RU2197958C2 - Pure 3r-3′r-stereoisomer of zeaxanthin for treating yellow spot degeneration in people - Google Patents

Abstract

FIELD: medicine, ophthalmology. SUBSTANCE: it is suggested new medicinal product (and composition on its basis) and new food additive for treating yellow spot degeneration in people. The product is a pure 3R-3′R-stereoisomer of zeaxanthin. This product is more reliable in comparison to beta-carotene or the mixture of lutein and zeaxanthin to provide retinal tissue against phototoxic damage. The innovation expands the quantity of preparations of mentioned indication. EFFECT: higher efficiency. 21 cl, 9 ex, 2 tbl

Description

 The invention relates to biochemistry, in particular to a specific isomer of a yellow pigment called zeaxanthin (abbreviated ZX). When administered to people as a medicine or vitamin, this pigment can treat or prevent a disease called macular degeneration, which damages the retina and can lead to blindness.

 The retina is a tissue that lines the back of the eyeball. It has a complex structure and consists of many different layers. It is described and illustrated in many medical manuals, such as Gittinger, 1988 and Vaughn and Asbury, 1992 (a full list of links is provided below at the end of the description).

 In the center of the retina in humans, there is a special rounded region having a diameter of approximately 1-1.5 mm and called a yellow spot. The macula has two characteristic features that distinguish it from the rest of the retina. First, the macula contains relatively few sticks; most of its photoreceptors have the shape of cones (in the fossa located in the center of the macula, the rods are completely absent). Secondly, the yellow spot has a pronounced yellow color, which gives it two pigments called lutein and zeaxanthin. Both of these. pigments belong to a class of molecules called "carotenoids." The chemistry of these carotenoid pigments is described below, after a brief summary of macular degeneration features.

Macular degeneration
The term "macular degeneration" refers to any condition that includes progressive damage to retinal cells or photoreceptor cones in the macular region in the center of the retina. It is described and illustrated in many publications and manuals, such as Taylor, 1993, Gittinger, 1988, and Vaughan and Asbury, 1992.

 There are several types of macular degeneration. The most common type is called "age-related macular degeneration", commonly abbreviated as AMD (or in some ARMD publications). AMD can cause visual impairment ranging from low vision loss to complete blindness.

 There are two forms of AMD, often called “wet” and “dry” forms. With a wet form, there is an intensive growth of capillaries and other blood vessels in the retina up to the fact that blood vessels disrupt and destroy the corresponding structure of the layers of the retina. Although this form of AMD can sometimes be treated using a laser to clog newly formed blood vessels, such treatment can only slow down the growth of blood vessels for a while and usually cannot prevent almost complete loss of vision over time; wet AMD almost always results in complete or almost complete blindness. The wet form is found only in approximately 5-10% of patients suffering from AMD.

 Another form of AMD is called the "dry" form of AMD. Since it occurs in at least 90% of all cases, it is often referred to simply as AMD. Although this form of AMD usually does not lead to complete blindness, it can lead to serious impairment of the patient’s vision and to the fact that the patient is unable to read or recognize well-known objects or faces of people, such as faces of friends or relatives. In this case, the disease often leads to functional blindness, making people unable to drive a car or walk confidently in public places and unable to lead a normal active lifestyle.

 Several diseases are also known in which macular degeneration is manifested as a symptom, including Stargart disease, Best disease, Button disease, Sjogren-Larsson syndrome, dystrophy of cone-rods, and ovoid waxy lipofuscinosis. References to articles describing each of these diseases are given by Dorey et al., 1993. In addition, other diseases that are caused by accumulation problems in lysosomes (eg, Thay-Sachs disease), or progressive degeneration of nerve cells (eg, Alzheimer's disease) are also associated with macular degeneration.

 Many of these diseases have genetic components, which is proved by heredity; several genes causing these diseases have been isolated, and tests based on genetic screening can be used to determine if a patient has a defective gene. Every person who has, or is likely to have, such a gene that is established on the basis of genetic testing or family history, is at increased risk of macular degeneration.

 As a result of the gradual impairment of vision, AMD causes great suffering. It costs billions of dollars every year, which is expressed both in the form of loss of productivity, and in the heavy burden that rests with family members, insurance agencies, social services and others who must provide or help pay for medical care and other types of help to people, suffering from blindness or severe visual impairment.

 In light of the problems caused by AMD, scientists and doctors have been looking for decades to treat or prevent blindness and other visual impairment caused by macular degeneration. However, despite all these efforts for more than half a century, effective treatments are currently lacking.

Diagnosis: Druze and lipofuscin
Typically, macular degeneration is detected using special retinal photographs. During one of the diagnostic procedures, the patient is injected with a fluorescent medicine, after which, for some period of time, the medicine is allowed to penetrate into the patient’s circulatory system and an enlarged photograph of the retina called an angiogram is taken. The photograph is then analyzed to determine the presence and concentration of any one or both types of cell debris.

 One type of cell debris that has been known and studied for several decades is called Druze. It occurs in two different forms. Usually in the eyes of any person over 40 there is a small amount of solid drusen (small particles with a diameter of less than 63 microns). As long as their number does not exceed the normal level, the presence of solid drusen does not indicate damage to the retina.

 In contrast, the formation of a significant amount of large soft drusen (also called wet drusen) indicates that significant retinal damage has occurred or is occurring, as large soft drusen formations can destroy and disrupt the organization of the retinal layers and can prevent retinal cells from receiving the right amount of nutrients from the blood. A patient whose retina contains a significant amount of soft drusen usually refers to those suffering from macular degeneration.

 Another type of retinal debris that is commonly present in patients suffering from macular degeneration is called lipofuscin. The correlation between lipofuscin and AMD has only recently become apparent (see, for example, Weiter et al., And Dorey et al., 1993).

Carotenoid chemistry
"Carotenoids" include a large class of molecules; in nature, more than 600 carotenoids have been identified. These molecules have several characteristic features, which are the following.

 1. Carotenoids are formed by the fusion of isoprene molecules containing 5 carbon atoms. Since the “building block” contains 5 carbon atoms, most carotenoids contain many blocks of 5 carbon atoms.

 2. Carotenoids have many unsaturated bonds. This allows them to absorb high-energy light waves in the blue and near-ultraviolet parts of the spectrum.

 3. Since carotenoids absorb waves with a wavelength corresponding to blue and close to the ultraviolet parts of the spectrum, without absorbing longer waves in other regions of the spectrum, usually carotenoids are yellow, orange, brown or red. The name "carotenoid" comes from the word carrot ("carrot"); the first known carotenoids that were identified as pigments are those that give carrots an orange color. The color that the carotenoids give to the solution may depend on various factors, including the concentration and presence of other chemical compounds.

 4. Carotenoids have "conjugated" double bonds. This means that double bonds alternate with simple bonds, so each carbon atom in the chain is linked by a double bond to one other carbon atom, but not a single carbon atom is linked by a double bond to two other carbon atoms. This arrangement is shown in figure 1, which presents the structure of β-carotene, ZX and lutein.

 Different carotenoids have different levels of conjugation, and more highly conjugated molecules generally provide better protection against "phototoxic" damage by high-energy light radiation. For example, in the three carotenoids shown in FIG. 1, the entire part of the straight chain is alternately conjugated by double and single bonds. In β-carotene and ZX, conjugation extends to the first bonds on both end rings. In contrast, lutein has a lower level of conjugation, since the double bond in one of its end rings is not located in such a way as to ensure complete conjugation. The only difference between ZX and lutein is the arrangement of the double bond in one (and not both) of the end rings.

 Since carotenoids are created and selected (by evolution) in such a way as to absorb the potentially harmful energy of blue and close to ultraviolet light, in natural conditions they are used as protective pigments. They are found in large quantities in plants, since one of the main functions of plants is to absorb sunlight as much as possible, while minimizing damage to cells by blue, ultraviolet and close to ultraviolet radiation. Damage by ultraviolet radiation to plants is an important problem and carotenoids help minimize such damage.

 Since carotenoids are well adapted to protect against phototoxic damage, animals have also acquired certain abilities (during evolution) to use carotenoids as light-protective pigments. Animals cannot synthesize carotenoids in their organisms, so they must absorb carotenoids (or carotenoid precursors) from plant sources. One example is β-carotene; mammals should get it from plants or from food. Once in the body of a mammal, β-carotene is converted into other molecular forms, including vitamin A (retinol), which is formed by the splitting of β-carotene into two halves.

 Carotenoids are divided into two main classes: carotenes and xanthophylls. Carotenes do not contain oxygen atoms and are true hydrocarbons consisting only of carbon and hydrogen. In contrast, xanthophylls (such as ZX and lutein) also contain oxygen.

 In FIG. 1 shows the numbering of carbon atoms in the left and right end rings ZX. It is customary to assign carbon atoms in the left end ring from 1 to 6, and in the right end ring they are assigned numbers “with a stroke”, for example, a 3'-carbon atom (read “three strokes”). Since ZX is completely symmetrical with respect to the left and right ends, the concepts of “left” and “right” are purely conditional and serve to simplify the description. However, it should be noted that lutein is not symmetrical; the position of the double bond in the "left" ring is not the same as the position of the double bond in the "right" ring.

 Since ZX is formed by adding two hydroxyl groups to the carbon atoms in position 3 at both ends in the structure of β-carotene, it has the chemical name 3,3'-dihydroxy-β-β-carotene; some chemists call it carotindiol. This molecule was given the name zeaxanthin because it was originally identified as the pigment that gives the corn its yellow color, and the scientific name of the corn is Zea mays.

 As noted above, more than 600 carotenoids have been identified in nature. Several tens have important biochemical and commercial value. ZX and lutein are especially important for the present invention, as they are present in the retinas of mammals and most other animals.

 Lutein is commercially important because it is widely used as a feed additive (in the form of plant extracts, mainly based on calendula) for chickens, which gives their skin and egg yolk a more yellow color, which attracts sellers and consumers.

 ZX can have the same effect and is stronger than lutein, but ZX sources are too expensive to use in poultry feeds. US patents 5308759 and 5427783 (addressed to Gierhart, assigned to Applied Food Biotechnology, Inc., of the same company, which is the assignee and applicant of this application) were aimed at solving the problem that ZX is too expensive to apply in feed for animals. These patents relate to the use of bacteria for the production of ZX in industrial quantities so that it can be added to feed for poultry and fish.

