that Fisher Scientific company produces) pack into 1 " (internal diameter) * 26 ".The sample introduction material (mainly is made up of from the xenthophylls flow point of aforementioned chromatographic column a part, dry weight 1.57g, the xenthophylls concentration that records with aforementioned official method 970.64 is 5, the 150mg/ pound) be dissolved in 96: 4 hexane/isopropyl alcohol of 15g, be adsorbed onto on the chromatographic column then.Then carry out wash-out with 96: 4 hexane/isopropyl alcohol (518g), 90: 10 hexane/isopropyl alcohol (174g) and Virahol (174g).Claim to such an extent that the dry weight of xenthophylls flow point is 0.35g, the xenthophylls concentration that records with aforementioned official method 970.64 is 9, and the 307mg/ pound promptly, is compared with the amount of xenthophylls in the hominy chop powder of initial (initial), and xenthophylls concentration has improved about 55 times.

Invention has been described though the front is with some features and in conjunction with its some preferred implementation, but those skilled in the art can understand that these features described and embodiment can have many variation, improvement and replacements that do not exceed scope of the present invention and essence.Therefore, all these improvements and changes should think that the present invention only is subjected to the qualification of the scope of following claims within described herein and claimed scope of the present invention, should give wide explanation rationally to such claims.

Claims

1. method that from the hominy chop powder, reclaims xenthophylls, this method comprises:

(a) the hominy chop powder of water content less than 30 weight % extracted with the organic solvent or the organic solvent mixed solution that contain antioxidant, the amount of contained antioxidant is less than 0.5 weight % of the xenthophylls total amount in the hominy chop powder, and organic solvent or organic solvent mixed solution were at least 2: 1 with the ratio of hominy chop powder;

(b) collection is extract obtained, obtains xenthophylls.

2. the method for claim 1, wherein the water content of hominy chop powder is mixed this hominy chop powder 10-90 minute at 10-80 ℃ with organic solvent or organic solvent mixed solution less than 20 weight %.

3. method as claimed in claim 2, wherein, the water content of hominy chop powder is less than 12 weight %.

4. method as claimed in claim 3, wherein, the water content of organic solvent is less than 20 weight %.

5. method as claimed in claim 4, wherein, organic solvent is methyl alcohol, ethanol, propyl alcohol, butanols, octanol or ethanol-hexane mixed solution.

6. the method for claim 1, wherein antioxidant is Santoflex, propyl gallate, butylated hydroxy anisole (BHA), Yoshinox BHT, Tert. Butyl Hydroquinone or tocopherol.

7. method as claimed in claim 6, wherein, organic solvent is ethanol or ethanol-hexane mixed solution.

8. method as claimed in claim 7, wherein, ethanol or ethanol-hexane mixed solution contains less than the antioxidant below the 0.3 weight % of the water of 10 weight % and the total amount that is equivalent to the xenthophylls in the hominy chop powder.

9. method as claimed in claim 8, wherein, antioxidant is a Santoflex.

10. method as claimed in claim 9 wherein, is mixed with ethanol or ethanol-hexane mixed solution the hominy chop powder at 50-70 ℃.

11. method as claimed in claim 4 wherein, is extracted and is undertaken by a series of two or more independent extractions or countercurrent extraction.

12. method as claimed in claim 6 wherein, is extracted and is undertaken by a series of two or more independent extractions or countercurrent extraction.

13. a method that reclaims xenthophylls from the hominy chop powder, this method comprises:

(b) collection is extract obtained, obtains thick xenthophylls;

(c) thick xenthophylls is carried out purifying;

(d) collect the xenthophylls that purifying is crossed.

14. method as claimed in claim 13, wherein, the water content of hominy chop powder is mixed this hominy chop powder 10-90 minute at 10-80 ℃ with organic solvent or organic solvent mixed solution less than 20 weight %.

15. method as claimed in claim 14, wherein, the water content of hominy chop powder is less than 12 weight %.

16. method as claimed in claim 15, wherein, the water content of organic solvent is less than 20 weight %.

17. method as claimed in claim 16 wherein, is removed deproteinize from the extract that contains thick xenthophylls, obtain refining xenthophylls, should make with extra care xenthophylls and carry out purifying by the positive adsorption chromatography.

