TW208718B - - Google Patents

Description

A6 B6 08718 Fifth, the description of the invention (1) The field of the invention (please read the precautions on the back before filling in this page) The present invention provides a method for manufacturing zeaxanthin, which includes a method for manufacturing biological withdrawal, and these Withdrawing organisms can produce zeaxanthin. The present invention further provides compositions (these compositions contain corn yellow produced by the method of the present invention) to improve the coloring effect and reduce the damage caused by the active gas species and the light-damaging molecules. BACKGROUND OF THE INVENTION Zeaxanthin, or 3, -2 &-/ S-carotene, is a natural carotenoid ethanol and is very useful for food and cosmetics. For example, zeaxanthin imparts a desirable yellow to reddish yellow color on the meat, skin, and eggs of poultry and fish. Zeaxanthin is a food pigment that is much better than chemically synthesized pigments. Many chemically synthesized pigments have been banned by government agencies because of the possibility of mutagenesis and carcinogenesis. Natural carotenoids obviously have no toxic effects even in high units, so they can replace yellow and red pigments that have been shown to be harmful to animals. Some glucosinolates, such as zeaxanthin, are pseudo-effective anti-gasification agents because they have paired chemical structures. As a result, huconin can also be used to reduce tissue damage caused by airless groups, prevent certain cancers, and stabilize vaporized compounds, except when exposed to light. • Order · The Ministry of Economy's Shiyang Biao Bureau's Zhen Gong Consumers Cooperate with Du Printing. Line · Zeaxanthin is used to improve the coloring of poultry and fish than other types of huosu, it is because Its potency, it can provide true colors, and it can be evenly distributed on meat and eggs. Zeaxanthin is at least 1.5 times more potent than lutein. When used in high doses in poultry, the currently used carotenoids or compounds containing glucocorticoids, such as canthaxanthin, alfalfa, and cayenne pepper, can cause abnormalities in meat Red or purple and cause the yolk color to be striped. The use of high doses of lutein will cause poultry meat and egg yolk to appear green. / 9-Carotenoids causes poor deposition in poultry meat, while canthaxanthin deposits in the iris of animal eyes. In contrast to zeaxanthin, according to the corn feeding report with high zeaxanthin content, high-dose zeaxanthin can evenly apply a yellow-red color to poultry meat and deposit well. Furthermore, at least some fish and crustaceans (such as shrimp, goldfish, and carp) can convert zeaxanthin to red astaxanthin. This point is showing that feeding these fish and crustaceans with zeaxanthin will increase the red coloring satisfactorily. Zeaxanthin is produced together with other carotenoids in certain plants (such as corn, calendula, and alfalfa). Although farmers use these plants to feed poultry, this food supplement is impractical because of the high cost and uneven quality of the corn yellow produced by these plants. This paper scale is suitable for China National Standards (CNS) A 4 specifications (210x297 mm) _ 3-A6 B6 08sn3 V. Description of invention (2) (please fill in this page before paying attention to twitlr.) Zeaxanthin is purified from natural resources, but the quality of zeaxanthin produced is very low. Furthermore, the high cost of chemically synthesizing zeaxanthin using off-the-shelf technology becomes a major obstacle. Especially because synthetically manufactured compounds have health risks, it is best to use natural methods to make food additives (for example, bio-synthesis methods). ^ The official publication of the American Feeding Control Association lists the "Generally Recognized Safety" (GRAS) approved by the US Food and Drug Administration (FDA), which can be used as food additives and used to make food additives. Because food additives must be approved by government agencies, it is best to use these "recognized as safe" organisms to make zeaxanthin. However, current inventors cannot find enough zeaxanthin to be used in commercial production from the "recognized as safe" organisms. • Playing · Among the "recognized safe" ® species approved by the FDA, there is Spongiococcum, which is the only algae that has been "recognized as safe" so far and can be used to feed poultry to enhance the yellow color. (21 C.F.R. § 73.275) Some researchers have also tried using Spongiococcum and other algae biosynthesis to synthesize carotenoids, but no one has been able to successfully isolate commercially viable zeaxanthin. In fact, the researchers ’reports only say that algae produce low amounts of lutein groups, and zeaxanthin is only a part of the lutein group: (Lutein group is rich in many oxygen-containing huskulins, such as astaxanthin, eye corners Flavin, cryptoxanthin, lutein, rosoflavin, anthocyanin, tung lutein, and zeaxanthin) such as Kathrein's US Patent 3,108,402 (October 29, 1963) using a Spong 丨The ococcum biotech produces a total of 300 mg (mg) of flavonoids in each liter of fermentation medium. Similarly, Kathrein ’s U.S. Patent 2,949,700 (August 