 In addition to plants, some carotenoids are synthesized by certain bacteria. The evolution of these bacteria occurred in places exposed to direct sunlight, and their carotenoids perform the same light-protective function as in plants. Publications that describe carotenoids from bacteria include McDermott et al., 1972, US patent 5429939 (Misawa et al., 1995) and other articles referenced therein.

 US Pat. No. 5,429,939 (Misawa et al., 1995) lists the DNA sequences of a large number of genes that encode enzymes involved in the biosynthesis of various carotenoids, including ZX. The role of each of the major genes (including crtE, crtB, crtI, crtY, and crtZ) in the creation of ZX is also described. Cells that contain plasmids carrying these genes were deposited with the American Type Culture Collection (such as Erwinia uredovora. ATCC 19321, and Erwinia herbicola. ATCC 39368) and the Fermentation Research Institute in Japan (e.g., E. coli FERM BP 2377).

Stereochemistry and isomers of zeaxanthin
Important concepts in carotenoid chemistry are “stereochemistry” and “stereoisomers”. They are explained in any textbook on organic chemistry.

 If an organic molecule has a carbon atom to which four different types of atoms or molecular groups are attached, then such a carbon atom is called a "chiral" carbon atom.

 If a chiral carbon atom is present in an organic molecule, then four different groups that are attached to such a chiral carbon atom can be arranged in either of two ways. These two different schemes are called stereoisomers. One of these stereoisomers will rotate the plane of polarization of light “clockwise”, and the other stereoisomer will rotate the plane of polarization of light “counterclockwise”. The isomer that causes clockwise rotation is called the R-stereoisomer (it is also called the D-stereoisomer). An isomer that causes counterclockwise rotation is called the S-stereoisomer (it is also called the L-stereoisomer).

 Since both carbon atoms at position 3 and 3 'in ZX are chiral, there are four possible stereoisomers. In the 3R-3'R isomer, both carbon atoms at position 3 and 3 'have an R configuration. In the 3S-3'S isomer, both carbon atoms at position 3 and 3 'have an L configuration. For convenience, these two stereoisomers are referred to herein as the R-R isomer and the S-S isomer.

 The third and fourth isomers are “mixed” or “meso” (one R- and one S-) isomers: the 3R-3′S-isomer and the 3S-3′R-isomer. Since ZX is completely symmetrical about its middle, the two isomers are identical in every respect; if you draw a 3R-3'S-isomer on a piece of paper, then by turning a piece of paper, you can turn it into a 3S-3'R-isomer. In fact, the simple "meso" isomer consists of both S-R and R-S isomers.

 If standard chemical synthesis methods are used to obtain ZX, the content of each of the four possible isomers will be approximately 25% of the total. However, since the S-R and R-S isomers are actually identical, their "meso" isomer will be 50% of the total, while each of the R-R and S-S isomers will account for approximately 25%. A mixture of all three stereoisomers is called a "racemic" mixture.

 However, the specificity of carotenoids with respect to the cell and the enzyme in the retinal tissue is so precise that the various isomers or stereoisomers are not interchangeable. Lutein and ZX are perceived by retinal cells as completely different and different molecules, despite the fact that, in conventional chemical terminology, they can be considered as isomers of each other (since they have the same number of carbon, hydrogen and oxygen atoms).

 Due to the presence of biological factors, the only isomers that are contemplated herein are stereoisomers. Any reference in the present description to the "isomer" of zeaxanthin refers to a particular stereoisomer ZX and does not include lutein. Lutein and ZX are regarded as completely different carotenoids.

 The stereoisomeric differences in carotenoids, which may appear small, barely distinguishable and insignificant, are actually extremely important when it comes to retinal tissue. Apparently, the only stereoisomer of ZX that is appropriately perceived and used by human retinal cells is the R-R isomer (3R-3'R stereoisomer).

 There are reports in the literature that trace amounts of the meso-isomer (R-S-isomer) ZX have been detected in retinal tissue. However, these trace amounts are apparently due to certain molecular transformations that can occur spontaneously under certain conditions, leading to the formation of mesozeaxanthin from lutein precursors (Bone et al., 1993 and 1994).

 Under laboratory conditions, ZX stereoisomers can be separated from each other using methods such as chiral column chromatography (Bone et al., 1993) or circular dichroism analysis (Britton, 1994).

Zeaxanthin and Lutein in the macula
By 1970, the role of carotenoids in protecting plants from phototoxic damage was well known. It was also known that carotenoids are present in animal tissue and that all carotenoids present in animals are from plants because animals cannot synthesize carotenoids. Based on this information, various publications noted the similarity between carotenoids in animals and plants and concluded that carotenoids protect animals from phototoxic damage.

 After the photoprotective role of carotenoids in animals was established, the study of the chemistry and role of carotenoids in the retina began. In one series of experiments, tests were performed in which animals were given food that did not contain any carotenoids and prepared from grains or seeds that did not contain any carotenoids (such as millet seeds). The results showed that no macula was formed in the retina of laboratory animals lacking carotenoids, and these retinas had abnormally high levels of soft drusen, indicating retinal damage (Malinow et al., 1980, Kirschfeld, 1982, Nam et al., 1984 and Snodderly et al. 1984). In light of these findings, it has been suggested that carotenoids appear to be essential for a healthy retina. Molecular confirmation has been obtained of the old truth that carrots and green vegetables are good for the eyes. However, it is still not known whether the yellow pigments in the retina should be obtained with food in their final form, or whether they can be synthesized in animals from other precursors, such as β-carotene or lycopene.

 Lutein was identified in 1949 as one of the yellow macula pigments (Wald, 1949). ZX was identified as another macular pigment only many years later (Bone et al., 1985). Publications that summarize known knowledge of yellow spot pigments by the mid or late 80s include Handelman and Dratz, 1986, Werner et al., 1987, Pease et al., 1987, Haegerstrom-Portnoy, 1988, and Handelman et al., 1988. In addition, according to these publications, it was found that ZX (which is fully conjugated, and therefore provides slightly better protection against damage caused by light radiation than lutein) is the dominant pigment in the fossa, a small area in the very center of the macula. The amount of ZX gradually decreases, and the amount of lutein increases as the radial distance from the fossa to the outer edges of the macula increases, so lutein is the dominant yellow pigment on the outer periphery of the macula. More recent publications that address various aspects of retinal aging and damage and which specifically discuss the role of carotenoids as protective agents in the retina include Sperduto et al., 1990, Gerster, 1991, Schalch, 1992 and Seddon et al., 1994.

 In general, for more than 10 years it has been known that lutein and ZX are two pigments present in the macula, and for more than ten years, the scientific community has been debating that these pigments can help protect the macula from phototoxic damage.

 However, despite the fact that these discoveries were made and hypotheses were made more than 10 years ago, no type of medicine, food supplement or food supplement or other form of treatment has been developed to date that would be effective in effectively preventing or slowing down (not to mention reversion) the gradual development of macular degeneration.

 The preceding phrase requires some clarification, since it is known that β-carotene, and vitamin A, and vitamin E can have some beneficial effect, contributing to the protection of retinal tissue (see, for example, US patent 5310764, Baranowitz et al., 1994 and two Eye Disease Case Control Study Group articles listed below). These patents and articles state, respectively, that both β-carotene, and vitamin A, and vitamin E can have a significant effect in preventing or reducing damage associated with macular degeneration.

 Such statements may be correct, given the general antioxidant role of carotenoids, vitamin A, and vitamin E. However, unfortunately, it is also true that the beneficial effects of β-carotene, vitamin A, and vitamin E on the retina are very limited. and does not reach the level corresponding to effective treatment. In all practical situations, macular degeneration cannot be prevented, stopped, and reversed. Any broad-spectrum antioxidants (such as β-carotene, vitamin A and vitamin E) are only palliative agents. Since there was no truly effective medicine available, these vitamins (with very limited and unsatisfactory success) were used to try to slow down the inevitable damage caused by macular degeneration.

 In accordance with the prior art, it should also be noted that many stores sell carotenoid preparations with labels indicating that they are useful for the eyes and eyesight. Such labeling on carotenoid mixtures may be acceptable since (as indicated above) it is known that β-carotene and vitamin A are generally useful as common antioxidants. However, none of the commercially available carotenoid mixtures contains ZX in quantities greater than the exceptionally small "trace" amounts. The vast majority of carotenoids in commercially available carotenoid mixtures are non-zeaxanthin-type carotenoids (mainly β-carotene and vitamin A).

 Close attention also deserves the position and research goals of some important government agencies and research consortia. In the United States, the National Institutes of Health (through the National Eye Institute (NEI)) and the National Advisory Eye Council recently published two reports entitled "Vision Research: A National Plan 1994-1998 ", NIH Publication 93-3186 (1994) (see, in particular, pages 55-65), and" Age related eye disease study ", NIH Publication 93-2910 (1993). In both publications and studies described therein focus on β-carotene (rather than ZX) as the compound most preferred for AMD: According to the information available to the applicant, after discussing the matter with officials from NEI, neither NEI nor any other organization affiliated with the National Institutes of Health is willing to or has not recently financed any studies of zeaxanthin as Potential Drug for AMD: Instead, NIH and other government agencies have allocated millions of dollars to research β-carotene as the most promising potential agent for treating or preventing AMD.

 Other prominent researchers who deserve special attention are members of the Eye Disease Case Control Study Group. This group recently published two articles entitled "Antioxidant status and neovascular age-related macular degeneration", Arch. Ophtalmol., 11: 104-109 (1993), and "Risk factors for neovascular age-related macular degeneration", Arch. Ophtalmol., 10: 1701-1708 (1992). Like the official NIH reports, none of these articles discuss or suggest the use of zeaxanthin as a medicine for AMD, and this consortium has also stopped or refused to fund any ZX studies as a potential agent for treating or preventing AMD.

 It should also be noted that carotenoids are of great interest for the treatment or prevention of cancer (starting with Peto et al., 1981) and for preventing the formation of cholesterol and reducing plaque deposits in arteries (Jialal et al., 1991). There is a huge amount of scientific literature that studies various types of carotenoid activity, and there is great interest in the development of methods for the chemical synthesis of carotenoids, including lutein and ZX. However, despite all the research in the field of carotenoids and the efforts to develop synthesis made in recent decades, not a single effective method of treating or preventing macular degeneration has been published. Given the enormous costs and suffering caused to people and society by a disease associated with macular degeneration, it should be recognized as a huge problem that needs to be addressed.