18. method as claimed in claim 17, wherein, remove deproteinize by precipitation or membrane filtration, used sorbent material is silica gel and magnesian mixture in the chromatography, in this chromatography, with hexane, hexane/isopropyl alcohol or Virahol refining xenthophylls is adsorbed on the sorbent material, the xenthophylls of purifying being crossed with ethanol, methyl alcohol, hexane, hexane/acetone, acetone, hexane/acetone/methyl alcohol, hexane/ethyl acetate, ethyl acetate or hexane/isopropyl alcohol elutes from chromatographic column.

19. method as claimed in claim 16 wherein, is carried out purifying by distributing (anti-phase) adsorption chromatography, ion exchange chromatography, size exclusion chromatography or counter current chromatography to thick xenthophylls.

20. method as claimed in claim 17, wherein, organic solvent is methyl alcohol, ethanol, propyl alcohol, butanols, octanol or ethanol-hexane mixed solution, and antioxidant is Santoflex, propyl gallate, butylated hydroxy anisole (BHA), Yoshinox BHT, Tert. Butyl Hydroquinone or tocopherol.

21. method as claimed in claim 19, wherein, described organic solvent is methyl alcohol, ethanol, propyl alcohol, butanols, octanol or ethanol-hexane mixed solution, and antioxidant is Santoflex, propyl gallate, butylated hydroxy anisole (BHA), Yoshinox BHT, Tert. Butyl Hydroquinone or tocopherol.

22. method as claimed in claim 20, wherein, organic solvent is to contain less than the water of 10 weight % and contain the ethanol or the ethanol-hexane mixed solution of the Santoflex below the 0.3 weight % of the total amount that is equivalent to the xenthophylls in the hominy chop powder.

23. method as claimed in claim 21, wherein, organic solvent is to contain less than the water of 10 weight % and contain the ethanol or the ethanol-hexane mixed solution of the Santoflex below the 0.3 weight % of the total amount that is equivalent to the xenthophylls in the hominy chop powder.

24. method as claimed in claim 22 wherein, is mixed with ethanol or ethanol-hexane mixed solution the hominy chop powder at 50-70 ℃.

25. method as claimed in claim 23 wherein, is mixed with ethanol or ethanol-hexane mixed solution the hominy chop powder at 50-70 ℃.

26. method as claimed in claim 24 wherein, is extracted and is undertaken by a series of two or more independent extractions or countercurrent extraction.

27. method as claimed in claim 25 wherein, is extracted and is undertaken by a series of two or more independent extractions or countercurrent extraction.

28. method as claimed in claim 26, wherein, the hominy chop powder is mixed at 50 ℃ with ethanol or ethanol-hexane mixed solution, remove deproteinize by precipitation or membrane filtration, used sorbent material is silica gel and magnesian mixture in the chromatography, in this chromatography, with hexane, hexane/isopropyl alcohol or Virahol refining xenthophylls is adsorbed on the described sorbent material, the xenthophylls of purifying being crossed with ethanol, methyl alcohol, hexane, hexane/acetone, acetone, hexane/acetone/methyl alcohol, hexane/ethyl acetate, ethyl acetate or hexane/isopropyl alcohol elutes from chromatographic column.

29. method as claimed in claim 27 wherein, is mixed with ethanol or ethanol-hexane mixed solution the hominy chop powder at 50 ℃.

30. method as claimed in claim 28 wherein, is adsorbed on the sorbent material refining xenthophylls with hexane, the xenthophylls of purifying being crossed with ethanol elutes from chromatographic column.

31. a method that reclaims xenthophylls from the hominy chop powder, this method comprises:

(a) with hominy chop powder samples dried to water content less than 30 weight %;

(b) add antioxidant in organic solvent or organic solvent mixed solution, the amount of the antioxidant of adding is less than 0.5 weight % of the xenthophylls total amount in the hominy chop powder sample;

(c) hominy chop powder sample is extracted with organic solvent or organic solvent mixed solution, extracting method is to mix 10-90 minute at 10-80 ℃ with 1 part of hominy chop powder at least about 2 parts organic solvent or organic solvent mixed solution;

(d) collection is extract obtained, obtains thick xenthophylls;

(e) thick xenthophylls is carried out purifying;

(f) collect the xenthophylls that purifying is crossed.