23, 1960) disclosed the use of non- "recognized safe" Chlorella and Chlorococcum streams to produce a total amount of lutein groups up to approximately L80 mg per liter of fermentation medium . • Line · Researchers have also reported the use of several non- "recognized safe" bacteria, fungi, and sponges to make zeaxanthin, but no method has been put into commercial use. For example, U.S. Patent 2,974,044 (1961; July 7) of Farrow et al .; U.S. Patent 3,841,967 (October 15, 1974) of DaseK et ak ,; U.S. Patent 3,891,504 (June 24, 1975) of Schocher. ); Shepherd et al. U.S. Patents 3,951,742 and 3,951,743 (April 20, 1976); Misawa et al. European Patent Publication 393690 (October 24, 1990); Gierhart ’s PCT International Publication W0 91/03571 No. (Announcement on March 21, 1991). The ft scale of this paper is applicable to China's National Standards (CNS) A4 specifications (2 丨 0x297mm) -4 08718 A6 B6 V. Description of the invention (3) The summary of the present invention is directed to a method for manufacturing zeaxanthin The method includes ①mutating algae withdrawing organisms, ②selecting a cultivating zeaxanthin from those mutated organisms, ③cultivating the selected cultivating organism in an effective medium to produce zeaxanthin, ④recovering Zeaxanthin produced by the selected bio-organism. The present invention further provides a biological substance capable of producing zeaxanthin, provides a composition encapsulating the zeaxanthin produced by the method of the present invention, and uses these compositions to improve the color of the cerebrum and reduce the reactive oxygen species and light Damage caused by damage molecules. According to one embodiment of the present invention, a method for producing zeaxanthin includes cultivating a zeaxanthin capable of producing 0.35 mg of zeaxanthin per gram of stem cells in an effective medium, and then recovering the corn produced by the microorganism Yellow matter. The better organism is Spongiococcum strain. In one embodiment, a biopill that can produce at least about 0.35 mg of zeaxanthin under the weight of each gram of stem cells is cultivated in continuous fermentation. A preferred tandem fermentation is carried out in an effective medium, the temperature is between about 30 degrees Celsius and 38 degrees Celsius, the acid age is between about 5.5 and 6.5, and the dissolved oxygen concentration is at least ten percent. And the pressure of the master part of the dioxide is pseudo-less than 0.04 atm. In an embodiment of the present invention, the method for recovering zeaxanthin is to separate the zeaxanthin-producing organisms from the effective culture medium to form a zeaxanthin-containing bio-periphery. The recovery method may include the step of rinsing the zeaxanthin-containing biomass at least once to remove at least a portion of the chlorophyll contaminants, thereby reducing the undesirable marginal color. In another embodiment, zeaxanthin is pseudo-extracted from organic organisms that can produce zeaxanthin using organic solvents. The purity of zeaxanthin recovered by this method is proven to be at least about 90%, and can be as high as about 100%. In an embodiment of the present invention, zeaxanthin is manufactured by an extractor capable of producing at least about 0.35 mg zeaxanthin per gram of stem cell weight, wherein zeaxanthin accounts for at least 5% of the total carotenoids production %. Furthermore, it is best to remove at least 1.75 mg of zeaxanthin per gram of stem cell replacement, that is, zeaxanthin accounts for at least about 25% of the total carotenoids. In another embodiment, zeaxanthin is made from an extract that can produce at least about 2.8 mg zeaxanthin per gram of stem cell weight, that is, zeaxanthin accounts for at least about 40% of the total carotenoids %. According to another embodiment of the present invention, zeaxanthin algae biomolecules can be produced by mutating the parent algae biomolecule and selecting one of the mutant biomolecules to produce at least about 0.35 mg of corn zeax per gram of stem cell weight quality. The preferred parent microorganism is Spongiococcum. Most of the best Xie organisms have the classification of Spongiococcum excentricum ATCC No. 40335 and its mutants. In one embodiment, the algae biological system is exposed to a mutagen (mutagenic factor) to produce mutations, such as nitrogen-methyl- 気 -nitro-atmo-nitrosoguanidine, ultraviolet, X-ray, gamma Ray, ethylmethane sulfonate This paper scale is applicable to China National Standards (CNS) A 4 specifications (210x297 mm) _ 5 _ (please read the precautions on the back before filling this page) Pack- • Order… ： 人 • 绿_, 0871 ^ A6 13 6 5. Description of the invention (4), nitrous acid, or a mixture thereof. After the mutation procedure, we exposed the mutant organisms to a metabolic inhibitor (factor) to select the organism capable of producing zeaxanthin, and the metabolic inhibitor was selected from the isoprenoid pathway inhibitor, chrysanthemum Selected from biosynthesis inhibitors, free radical generators, and their mixtures. In one embodiment, the mutated carrier is exposed to an isoprenoid pathway inhibitor, such inhibitors such as nystatin, anti-embryonic