 Therefore, the present invention allows a significant step forward in the creation of both (1) a safe and effective medication for treating patients who have been diagnosed with macular degeneration, and (2) a dietary supplement such as pills with vitamins that can be taken by every person who wants to reduce the risk of macular degeneration when reaching or exceeding middle age.

Synthesis of Lutein and Zeaxanthin: Prototypes
Various works related to the prior art describe methods for producing ZX. These works can be grouped into two categories: enzymatic methods in which microbes play a key role in the production process, and non-enzymatic synthesis methods that use purely chemical reactions. In most of these works, it is assumed that ZX should be used for known purposes, for example, in the form of feed additives for poultry or fish in order to darken the meat and make it more attractive. Obviously, none of these methods led to the production or sale of ZX in industrial quantities.

 Back in October 1995, the only way to purchase ZX either in purified form or in semi-concentrated form, where ZX was more than about 5 wt%, was to purchase milligram quantities of ZX from specialized chemical companies such as Atomergic Chemicals Corporation (Farmingdale, NY ) or Spectrum Chemical Manufacturing Company (Gardena, CA). In 1995, the prices of these specialized manufacturers for purified ZX in the form of synthesized racemic mixtures containing undesirable S-S and S-R isomers ranged from $ 90 to $ 125 per milligram. This corresponds to approximately $ 100,000 (in US dollars) per gram of ZX as a racemic mixture. Obviously, such preparations potentially do not have a real possibility for their use as drugs or food additives, both because of their cost and because they contain large quantities of undesirable and possibly dangerous S-S and meso isomers. Prior to the present invention, purified or semi-purified R-R-zeaxanthin was simply not available in any form.

Among the well-known publications that describe the preparation of ZX using microbial fermentation, the following should be mentioned:
(1) Courington and Goodwin, 1955, the earliest known work describing the production of ZX using bacteria from the genus Flavobacter.

 (2) U.S. Patent 3,891,504 (Schocher and Wiss, 1975, assigned to Hoffman LaRoche), which also describes the preparation of ZX using Flavobacter cells. These cells containing ZX were added to the chickens food, causing the desired color.

 (3) U.S. Patent 3,841,967 (Dasek et al., 1974) and U.S. Patent 3,951,743 (Shepherd et al., 1976). Both patents are assigned to Nestle. They describe methods and nutrients that can be used to increase the amount of ZX produced by bacteria.

 (4) Two more recent US patents (US 5308759 and 5427783, both in the name of Gierhart) are assigned by Applied Food Biotechnology, Inc., the same assignee and applicant, as in the case of this application. These patents describe a bacterial strain (Flavobacterium multivorum) isolated from the Missouri River. It was found that these bacteria produce ZX without producing significant amounts of other carotenoids. This was important when creating food for poultry and fish, which would make it possible to obtain meat and egg yolk of a darker color, since carotenoids compete with each other when they enter the bloodstream after animals enter the body. Therefore, the absence of other carotenoids produced by the strain F. multivorum, which was established by Gierhart, can make ZX more suitable as a pigment for animal tissue and, therefore, increase its potential capabilities and productivity.

 US Pat. No. 5,308,759 describes methods for producing ZX for feeding poultry and fish using F. multivorum from AFB. US 5,472,783 describes feed mixtures. In both patents, the use of ZX is limited to its use in feed for poultry or fish, and no proposal has been made in any of them to use ZX for treating humans.

 None of the patents authored by Gierhart expressed any considerations regarding specific ZX stereoisomers for two reasons: (1) the characteristics of the ZX stereoisomers produced by AFB F. multivorum were not known in 1989 when applications were filed. , and (2) since the patents exclusively discuss the production of ZX for use in feed for poultry or fish, there was no apparent reason for considering the various stereoisomers.

 AFB wild-type strain F. multivorum was deposited with ATCC and assigned ATCC registration number 55238. Since these bacteria produce a specific type of lipid called sphingolipids, ATCC reclassified these bacteria as Sphingobacterium multivorum and added these cells to its catalog under this name. The name Sphingobacterium, which appears in the ATCC catalog, has not yet appeared in any of the cited works that refer to the official guidelines on the taxonomy of microorganisms: Bergy's Manual of Systematic Bacteriology, supplemented and revised in the International Journal of Systematic Bacteriology.

 Work on the production of ZX by standard chemical synthesis (without the use of microorganisms) has been reported over the past 20 years, including in US patents 4,153,615 (Sausy, 1979), 4,952,716 (Lukas et al., 1990) and 5,227,507 (Lukas et al. , 1993). However, these methods have serious disadvantages.

 Usually they require numerous reaction stages and at each stage a yield of less than 100% is achieved, so the final ZX yield at the end of the multi-stage process is relatively small. In addition, chemical synthesis usually produces the undesired S-S and S-R stereoisomers of ZX, as well as various conversion or decomposition products, such as oxidized zeaxanthin and zeaxanthin molecules, which have lost one or more double bonds in the straight chain and / or in the end rings.

 In general, prior to the creation of the present invention, the source of purified R-R-zeaxanthin, suitable for human consumption, either as a medicine or as a dietary supplement, was not known.

 Therefore, one of the objects of the present invention is the discovery of the fact that the AFB strain F. multivorum (ATCC registration number 55238) and its mutated descendants produce the ZX R-R stereoisomer as the only detectable isomer without detectable amounts of undesirable S-S or S-R stereoisomers.

 Another object of the present invention is a method for preparing a medicament for treating patients who have been diagnosed with macular degeneration, in particular age-related, based on the use of cells derived from AFB strain F. multivorum (ATCC registration number 55238).

 Another object of the present invention is a method of manufacturing a food supplement for people in the form of pills with vitamins or as an additive to food products, such as margarine, to reduce the risk of macular degeneration at a later age, based on the use of cells derived from the strain F. multivorum company AFB (registration number ATCC 55238).

 Further, an object of the present invention are preparations based on zeaxanthin that contain the RR stereoisomer as the only or clearly dominant isomer, in the form of compositions intended for oral administration by humans, either as a medicine for treating diseases or degeneration of the retina, or as a dietary supplement to reduce risk of vision loss in old age.

 These and other objects are explained in more detail in the sections "Summary" and "Description of the invention" below.

Summary of the invention
The present invention provides a process for the preparation of zeaxanthin that contains the 3R-3'R stereoisomer of zeaxanthin (also called the RR isomer or RR-zeaxanthin) as the only detectable or clearly dominant isomer intended for human ingestion as a medicine or food supplement. ZX, a yellowish pigment found in the cells of the macula in the human retina, absorbs blue and near-ultraviolet light, thereby protecting the retina from phototoxic damage. ZX preparations that contain only the desired RR isomer are produced by the Flavobacterium multivorum cell strain (ATCC registration number 55238). These bacteria do not produce any appreciable amounts of unwanted SS- or SR-stereoisomers and do not synthesize significant amounts of other carotenoids, such as β-carotene or lutein, which can compete with ZX for alimentary absorption after oral administration. After synthesis using these bacteria, ZX can be purified by methods such as solvent extraction and can be taken orally, either as a therapeutic drug by patients suffering from macular degeneration, or as a dietary supplement by a person who wants to reduce the risk of age-related macular degeneration spots, which is widespread among people aged about 50 or 60 years. Oral compositions are also available, such as (1) waterproof capsules containing RR-zeaxanthin mixed with a carrier such as vegetable oil, (2) various foods (such as margarine, dairy products, syrup, homemade dough biscuits and semi-finished meat products that are not subjected to strong heat treatment) containing RR-zeaxanthin as an additive, and (3) granular compositions that can be added to soups, salads, drinks or other foods.

 In FIG. Figure 1 shows the structures of β-carotene, lutein, and zeaxanthin molecules; the structures of all three carotenoids and the numbering system for end rings are shown. These buildings are known from analogues.

 Figure 2 presents a flow chart describing the stages of fermentation and purification of ZX produced by microorganisms that synthesize the pure 3R-3'R stereoisomer of zeaxanthin.

 This application describes a method of manufacturing a medicinal product or food composition intended for administration to people to prevent or reduce macular degeneration, a painful condition that impairs vision and can lead to blindness. Zeaxanthin-based formulations intended for oral administration by humans should contain the ZX 3R-3'R stereoisomer (also referred to as the R-R isomer or R-R-zeaxanthin for convenience) as the “clearly dominant” isomer. As used herein, a “clearly dominant isomer" refers to a preparation that contains at least 90% or more of the R-R isomer, and undesired S-S or S-R isomers comprise less than 10% of the total amount of zeaxanthin in the preparation. Preferably, any formulations intended for human use should contain the ZX R-R isomer as the only detectable isomer and not contain appreciable amounts of undesired S-S or S-R isomers. Such formulations are provided herein.

 Currently, using analysis based on chiral column chromatography (as described by Bone et al., 1993), it was confirmed that the strain of the bacterium F. multivorum (ATCC registration number 55238), isolated by Applied Food Biotechnology (AFB), produces RR -isomer in the form of the only detectable ZX isomer during fermentation, as described in the examples. In accordance with Example 4, an analysis made by Professor Landrum showed that no significant amounts of either the S-S isomer or the R-S mesoisomer were found in ZX preparations obtained by fermentation using these cells from the indicated F. multivorum strain.

 The differences between the RR, RS, or SS isomers of zeaxanthin can be very important if ZX-based drugs are administered to humans as a medicine or food supplement, since the only ZX isomer that is found naturally in the human retina is the RR isomer . It is likely that the administration of significant amounts of RS and SS isomers will be very undesirable and harmful from a medical point of view, since (1) RS and SS isomers are not found in vivo in the human retina except for the possibility of their presence in extremely small trace amounts as by-products that result from the decomposition of lutein inside the retinal cells, and (2) the RS and SS isomers can competitively replace the desired RR isomer in the retinal tissue, possibly leading to serious cell damage and to medical complications.

 Stereoisomerically pure ZX preparations obtained by fermentation using the F. multivorum strain as described herein are extremely valuable since the separation of ZX stereoisomers obtained by chemical synthesis is very complex and expensive. Although the separation of stereoisomers can be carried out in small quantities in laboratory facilities, it is prohibitively expensive in industrial production.

Use as a medication for patients with AMD
According to a first aspect of the present invention, an RR-zeaxanthin preparation as provided herein can be manufactured and used as a medicine, i.e. as a medicine that can be prescribed by attending physicians to treat patients who have been diagnosed with macular degeneration or a disease that can cause macular degeneration as a symptom or manifestation, for example, diseases such as Stgart’s disease, Best’s disease, Button’s disease , Sjogren-Larsson syndrome, dystrophy of cone-bacilli, sheep's waxy lipofuscinosis, or a disease associated with accumulation in lysosomes, for example, Thay-Sachs disease.

 When used for such treatment, a ZX-based preparation should include a sufficient amount of the Z-R-R isomer of ZX to achieve the level of therapeutic agent in a carrier or in a form (such as a capsule) suitable for administration to humans, as described below. In a preferred embodiment, the ZX intended for medical treatment is packaged in unit dosage form, such as capsules or tablets, with each dose preferably containing at least about 1 milligram (mg) of RR-zeaxanthin and may, if necessary, achieve the best therapeutic efficacy contains zeaxanthin in the range of from about 3 mg to about 10 mg.

 In accordance with a second aspect of the present invention, an RR-zeaxanthin-based preparation described herein can be manufactured and used as a prophylactic drug for patients who have been diagnosed with increased sensitivity to macular degeneration due to both a family history and a genetic history. diagnosis of any of the above diseases. Unit doses prepared for administration to those patients who are at increased risk of AMD disease but who are not yet suffering from acute AMD may contain lower amounts, such as from about 0.1 mg to about 2 mg per dose.

 Using this description, any of these doses may have an affordable commercial price. Capsules containing 25 milligrams (or any smaller amount) in an oily liquid carrier can be made economically using microbial fermentation in combination with a solvent extraction step. Powdered compositions containing even higher amounts (e.g., 100 mg or more per dose) can also be made using more extensive purification, for example, using the methods described in example 4.

Use as a vitamin or food supplement
In accordance with a third aspect of the present invention, ZX can be made and packaged as a vitamin or food supplement or food supplement for use by people who currently do not suffer from macular degeneration but who want to reduce their risk of macular degeneration in older age. When administered for these purposes, the corresponding doses should significantly exceed the trace amounts contained in the powders that are currently sold in stores, but they should be lower than when ZX is used as a therapeutic drug for a person in respect of whom it was found that he is suffering from AMD. Such dosages recommended for oral administration as daily doses should probably be in the range of about 0.05 mg to about 5 mg. For example, a dose of 0.05 to 1.0 mg may be acceptable when RR-zeaxanthin is one of ten or more agents in a multivitamin capsule or tablet, while a dose of 1 to 5 mg may be acceptable for retail sale to people. wanting a higher dose.

 Regardless of whether it is used as a therapeutic drug or as a nutritional supplement, a ZX-based preparation intended for human use should contain the R-R isomer as the sole or “clearly dominant stereoisomer” of ZX. In the context of this description, the term “pronouncedly dominant isomer” is used to describe a ZX-based preparation in which the desired RX isomer of ZX is at least about 90% of the total amount of ZX in the mixture and the undesired SS or SR isomers are less than approximately 10%.

 Preferably, the R-R isomer should be the only detectable ZX isomer in any formulation intended for human ingestion. In the implementation of the present invention, this has become possible in industrial volumes and at an affordable price, since the F. multivorum bacteria line described herein produces the R-R isomer as the only detectable ZX stereoisomer. If any of the S-S or S-R isomers is present in the fermented mixtures after purification, their quantities are too small to be detected by the methods described in example 4.

 In addition, unlike most bacterial strains, F. multivorum cells described herein do not produce a mixture of carotenoids; these cells form R-R-zeaxanthin as the only detectable carotenoid. Since ZX must compete with other carotenoids for alimentary uptake and tissue uptake, it may be useful to increase ZX uptake and retinal deposition after oral administration, especially when ZX is used as a medication to treat diagnosed macular degeneration.

Industrial production using bacterial fermentation
As is known to those skilled in the art, methods that are used for bacterial fermentation under laboratory conditions can be very costly and difficult to control when adapting to large-scale production. Therefore, according to the invention, improved nutrient media and methods for the industrial use of F. multivorum cells identified by the authors have been developed. Improved culture media and methods are substantially easier to apply, and they are substantially cheaper per gram of ZX obtained compared to the media and conditions described previously in US Pat. Nos. 5,308,759 and 5,427,783. Preferred culture media and conditions are described in Example 1.

 After fermentation, one or more stabilizing compounds can be added to the cells in order to prevent the decomposition of ZX during purification. Stabilizers can be added while the cells are still in the fermentation vessel, prior to pasteurization or other processes. Applicants have studied various potential stabilizers. Currently, the best results are obtained using a combination of stabilizers specified in example 2.

After adding stabilizers, the bacteria can be pasteurized by heating to 55 ^{° C.} for 25 minutes in order to kill the bacteria without damaging the ZX. The cultures are then cooled to room temperature and, by mechanical means, such as cross-flow microfiltration, fluid is removed from the cell culture. This can increase the concentration of cells and solid particles from an initial value of approximately 10 vol.% To approximately 60-80 vol.% In the filtrate. This process allows you to get cell paste.

 It is likely that intact and viable F. multivorum cells may be suitable for direct ingestion by humans as well as other food products (cheese, yogurt, beer, etc.) that contain viable or killed, but undamaged cells microorganisms. For F. multivorum cells, there is no evidence of their pathogenicity. They were isolated from a cold watercourse and, since they are adapted to life in cold water, they cannot survive or reproduce normally at the temperature of the human body. In addition, these cells do not have any known toxic constituents; they are gram-negative and do not have the structure of cell walls characteristic of gram-positive bacteria. When directly fed to birds or fish in the form of a cell paste, bacterial cells are likely to be acceptable as good carriers. ZX was released upon animal uptake of cells and was absorbed into the bloodstream and deposited in appropriate places in various tissues (including the retina).

 Thus, intact cells of F. multivorum. containing RR-zeaxanthin may be suitable for direct human administration, if necessary, in the form of any of three forms: (1) as an intact viable form, (2) as an intact dead form after pasteurization, or (3) as a composition in which bacterial cells were killed and their membranes were destroyed in order to open the cells and make ZX more accessible. This can be accomplished by methods such as irradiation with ultrasound (using high frequency sound waves), high pressure treatment or grinding. In another embodiment, this step may be omitted if a solvent extraction process is used that destroys cell membranes.

 If necessary, the method of producing ZX may include a stage of washing the cells, during which, after fermentation, residues of the nutrient medium and waste resulting from metabolism are removed by washing the cells with a solution containing any desired ingredients, such as stabilizers, preservatives, flavoring agents, etc.

Cleaning
If necessary, the cell paste (consisting of either intact or destroyed cells) can be dried in order to further concentrate the cells and increase the concentration of ZX in the dry mass. This can be done mechanically, such as spray drying (using heat) or lyophilization (drying by freezing under vacuum). If drying is used, the resulting solid residue is usually called dried biomass, and it usually contains about 1-10% by weight of ZX along with other solid particles of the cells, residual solid particles of the fermentation medium and stabilizers described above.

 To concentrate ZX, which mainly accumulates in cell membranes, an extraction step may be performed before or after (or instead of) disruption or drying. Suitable solvents for extraction typically include polar organic solvents. According to the data obtained, tetrahydrofuran (THF) is the best solvent, which has an active effect on cells and makes a separate stage of membrane destruction unnecessary. Although mixing is not necessary when using THF in a laboratory process, it is likely that in industrial production mixing during the solvent mixing step is necessary.

 Other solvents have also been investigated, and they continue to be studied and evaluated, but to date no more acceptable solvent has been identified than THF. The organic solvents studied so far that do not have a cyclic structure (such as acetone and diethyl ether) had lower solubility levels in them ZX, and other solvents such as methanol, ethanol and hexane had even lower solubility levels in them. ZX.

 The solvent is mixed with cell paste or with dried biomass under conditions in which the solvent is capable of dissolving the maximum possible amount of ZX. The dissolved liquid fraction is then separated from the solid particles using methods such as centrifugation or filtration. Solids can be discarded or can be used as starting material for other stages of the process (including, if necessary, repeated solvent extraction cycles). The liquid fraction is treated to remove the solvent, usually by evaporation. This leaves a viscous oil containing R-R-zeaxanthin, as well as other soluble components, extracted from the cell paste with a solvent. When THF is used for a single extraction of cells containing 1-3% by weight of ZX, and when further THF is removed by evaporation, the resulting liquid contains from about 5 to 20% by weight of ZX.

Another type of solvent extraction, for which preliminary good results have been obtained, involves the use of a supercritical fluid (i.e., a compound that is usually a gas at atmospheric pressure but turns into a solvent acting at elevated pressure). Carbon dioxide is the most widely used solvent for supercritical extraction, and extraction systems based on the use of CO ₂ on an industrial scale are the most affordable. In such systems, liquefied carbon dioxide is mixed with cell paste or with dried biomass in a pressure vessel. Then the fluid is passed through a series of chambers in which the pressure decreases in a stepwise manner. ZX precipitates from the solution at a rather high pressure, so it can be collected at an early stage of pressure reduction, while the main part of the impurities remains dissolved in carbon dioxide and must go into other reaction chambers with even lower pressure. Extraction efficiency with a supercritical solvent can be further enhanced using selectively capturing agents (such as ethanol, propylene glycol or ethyl acetate). Some of these selectively capturing agents have been previously investigated and it has been shown that they significantly increase the solubility of ZX in a supercritical solvent, carbon dioxide.

 Although carbon dioxide is widely used for supercritical extraction, other compounds are also used (including various nitrogen or chlorofluorocarbon compounds). In principle, any solvent that has a gaseous or liquid state depending on pressure can be examined to determine if it is suitable for purification of ZX from bacteria in accordance with the present description.

 If necessary, the oily liquid containing ZX obtained after solvent extraction or by supercritical extraction can be mixed with a carrier, such as vegetable oil, and then included in a capsule intended for human ingestion, which does not require any additional ZX purification. This method is a cost-effective way to obtain a semi-purified, easily digestible form of RR-zeaxanthin, suitable for human use, either as a medicine for people suffering from macular degeneration, or as a food supplement for people who want to reduce the risk of yellow macular degeneration spots at an older age.