acid, mevinolin, saponin, amphotericin B, dish salt farnesyl compound, azasqual ^ es, allyl derivatives, thiocarbamate, pyrimidine, squint vl, morpholines, or mixtures thereof. In another embodiment, the mutated biopsy is pseudo-exposed to a carotenoid S biosynthesis inhibitor, such as norflurazon, metf lurazon, benzophenone Bf |) ketone, diphenyl, niacin, oxyfluorfen, fluorfen , / 3-ion ketone, or a mixture of them. In another embodiment, the mutated bio-image is exposed to a fluid-based generator, such as hard electricity ^ other 4 types, peroxide, ultraviolet light, X-rays, gamma rays, odor, or a mixture thereof to produce corn Microbes of xanthin can also be separated based on the i color, where the color pseudo-represents zeaxanthin. The present invention produces algae (preferably Spongiococcum) withdrawn organisms. These organisms can produce at least about 0.35 mg of zeaxanthin per gram of stem cell weight, and zeaxanthin preferably accounts for at least the total carrot-like biomass 5% of prime production 1. In another embodiment of the present invention, algae (preferably Spongiococcum) can produce at least about 1.75 milligrams of zeaxanthin per gram of dry fines, which means that zeaxanthin accounts for at least about 25% of carotenoids production. In another embodiment, zeaxanthin is produced by algae (preferably Spongiococcum) that produces at least about 2.8 mg zeaxanthin per gram of stem cell weight, that is, zeaxanthin accounts for at least about When withdrawing 40% of the total carotenoids production. Printed by the Employees Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) Ding ’s best Spongiococcum strain is ① with Spongiococcum excentricum ATCC Ho.74108 (hereinafter referred to as HZ1236 / 274) and its mutation The classification of organisms, in which mutants can produce 3 zeaxanthin less than about 0.35 milligrams per gram of stem cell weight; ② Youzhi Spongiococcum excentricum ATCC No. 74109 (known as HZ1236 / 437) and its variants , Wherein the variants can produce at least about 0.35 mg of zeaxanthin per gram of stem cell weight; and ③ Yougong Spongiococcum excentricum ATCC Νο.74ί〇7 (hereinafter referred to as YaoHZ1236 / 538) and the classification of its variants is pending, of which The variant can produce at least about 0.3 mg of zeaxanthin per gram of stem cell weight. A preferred embodiment of the present invention is a method for pseudo-manufacturing zeaxanthin, which method includes (a) making Spongiococcum barley seeds & withdraw biological mutations, (b) selecting from mutant organisms that can produce at least one gram of stem cell weight per gram About 0.35 milligrams of zeaxanthin organisms, (c) cultivating the selected microorganisms in an effective medium to produce a satisfactory amount of three beiges, and (d) recovering the corn produced by the selected ceresin Yellow matter. A better example includes the selection and preparation of this paper. The Chinese standard (CNS) A4 specification (210X297) * is used for this paper size-6-A6 B6, 08 'plus five, description of invention (5 Please read the notes on the back first Fill in the page pack and use at least about 1,75 mg of zeaxanthin per gram of stem cell weight. Other preferred embodiments include selecting zeaxanthin that can produce at least about 2.8 mg per gram of stem cell weight. According to another embodiment of the present invention, a § quasi-biological (preferably pseudo Spongiococcum) pseudo-organism (preferably pseudo Spongiococcum) capable of producing at least about 0.35 milligrams of zeaxanthin from each stem cell replacement is used to manufacture zeaxanthin. The manufacturing method includes culturing The Bio-E manufactures and recycles zeaxanthin. In a preferred embodiment, we selected a mutant algae organism to manufacture because it can produce at least 0.35 mg of zeaxanthin per gram of stem cell weight. The microorganism The best is Spongiococcum. The present invention 3-Example Ye a composition containing zeaxanthin, the composition includes algae biomolecules (preferably Spongiococcum), the biological energy Each gram of stem cells is reset to produce at least about 0.35 milligrams of zeaxanthin, of which zeaxanthin accounts for at least about 5% of the total production of Hutuoxin in this bio. In a preferred embodiment, one contains corn I: The qualitative composition includes zeaxanthin biomass and animal feed for feeding food animals to enhance the color effect. Preferred food animals include poultry, fish, and crustaceans. Other embodiments of the invention include the use of Zeaxanthin produced by algae organisms (preferably Spongiococcum) is used to enhance the color or reduce the damage caused by the active gas species and light-damaged molecules. Bio-organisms, methods for manufacturing such bio-organisms, and the use of such bio-organisms to produce large quantities of commercially viable zeaxanthin. The present invention further provides a composition comprising zeaxanthin (the zeaxanthin It is produced by the method of the present invention), and the composition is used to enhance the coloring effect and reduce the damage caused by active oxygen species and light-loss molecules. Production can produce jade The xanthin is produced