 Alternatively, R-R-zeaxanthin in the semi-purified oily liquid may be further purified to increase the concentration of ZX and remove any impurities. This can be accomplished by methods such as (1) using two solvent systems that use a combination of two different solvents, (2) adsorbing onto a substrate (such as a filter layer of tissue) that promotes crystallization of ZX, or (3) chromatography in countercurrent. The chromatography method used for purification of ZX and allowing to obtain ZX with a purity of approximately 98% is described in example 4.

 Methods for purifying other carotenoids are described in US Pat. Nos. 5,382,714 (Khachik, 1995) and 4,851,339 (Hills, 1989). Given their chemical similarity, any method suitable for purification of β-carotene or lutein can probably give good results in purification of ZX.

Administration Methods
Oral administration is the preferred method of administering ZX to people to protect the retina using oral forms of administration such as capsules intended for daily or weekly administration, or using ZX as food products supplemented with ZX or food additives, as described below. Treatment does not require regular ingestion at regular intervals (as in the case of pills intended for daily or weekly use), but instead involves occasional intermittent ingestion, in which a certain period of time must elapse between doses (for example, one or several days , preferably less than a week), which allows small amounts of ZX to gradually accumulate in the macula tissue. As with any vitamin supplement, a single dose may also be acceptable, but a single dose will not be as effective as taking small doses periodically for several years. Studies of carotenoid absorption in mammals have shown that daily ingestion is preferable to weekly or other random doses due to “loading” factors, which are confirmed by blood concentrations.

 Since the uptake of carotenoids after oral administration is usually relatively low for patients suffering from severe macular degeneration, it may be necessary to use other forms of administration, such as intramuscular or intravenous injection or implantation of a device with a slow release of the drug. Carriers for injection compositions may include water, a buffering agent, and an organic compound having several hydroxyl groups, for example, compounds such as propylene glycol, dextran, or cyclodextrin.

 For oral administration, various packages can be used to protect the ZX from oxidation for a long time and correspond to the oily nature of ZX. Examples of suitable compositions for oral administration are as follows.

 (a) An easily digestible waterproof capsule with a liquid enclosed in it, the capsule and liquid having a size that allows them to be swallowed without damage, they are pharmacologically acceptable, and the liquid contains RR-zeaxanthin mixed with a suitable carrier or solvent, such as a vegetable oil. If necessary, fluidized ZX can be microencapsulated or incorporated into micelles, as described in examples 9 or 10, in order to protect ZX from degradation in the stomach. Such capsules can be made from a relatively hard, inelastic material or from a plastic material that is usually used for capsules containing vitamin E. If the capsule is made from a material that is resistant to the acidic environment of the stomach and is digested by intestinal enzymes, ZX can be protected from degradation in the stomach as well. bioavailability of ZX may be increased. However, it is known that at least part of the ZX that enters the stomach as a component of a chewed plant mass can pass through the stomach without change; therefore, protecting ZX from the acidic environment of the stomach is not critical, and the choice of capsule material should primarily be cost-effective, and not scientifically sound.

 (b) A tablet for oral administration by a person, the tablet containing RR-zeaxanthin and a compressible binding agent compatible with zeaxanthin and allowing the tablet to maintain its shape after compression under appropriate pressure, the tablet being pharmacologically acceptable and has a size, allowing you to swallow it without damage. If necessary, the tablet may be coated to protect the ZX from the acidic environment of the stomach.

 (c) A composition comprising a food product that is intended for human consumption and which is acceptable for use as food and tastes good, is also suitable as a carrier of zeaxanthin and contains R-R-zeaxanthin as an additive. ZX is a yellow-orange pigment with the same general hydrophobic characteristics as vegetable oil, shortening (a blender for the baking industry) or chicken fat; it is also similar to other carotenoid food colors, such as β-carotene.

 Thus, it can be added as a food coloring to various food products, such as margarine, dairy products, syrup, baked goods, homemade cookie dough, fried dough, semi-finished meat products that are not heat-treated, and ingredients for soups . Other suitable food products may include granular compositions, such as mixtures of salting or flavoring agents used as additives in soups, salads, cookies, etc. Granular compositions may optionally have a protective coating to reduce the decomposition of ZX by the acidic environment of the stomach. Numerous examples of the use of β-carotene and other carotenoids as food colors and food additives are known; they are described by Klaui et al., 1970; Klaui and Bauernfeind, 1981; Colombo and Gerber, 1991; and U.S. Patents 4,522,743 (Horn et al., 1985), 5,180,747 (Matsuda et al., 1993), 5,350,773 (Schweikert et al., 1994) and 5356636 (Schaneider et al., 1994). Owing to their identical chemical characteristics, any method of adding β-carotene or lutein to a food product intended for humans can probably also be directly applied to R-R-zeaxanthin.

 (d) A composition comprising a food product intended for human consumption, the food product comprising microorganism cells that are safe for humans and which contain the R-R isomer of zeaxanthin. Food products can be selected from cheese, yogurt, milk and beer. If necessary, cells of the microorganism can be viable, or they can be killed by methods such as pasteurization or fragmentation.

 Other forms of packaging may also be used, and they may be preferred for various purposes.

Animal RR Zeaxanthin Testing
ZX, which was synthesized using F. multivorum cells obtained from the ATCC 55238 line, was tested for its ability to protect the retina in birds of the species Coturnix coturnix japonica, commonly called Japanese
partridge. These species are a convenient animal model for studying macular degeneration in humans due to a number of factors listed below.

 (1) The entire retina of partridges is similar to the human yellow spot in a number of important ways. For example, the partridge’s retina contains both ZX and lutein and, like the human macula, it has more photoreceptors in the form of cones than in the form of rods.

 (2) Some manifestations of the pathology characteristic of the human retina are characteristic of the Japanese partridge retina, for example, the Japanese partridge retina accumulate soft drusen and lipofuscin, which largely correlates with the initial manifestations of AMD in humans.

 (3) Although the partridge's retina is substantially smaller than the human retina, the entire partridge's retina is yellow due to the presence of ZX and lutein. This allows you to effectively use the entire retina of the partridge as a model of a small area of the yellow spot in the center of the human retina, which greatly facilitates analysis and observation.

 (4) The retina of a partridge is vascular-free and has a structure similar to that of the fossa of the human retina.

 (5) The life span of female partridge is approximately 1-1.5 years, and of males is 3-4 years. This allows us to study the aging process, which can be very difficult for other species with a long life span.

 These factors are discussed in more detail in Fite et al., 1991 and Fite et al., 1993.

 These experiments are described in examples 5-8. Excellent results have been obtained that clearly show that R-R-zeaxanthin produced by F. multivorum cells (1) is properly deposited in the retina after oral administration and (2) is highly effective in protecting retinal cells from phototoxic damage.

 In addition, as described in Example 8, preliminary results show that R-R-zeaxanthin is significantly more potent and effective than β-carotene in protecting the retina from phototoxic damage. When β-carotene is fed to experimental animals at a high dose, the insignificant protective effect of β-carotene does not even reach the level of statistical significance. In contrast, when R-R-Zeaxanthin is fed to experienced animals at the same dose, it completely blocks and prevents measurable manifestations of retinal damage.

Microbial sources of RR-zeaxanthin
The Flavobacterium multivorum cells described herein are deposited in ATCC (ATCC registration number 55238; as noted above, they correspond to Sphingobacterium multivorum in the registry catalog. However, their name was not changed in Bergy's Manual). This cell line provides specialists in this field several ways of microbiological synthesis of isomerically pure RR-zeaxanthin.

 First, the immediate and unmodified descendants of these cells can be used to synthesize R-R-zeaxanthin without noticeable amounts of other undesirable stereoisomers. Of the total number of carotenoids produced by these cells, more than 90% are necessary carotenoid ZX.

 Secondly, the offspring of strain ATCC 55238 can be used after its modification by methods that increase the production of the R-R isomer ZX. Mutant or other altered cell lines can be created using any of several methods, such as (1) treating the offspring of the wild-type strain ATCC 55238 with mutagenic agents such as ultraviolet radiation or X-ray radiation, or known chemical mutagens such as N-methyl -N'-nitro-N-nitrosoguanidine, (2) obtaining sexual combinations by mixing F. multivorum cells with other types of bacteria, which actively enhances the conjugation and DNA exchange between bacterial cells, (3) processing of F. multivorum bact serial transposons or viruses that can cause rearrangement of relatively large sections of DNA. These methods allow for random changes in progeny cells, and then progeny are analyzed using screening methods to identify and isolate progeny cells that produce higher levels of ZX.

 Screening methods can be facilitated by chemicals (such as diphenylamine, nicotine, or lovastatin) that inhibit one or more of the enzymes involved in the biosynthesis process that produces ZX. It should be explained to a layman that these suppressor poisons create obstacles or interferences that can only be overcome by mutant cells producing abnormally high amounts of ZX. However, experiments that can be used to identify highly productive mutants or variants are simple, quick, and easy to implement. Since ZX is a yellow pigment, a simple visual examination of the culture plate can be used to identify mutant colonies that have the required characteristics (1) good cell growth rate and (2) the ability to produce abnormally high amounts of yellow pigment. Screening methods after processing with a mutagen may also use an automatic device if necessary (such as an automatic tablet reader or devices for automatically sorting cells connected to flow cytometers).

 These mutagenesis and screening techniques are generally accepted and well known in the art. Any cells that are direct descendants of the wild-type strain of ATCC 55238 are considered to be offspring of these cells, even if they have been modified, mutated or sexually combined with other cell lines using any of the methods listed above.

 In a third alternative approach, non-progeny microbial cells can be created that contain genes isolated or derived from an ATCC 55238 cell line that express enzymes that promote R-R-zeaxanthin synthesis. Such genes can be isolated and identified using known methods. For example, the DNA sequences of the carotenoid producing crt strain gene genes described in US Pat. ATCC 55238. Carotenoid-producing genes isolated from these cells can then be inserted into plasmids, cosmids, phages, or other acceptable vectors that can be used to genetically transform any desired type of host cell on, such as E. coli cells, yeast cells, insect cells or mammalian cells. Controlled genetic engineering of this type can ensure the production of transformed R-R-zeaxanthin cells using genes derived from ATCC 55238 cells.

 In addition, protein-coding parts of genes producing ZX from ATCC 55238 cells (i.e., parts of genes that are transcribed into messenger RNA and then translated into ZX-synthesizing enzymes) can be placed under the control of strong and / or inducible promoters. Such “chimeric” genes containing gene promoters derived from various genes can be used for various purposes, such as (1) suppressing ZX production during cell growth and reproduction, and then dramatically increasing ZX production by cells during fermentation and (2) insertion of genes into new types of host cells that may be preferable for industrial use, such as E. coli cells or yeast cells, which can be used for well-known and largely optimized methods of fe mentation, manufacturing and purification.

 Genes producing ZX and isolated from cells of the ATCC 55238 line can also be amplified using other well-known methods. For example, bacterial genes often use "non-preferred" codons, which reduce and regulate the amount of protein synthesized by the gene. In order to reduce these limiting mechanisms, non-preferred codes in the ZX synthesizing gene in ATCC 55238 cells can be replaced by “preferred” codons, which can increase the expression of the ZX-producing enzyme in the selected host cell.

 In another example, cysteine residues can interfere with enzyme activity or stability by forming unnecessary disulfide bridges with other cysteine residues in either the same or other protein molecules. Therefore, the activity or stability of the enzyme can sometimes be increased by replacing one or more cysteine residues with other amino acid residues (for example, see US Pat. No. 4,737,462 to Mark). In addition, protein expression can often be increased by embedding codons for common amino acids, such as glycine, instead of codons that encode methionine and tryptophan, which are less common and which tend to slow down and decrease protein expression. After creating a synthetic gene that leads to the replacement of an amino acid of this nature, the modified protein can be examined to determine whether it retains the desired enzymatic activity, whether it is expressed in large quantities or in a more stable form.

 The above are examples of known genetic engineering methods that can be evaluated on genes producing ZX isolated from ATCC 55238 cells in order to determine if any modification will enhance ZX production by F. multivorum cells or other types of host cells.

 Used in the claims, the concept of "cells that have been genetically engineered to create at least one gene synthesizing zeaxanthin and containing a DNA sequence obtained from a strain of Flavobacterium multivorum. Which was assigned registration ATCC 55238", includes cells containing genes having sequences Chemically synthesized DNA using a DNA or mRNA sequence that has been determined by analysis of ATCC 55238 cells or their progeny. Automatic DNA synthesis devices are well known and can be used to copy any known gene sequence without the need to replicate the original host cell. The term “zeaxanthin synthesizing genes” includes any genes expressing an enzyme or other protein that is involved in the ZX biosynthesis pathway and which can be used to increase ZX production when inserted into suitable host cells, regardless of which particular enzyme in the ZX biosynthesis pathway encodes this gene.

Examples
Example 1. Fermentation on an industrial scale
The culture medium, which according to the applicants was preferred for the initial small-scale testing of Flavobacterium multivorum in the laboratory, is a culture medium E, described in example 3 in US patent 5308759 (Gierhart, 1994) and 5427783 (Gierhart, 1995). This culture medium contained several ingredients that were expensive and difficult to work with. To reduce costs and increase convenience, an extensive study was conducted after the filing date of these applications to create a breeding ground more suitable for industrial use. Among the nutrient media that are currently preferred for industrial-scale fermentation, corn flour and several other ingredients have been excluded. These preferred media contain either high maltose corn syrup or beet sugar molasses in a concentration range of 1 to 10% w / v. along with an extract of soaked corn in a concentration of 0.5-4% wt./about .; ammonium sulfate heptahydrate at a concentration of 0.5% w / v; sodium chloride at a concentration of 0.5% wt. /about. ; magnesium sulfate heptahydrate at a concentration of 0.1% w / v; sodium acetate at a concentration of 0.1% wt./about .; iron sulfate heptahydrate in a concentration of 0.001% wt. /about.; yeast extract at a concentration of 0.2% wt. /about. ; thiamine-Hcl at a concentration of 0.01% wt./about .; from 1 to 6% wt./about. hydrolyzed casein (e.g., NZ Amine HD available from Sheffield Products, Division of Quest International, Norwich, NY); and vegetable oil at a concentration of 1 vol. % After mixing these ingredients, an amount of NaOH sufficient to raise the pH to 6.5 is added; in contrast, when the pH of the growth medium was adjusted to 7.5, as described for laboratory experiments in US Pat. Nos. 5,308,759 and 5,427,783, too many particulate matter precipitated from the soaked corn extract.

The culture medium is autoclaved at 121 ^{° C.} for 30 minutes, then it is cooled to 27 ^{° C.} and inoculated with 5-10 vol.% “Liquid preculture” containing the F. multivorum strain, which produces RR-Zeaxanthin and does not produce SS - or SR stereoisomers. Cells that are used to obtain a liquid preculture are grown in a test tube with beveled agar designed to determine the number of microorganisms. These cultures, obtained on mowed agar, were inoculated with clonal colonies of F. multivorum obtained from the strain deposited by the applicants in ATCC (ATCC registration number 55238). After incubation for 48 hours at 28 ^{° C., the} starting cultures on mowed agar were stored in a refrigerator at 4 ^{° C.} until they were used as an inoculum for a liquid medium. Viable cells can also be frozen for long-term storage using conventional freezing chambers, dry ice, or liquid nitrogen.

A liquid preculture is prepared using cells taken from mowed agar to inoculate 30 ml of the liquid medium obtained by the method described above and contained in a 300 ml septum flask. The following growth conditions are used: 28 ^{° C.} , pH 7.2 to 7.6, aeration by stirring at 250 rpm and cultivation for 24 hours. After the initial 24 hours of incubation, cells contained in one or more flasks for preliminary cultivation with a volume of 30 ml, is used to inoculate ten times more nutrient medium in a fermentation vessel of an appropriate size. The cells are then incubated for 48-72 hours at 28 ^° C. The pH is maintained at 6.80-7.20 using NaOH and / or phosphoric acid. The concentration of dissolved oxygen is maintained at a level of 30-40% of the saturating concentration by sparging filtered air through a vessel at a rate of 1 air volume per 1 volume of liquid per minute while shaking the vessel on a mixer at a speed of 400-1000 rpm. Experiments using periodic sampling and high performance liquid chromatography showed that the maximum amounts of ZX are usually produced within approximately 72 hours when the cells are fermented under the indicated conditions.

Example 2. Adding stabilizers
Formed in the fermentation process in accordance with example 1 ZX needs to be stabilized in order to facilitate subsequent cleaning and preparation of the composition and to guarantee cleanliness. Stabilizing compounds can be added to F. multivorum cells (or to a cell extract containing ZX) at any time during the preparation or purification; usually one or more primary stabilizers can be added to the cells while they are still in the fermentation vessel. Applicants have studied various potential stabilizers. To date, the best results have been obtained using a combination of stabilizers that are mixed with a small amount of an acceptable solvent before being added to the cells (for example, with about 2 ml of ethanol in a 20 L fermentation vessel). The preferred stabilizer mixture contains tert-butylhydroquinone (abbreviated as TBNX, also called 2- (1,1-dimethylethyl) -1,4-benzenediol) in an amount that allows, after mixing with the cells, a final concentration of about 250 μg / l (micrograms per liter) up to about 50 mg / l; ethoxyquin, the concentration of which after mixing is in the range from about 250 μg / l to about 250 mg / l; α-tocopherol at a concentration of from about 250 μg / L to about 250 mg / L; and EDTA (ethylenediaminetetraacetic acid) at a concentration of from about 500 μg / L to about 500 mg / L. Acceptable concentrations can vary significantly and should depend on various factors, such as subsequent purification steps and the intended packaging method and route of administration. The preferred concentrations of these stabilizers used when using a single extraction of THF followed by mixing with vegetable oil and encapsulation in a waterproof pill, similar to that used for vitamins, are approximately 25-50 mg / l TBNH, 250-500 μg / l ethoxyquin, 250- 500 μg / L α-tocopherol and 500-1000 μg / L EDTA.

After the addition of stabilizers, the cell culture is pasteurized by heating to 55 ^{° C.} for 25-50 minutes. This procedure kills bacteria without damaging the ZX they produced. The culture is then cooled to room temperature and cells containing ZX and other solid particles present in the culture broth are separated from the liquid phase using a cross-flow microfiltration system that increases the cell / solid concentration from an initial value of about 10-15 vol. % to a concentration in the filtrate of approximately 60-80 vol.%. This process results in cell paste, which also contains some residual solids from the culture medium.

To obtain preparations based on ZX, which were fed to a Japanese partridge for researching the retina, as described in examples 5-7, the cell paste was frozen to -70 ^° C, then dried by lyophilization at 25 ^° C in a high vacuum, to obtain dried biomass containing about 1-10 wt. % ZX In the above experiments, the amount of ZX in each portion was evaluated individually and the portions having different concentrations were combined and mixed in order to ensure the use of comparable concentrations in experiments on a Japanese partridge.

 To obtain ZX intended for human ingestion, solvent extraction is used to obtain a viscous oily liquid, as described in Example 3.

Example 3. Incomplete purification of oily liquid
After receiving the cell paste, analogously to example 2, it can be processed by any of the multi-member methods. As indicated above, if necessary, cell membranes can be destroyed in order to open the cells and make ZX more accessible using methods such as irradiation with ultrasound (using high-frequency sound waves), high pressure treatment or grinding, keeping the cell temperature below approximately 30 ^o C to prevent oxidation. However, this step is not necessary when tetrahydrofuran (THF) is used in the solvent extraction step, since THF very efficiently destroys cell membranes without mechanical intervention. Stirring is not necessary when THF is used in laboratory experiments; however, it is likely that in industrial production, mixing during the solvent mixing step may be appropriate.

In the experiments carried out, the extraction of THF included mixing about 8-20 volumes of purified filtered THF with one volume of cell paste containing 60-80% solid particles at a temperature below 25 ^o C for 2-24 hours. THF actively affects the cells, leading to obtaining a liquid in which flocculent solids are suspended. After centrifugation at 20,000 g for several minutes, most of the THF can be removed by decantation. The remaining THF can be evaporated in vacuo, resulting in a viscous oil. When a cell paste containing 1-3% ZX was treated with a single extraction of THF, the resulting oil typically contained about 5-20 wt.% ZX.

Example 4. Obtaining highly purified zeaxanthin in dry powder form with a 100% content of RR isomer
A preparation of highly purified ZX in dry powder form was obtained by treating a THF-extracted oily liquid described in Example 3 using liquid chromatography as follows. The oily liquid containing ZX was dissolved in hexane, then passed through a chromatographic column containing neutral aluminum powder. In order to remove impurities of carotenoids, such as β-carotene and lycopene, as well as lipids and other contaminants, hexane was used to wash the column in a volume equal to two column volumes. Then, to separate ZX, a hexane: acetone mixture was passed through the column in a ratio of 80:20. The resulting dissolved ZX was dried under vacuum. Chromatographic analysis showed that it is ZX with a purity of at least 98%; only trace amounts of any impurities were detected.

 After being stored for approximately six months in a relatively unprotected state (usually during normal freezing with an average sampling frequency and without the use of any antioxidants and without taking any precautions to prevent contact with atmospheric oxygen), a sample of this ZX preparation was sent for stereoisomeric analysis to Professor John Landrum (one of the co-authors of the articles Bone, Landrum, etc.) at Florida International University, Miami. Florida. His analysis using chiral chromatography on dicarbamate derivatized columns showed that the unprotected preparation, which had been stored for six months, contained 92% ZX. The impurities were probably mainly ketocarotinoids that eluted before ZX; ketocarotenoids have an additional oxygen atom attached to the carotenoid in one of the positions, and they are the usual by-products that appear during storage of carotenoids without protection against oxidation. A chiral analysis performed by Professor Landrum showed that the desired R-R isomer was 100% of the total amount of ZX in the preparation. No appreciable amounts of unwanted S-S or S-R stereoisomers were found.

 It was found that the chromatographic purification described above, although it is fully feasible and highly efficient, is not ideally suited to produce highly purified ZX in industrial quantities. As a method potentially acceptable for industrial production, we can name an alternative method developed for the purification of lutein and described in US patent 5382714 (Khachik, 1995; also see Khachik et al., 1991), which uses a mixture of cold ethanol: water in an extraction system consisting of two solvents, followed by lyophilization.

Example 5. Studies of zeaxanthin using a partridge group with a different diet as a model
All studies using Japanese partridge were performed at the Schepens Eye Research Institute of Harvard Medical School (Boston, Massachusetts) under a contract with Applied Food Biotechnology, Inc. (assignee of this application). The number of birds in all treated or in control groups was such that was necessary to obtain statistically reliable data. In most cases, the number of birds in the control groups corresponded to the number of birds in the treated groups.

 Carotenoid-deficient bird feeds were obtained from Purina Mills (St. Louis, Missouri). Such bird feeds were sold exclusively for experimental use and were obtained on the basis of grain (such as millet seeds), in which there are no carotenoids in vivo.

 All ZX-based formulations fed to partridges were dried biomass of F. multivorum cells, which were fermented, stabilized with the agents of Example 2, pasteurized to kill cells, and dried by lyophilization. These stages of fermentation and preparation were carried out by Applied Food Biotechnology, Inc. on her equipment in O'Fallon, Missouri.

 All experimental animals were hatched from carotenoid-deficient eggs. They were obtained by feeding the parental generation (designated as birds of the P1 generation) when the birds reached puberty exclusively using a diet with a carotenoid deficiency. Their eggs were broken and analyzed for carotenoid content until carotenoid deficiency was detected in the eggs. The eggs, which were then laid by birds of the parental generation with a deficiency of carotenoids, were used to breed birds of all experimental and control groups.

 Experienced and control birds were divided into four main groups, which were kept on different diets. These groups were designated as group C +, group C-, group BC +, group ZX (+5) and group ZX (+50) depending on which carotenoids they received in their diet.

 Birds of the C + group were kept on a standard commercial diet that included several carotenoids; this diet also contained synthetic α-tocopherol (vitamin E) as an additive.

 Group C birds were kept on a diet in which almost all carotenoids were absent, as described above. However, this diet included all other essential nutrients, and it included synthetic vitamins A and E. as additives.

 Birds of the BC + group were kept on a diet in which all carotenoids were absent, but β-carotene was added to it in the same dose as was used for the group on a diet with a high ZX content (i.e., 50 mg was added β-carotene per kilogram of feed). Birds of the BC + group were transferred to a diet containing β-carotene seven (7) days before the start of the procedure for retinal damage by light. This group allows a direct comparison between the group on the diet supplemented with β-carotene and one of the subgroups of birds on the diet supplemented with ZX, which was also transferred from a carotenoid-deficient diet to the diet supplemented with ZX seven days before the start damage to the retina by light. As described in Example 8, this direct comparison showed that ZX is highly effective in preventing phototoxic damage, while the protective effects of β-carotene were so weak that the results did not reach a statistically significant level.

 ZX + birds were kept on a diet that lacked all other carotenoids, but which included dried biomass containing R-R-zeaxanthin from AFB F. multivorum cells. The diet of these birds included two different doses of ZX, which makes it possible to evaluate the dose-dependent action and to reveal correlation with various manifestations of retinal damage. ZX group birds (+5) were kept on a diet with a relatively small amount of ZX, on average adding 5 mg of ZX per kilogram of feed. Since Japanese grouse eats approximately 25-35 g of feed per day, in the low-dose ZX group per bird per day, approximately 0.125-0.175 mg of ZX was consumed. Birds of the ZX group (+50) were kept on a diet with a tenfold increase in the amount of ZX (50 mg ZX per kilogram of feed), with each bird absorbing approximately 1.25-1.75 mg ZX per day.

 The concentrations of carotenoids in the control feed, in the carotenoid-deficient feed and in the feed for the ZX + group were analyzed using high-performance liquid chromatography (HPLC) using the methods described by Stacewicz-Sapuntzakis et al., 1993. The results are shown in Table 1.

 All birds were reared and kept in ordinary brood cages. Except for the cases indicated below, they were kept under normal lighting with a wide spectrum of waves, the duration of which ranged from 10 to 14 hours per day.

Example 6. Retinal deposition of zeaxanthin. orally administered
Chemically analyzed the concentrations of ZX (and other carotenoids) accumulated in the retinas of birds in each of the groups contained in the different diets described in example 5.

To carry out these analyzes, birds that hatched from carotenoid-deficient eggs and kept on an appropriate diet for at least 6 months were killed by twisting the neck. Retinal tissue was isolated by dissection enucleated eye, and tissue separate the retina was ground to near homogeneity into 250 .mu.l of distilled deionized water, using a glass pestle pestle or polytetrafluoroethylene (TEFLON ^®). 10 μl was taken from this homogenate and used to analyze the protein content of the homogenate in order to standardize the results obtained for various retinal samples. To the remaining 240 μl of a retinal tissue suspension, 250 μl of methanol containing 2% w / v was added. pyrogallol and 50 μl of potassium hydroxide at a concentration of 60% w / v. The mixture was kept in a water bath at 70 ^° C for 1 h, then 500 μl of 50 vol% ethanol was added, followed by 2 ml of hexane. The mixture was vigorously stirred for thorough mixing, then left to stand for separation at 5 ^° C. The epiphase (i.e., the lighter phase that floated to the surface of the remaining liquid) containing hexane and extracted carotenoids, tocopherols and retinols was removed. An additional 2 ml of hexane was added to the tissue homogenate and the mixture was vigorously mixed and allowed to separate again. This epiphase was removed and combined with the first. This process was repeated again using the third extraction cycle in order to ensure complete extraction of carotenoids, tocopherols and retinoids.

 The combined hexane extracts were then washed with 1 ml of water to remove residual potassium hydroxide. An additional 1 ml of hexane was added to the hexane-water mixture, then the hexane layer (epiphase) was carefully removed with a pipette. After that, hexane was evaporated in a constant stream of nitrogen gas. The resulting residue contained carotenoids, tocopherols and retinols and other unidentified hexane extractable compounds. This residue was then dissolved in a solvent (methanol: chloroform: triethylamine) and analyzed by HPLC as described above.

 The results are presented below in table 2, which also shows the levels of damage after high-intensity light exposure.

 A lutein-like compound was detected, which is probably not lutein, which was determined by the retention time during HPLC and scanning of the peak in question using a set of photodiodes.

 It should be noted that β-carotene was not found in all retinas taken from birds on any of the described diets.

 The concentrations of carotenoids in the retina shown in Table 2 indicate that when administered orally, RR-zeaxanthin obtained by fermentation of cells that are descendants of Flavobacterium multivorum (ATCC registration number 55238) is indeed deposited in the retinas of experimental animals that received RR-zeaxanthin in food diet as a dietary supplement. This is an important discovery because ZX must be digested in the usual way, must overcome the intestinal barrier, must enter the bloodstream, and must be absorbed by the retinal cells of birds in sufficient quantities to protect the retinal tissue from phototoxic damage. All of these barriers were overcome by using the preparations of R-R-zeaxanthin provided by bacterial fermentation as provided herein.

Example 7. Protection of the retina by RR-zeaxanthin
A part of the birds in each group on a different diet were exposed to high-intensity light from the visible part of the spectrum at 2000-3000 lux for 28 hours using cycles consisting of a one-hour light period followed by a two-hour period of almost complete darkness. After a period of cyclic illumination, the birds were placed in almost complete darkness for 14 hours, after which they were sacrificed. Such high-intensity light exposure, as was established in preliminary experiments, causes significant serious damage in birds of the carotenoid-deficient group and moderate damage in birds of the control group (which were kept on a normal diet). In preliminary experiments, it was also found that after irradiation with light, a 14-hour period is required to measure the maximum number of apoptotic cone nuclei in unprotected (carotenoid-deficient) birds.

 In birds, which were kept on the control diet, maximum apoptosis was detected approximately 24 hours after irradiation, while in birds, which were kept on the diet with the addition of ZX, maximal apoptosis was detected after a significantly longer period of time than 24 hours. An increase in the length of time before damage can be detected is a reliable indicator of ZX's protective actions.

The retinas of these birds were isolated by micro-dissection, fixed in xylene and dehydrated in ethanol before inclusion in the paraplast (Oxford, 56 ^o C). Then sections of retinal tissues located in the paraplast were made and stained according to the method of Gallyas, 1990, or with propidium iodide to visualize the pycnotic nuclei characteristic of apoptosis. The number of pycnosis nuclei, which can be detected in one field of view of the microscope at 400-fold (linear) magnification, was calculated. For each experimental group, cores were counted in at least 6-8 separate fields of view and the obtained values were averaged.

 The results shown in Table 2 clearly show that the death and damage of the retinal cells (1) was greatly reduced and / or slowed down using the RR-zeaxanthin synthesized by bacteria even at a low dose of ZX (+5) compared to birds that were kept on normal control diet, and (2) it decreased even more when using a higher dose of ZX (+50). The complete absence of any pycnotic nuclei in the retina of the ZX-treated group (+50) is unconditional evidence that RR-zeaxanthin from F. multivorum cells (ATCC registration number 55238) represents a significant step forward in the protection of retinal cells from phototoxic damage. According to the information available to the applicants and their conviction, no other agent ever studied allowed to achieve this level of protection or even to approach it.

Example 8. Direct comparison of the effectiveness of RR-zeaxanthin and β-carotene in protecting retinal tissue
As indicated above, the birds that were on the BC + diet received a dose of β-carotene (50 mg per kilogram of feed), which is equivalent to the dose of zeaxanthin in the ZX group (+50). This allows a direct comparison of the efficacy of β-carotene and ZX to protect retinal tissue from phototoxic damage.

 The results showed that phototoxic damage in the BC + group was reduced only to a very small extent (on average by about 10% or less). Compared with standard deviations in the control group, this decrease was statistically unreliable; the probability that small declines were due solely to random fluctuations was 0.12-0.14.

 This inability of β-carotene to provide better protection against phototoxic damage to retinal tissue, while R-R-zeaxanthin at the same dose caused an almost 100% decrease in the same indicator of damage and cell death, clearly demonstrates the importance of this discovery. R-R-zeaxanthin synthesized by microbial fermentation is a significant achievement superior to any previously known agents.

Example 9. Obtaining absorption enhancers
Zeaxanthin-containing "micelles" with a diameter of less than 1 μm, which include only the desired RR isomer of zeaxanthin, can be obtained either from a biomass extract obtained using a solvent or from the oily liquid described in example 3 using certain types of bile salts, as described by Olson, 1994. An oily liquid containing RR-zeaxanthin can be mixed with an acceptable bile salt, such as phosphate salts of glyco or taurocholate, available from Marcor Development Company of Hackensack, New Jersey, or using Hovhan gall bladder extracts containing salts of bile acids that are available from Saizman Corporation of Davenport, Iowa. This gallbladder product can be mixed either with an extract obtained with a solvent, or with an oily mass and with other specific salts, including sodium chloride, calcium chloride or potassium chloride. This mixture is then processed in a mechanical homogenizer that contains mixing devices, such as rotating blades, the speed and duration of rotation of which can be optimized using a standard experiment by analyzing the range of micelle sizes obtained using various combinations of blade size and shape, speed and duration of rotation. If necessary, the resulting micelles are dried, free from solvent, then diluted to any desired concentration using a carrier or dilution liquid, such as vegetable oil. Then this mixture can be included in a capsule or other device, which should help swallow and protect the micelles from decomposition in the acidic environment of the stomach.

 Other emulsifiers and lipids can also be used to obtain emulsions with small particle sizes. Nonionic surfactants, such as twins and spans, as described by Olson, 1994, as well as lipid materials such as phospholipids and sphingolipids, which can form small lipid vesicles (less than 1 μm), can be used.

Example 10. Microencapsulated Zeaxanthin
This example describes the preparation of zeaxanthin in microencapsulated form. Microcapsules are solid particles 10-1000 μm in size, consisting of a core material (such as RR-zeaxanthin) encapsulated with a coating or shell material that can be obtained from various compounds such as gelatin, gum arabic, starch, zein ( protein from corn), etc. In the process of manufacturing the shell material, other compounds can also be added to help maintain the shape, structure, stability, or other required characteristics of the resulting preparation. Such compounds may include emulsifiers, sorbitol, antioxidants such as TBNX or 2- (1,1-dimethylethyl) -1,4-benzenediol, or gelling agents such as carrageenan.

 Pure or partially purified zeaxanthin is dissolved in an appropriate solvent, such as ethanol, acetone or THF. In a solution, when dissolved zeaxanthin is added to water, microcrystals with a diameter of less than 10 microns are formed. This process is improved if, during the addition, water and zeaxanthin crystals in the solvent are sonicated at high frequency in the presence of emulsifiers such as Tween 80.

After the formation of microcrystals, shell material is added to a mixture of water, zeaxanthin and a solvent. For some shell materials, such as gelatin, it is necessary to maintain the temperature at 60 ^° C for about 2 hours. After complete dissolution of the shell material, the entire mixture is placed in an ultrasonic irradiation device (sonicator) for 5-10 minutes in order to re-emulsify the crystals.

 Microcapsules are formed by drying a mixture of core and shell material using any suitable drying method, such as spray drying, or using a spinning disc in accordance with Sparks et al. Described in US Pat. No. 4,675,140. Spray dryers are widely used in the food industry. and in the production of feed. A rotating disk is a device that consists of a disk (approximately 4 inches in diameter) that can be held at a certain temperature under controlled conditions. It can operate at various speeds ranging from 1000 to 10,000 rpm. A mixture of core and shell materials is added to the center of the disk as it rotates at a speed of, for example, 4000 rpm. Microcapsules are formed by contacting a liquid with a heated rotating disk. Microcapsules are discarded from the center of the disk by centrifugal force and collected on a flat surface that is pre-coated with a “collecting” or “trapping” agent, such as hydrophobic starch or dextrin. The microcapsules are then separated from the capture agent by sieving through a calibration sieve. Microcapsules are placed in a container to protect them from light and air and stored refrigerated until packaged in capsules for oral administration.

 Thus, the above described new methods of creating drugs that contain R-R-zeaxanthin, intended for oral administration by humans, as well as compositions that contain synthesized by microorganisms R-R-zeaxanthin and which are intended to prevent or treat macular degeneration. Although the present invention has been explained with specific examples and described with reference to certain specific embodiments, it will be apparent to those skilled in the art that various modifications, changes, and equivalent replacements are possible for illustrative examples. It is intended that any such changes which arise directly from the present description and which do not deviate from the essence and scope of the invention fall within the scope of the invention.

Claims (21)

 1. The use of the 3R-3'R stereoisomer of zeaxanthin as a medicine for the treatment of macular degeneration in humans.  2. The use of claim 1, wherein the drug is a unit dosage form suitable for administration to humans and containing a sufficient amount of zeaxanthin to provide a beneficial therapeutic effect for a person suffering from macular degeneration.  3. The use of claim 2, wherein the unit dosage form contains at least about 1 mg of the 3R-3'R stereoisomer of zeaxanthin.  4. The use of the 3R-3'R stereoisomer of zeaxanthin as a dietary supplement, which is intended for administration to humans in order to reduce the risk of macular degeneration.  5. The use according to claim 4, where the food supplement is intended for the treatment of a patient suffering from Stargart's disease, Best’s disease, Button’s disease, Sjogren-Larsson’s syndrome, dystrophy of rod cones, sheep’s waxy lipofuscinosis, a disease caused by accumulation problems in lysosomes or increased genetic sensitivity to macular degeneration.  6. The use of claim 4 or 5, wherein the nutritional supplement is formulated in a unit dosage form that contains at least about 0.1 mg of the zeaxanthin 3R-3'R stereoisomer.  7. The use according to any one of claims 1 to 6, wherein zeaxanthin is prepared by a method that includes culturing in a liquid medium and under conditions stimulating the synthesis of zeaxanthin cells that synthesize a 3R-3'R stereoisomer of zeaxanthin in an amount of at least 90 % of the total number of carotenoid molecules synthesized by cells.  8. The use according to claim 7, where the cells synthesize the 3R-3'R stereoisomer of zeaxanthin without synthesizing any noticeable amount of any other stereoisomer of zeaxanthin.  9. The use according to claim 5, where the cells are bacterial cells derived from a strain of Flavobacterium multivorum, which was assigned ATCC registration number 55238.  10. The use according to claim 5, where the cells were subjected to genetic engineering methods so that they contain at least one gene that synthesizes zeaxanthin and includes a DNA sequence obtained from cells that are descendants of the Flavobacterium multivorum strain, which was assigned a registration number ATCC 55238.  11. A composition for treating or preventing macular degeneration in humans, comprising a 3R-3'R stereoisomer of zeaxanthin in an amount that is therapeutically acceptable for humans, wherein the 3R-3'R stereoisomer of zeaxanthin is at least about 90% of the total the amounts of zeaxanthin, and the SS and SR stereoisomers comprise less than about 10% of the total amount of zeaxanthin in the composition and a physiologically acceptable excipient, solvent or carrier.  12. A composition for treating or preventing macular degeneration in humans, comprising a 3R-3'R stereoisomer of zeaxanthin in a carrier or solvent and for oral administration to humans in a therapeutically effective amount.  13. The composition of claim 12, wherein the 3R-3'R stereoisomer of zeaxanthin comprises at least about 90% of the total amount of zeaxanthin, and the S-S and S-R stereoisomers comprise less than about 10% of the total amount of zeaxanthin in the composition.  14. The composition according to item 12, which practically does not contain either the S-S- or R-S-stereoisomer of zeaxanthin.  15. The composition according to any one of paragraphs.11-14 in the form of a standard dosage form, each standard dosage form containing at least about 0.1 mg of the 3R-3'R stereoisomer of zeaxanthin.  16. The composition according to any one of paragraphs.11-14 in the form of a standard dosage form, each standard dosage form containing at least about 1 mg of the 3R-3'R stereoisomer of zeaxanthin.  17. The composition according to any one of paragraphs.11-14, where the carrier is a food product intended for oral administration by humans.  18. The composition according to any one of paragraphs.11-17, characterized in that it has a protective coating designed to reduce the decomposition of zeaxanthin in the stomach.  19. The composition according to any one of claims 11-17, comprising non-pathogenic microbial cells that contain a 3R-3'R stereoisomer of zeaxanthin at a concentration of at least 2 wt.%, As a fraction of the microbial cell mass.  20. The composition according to any one of paragraphs.11-19, where zeaxanthin is prepared by a method that includes culturing in a liquid medium and under conditions stimulating the synthesis of zeaxanthin cells that synthesize the 3R-3'R stereoisomer of zeaxanthin in an amount that is at least 90 % of the total number of carotenoid molecules synthesized by cells.  21. The composition according to p. 20, where the cells are bacterial cells that are progeny of a strain of Elavobacterium multivorum, which was assigned a registration number ATCC 55238.

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