by the Ministry of Economic Affairs, Central Bureau of Standards, and consumption of employees. The invention provides algae organisms capable of producing zeaxanthin. Here, an evacuated organism capable of producing zeaxanthin is capable of measuring the weight of stem cells per gram. Produce at least about 0.35 milligrams of zeaxanthin. A biomolecule that can make zeaxanthin is best to produce zeaxanthin that accounts for at least about 5% of all its carotenoid S. Yield here. Carotenoids refer to all carotenoids produced by the withdrawal organism, including but not limited to other xanthophyll groups (such as lutein). According to the one-shot embodiment of the present invention, the withdrawal organism for producing zeaxanthin Like by mutating the algae parent organism and selecting the organism that can make zeaxanthin. The parent microorganism here refers to any withdrawing organism with mutation, the purpose of which is to obtain an withdrawing organism that can produce more zeaxanthin than the parent. Maternal withdrawal includes but is not limited to natural products, variants, and evening paper standards. Chinese National Standard (CNS) Grade 4 specifications (210X297 mm) _ 7 _

A 6 B6 V. Description of the invention (6) Thoroughly mutated or previously selected. The parent and the zeaxanthin-producing microorganisms in the present invention may be recombinant or non-recombinant withdrawn organisms. (Please read the precautions on the back before writing this page.) In one embodiment of the present invention, the parent algae olive is pseudo-exposed to a selected culture medium, which enables the growth of zeaxanthin-producing biomass. Thus, natural mutants of the parent strain that can produce zeaxanthin can be isolated. In a preferred embodiment of the present invention, the parent biotic system is subjected to at least one chemical or physical mutation source to increase the rate of occurrence of mutations, thereby increasing the probability that we can obtain a biologic organism that produces zeaxanthin. The mutant microorganism here refers to a mutant parent organism, where the mutation is naturally occurring or the result of exposure to a mutagen (factor). The preferred parent organism of the present invention is algae, more preferably Spongiococcum algae. The best parental organism is Spongiococcum excentricura ATCC No. 40335, which is the subject of US Patent Application No. 07 / 474,248 (invented by Liao et al, filed on February 5, 1990) under pseudo-trial.

Spongiococcum algae has many advantages over other algae, including its ability to grow to a cell density that is at least 4 times higher than that of other leaf-making algae. Other algae such as Chlorella (Farraw et al. US Patent No. 3,280,50, 1990, October 10) September 25th), or some Flavobacter um raultivorum strains (Gerert PCT International Bulletin No. W0 91/03571, published on March 21, 1991). Higher cell density results in more production per unit volume, thereby reducing production costs. In addition, the intracellular products of Spongiococcum are philosophically available to animals because animals can digest the cell walls of Spongiococcum and obtain intracellular products. Therefore, the whole Spongiococcum cells can be directly used to feed animals. Conversely, most of the algae that make lutein groups (such as Chlorella) cannot be directly used to feed animals, because the cell walls of these algae are made of cellulose material and cannot be digested by most non-ruminant animals. Being able to feed necrotic cells to animals (such as poultry) can avoid additional downstream recycling procedures, which can reduce manufacturing costs and reduce zeaxanthin losses. Furthermore, because higher zeaxanthin production energy can be obtained according to the present invention, less total cell weight is required to produce the same amount of zeaxanthin. Printed by the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs.......... In one embodiment of the present invention, the algae (Spongiococcum algae most released) bees are exposed to a Chemical or physical Ϊ mutagens (factors) can be used to make zeaxanthin extracts. Preferred mutagens include, but are not limited to, nitrogen-methyl-nitro-nitro-nitrosoguanidine (NTG), ultraviolet light, X-rays, gamma rays, ethylmethane sulfonate and nitrous acid. These mutagen can be used alone or in combination. The best mutagen used in the present invention is NTG: In an embodiment of the present invention, Spongiococcum excentricum ATCC No. 40335 is contained in a buffer solution known to be exposed to biological images, and then the biological extract is rinsed and resuspended in a growth Culture medium (for example, a yeast extract / $ / glucose (YEP) medium), and cultivate overnight at about 35 degrees Celsius to obtain the mutant Spongiococcum, and use the Chinese paper standard (CHS) A4 specification (210x297 Long) -8-V. Description of the invention (7) A 6 B 6 The Ministry of Economic Affairs Central Standards Bureau employee consumer cooperative printed bio. The mutated organisms can then be grown on solidified growth medium at approximately 35 degrees Celsius for approximately 5 to 7 days to obtain a single flora from the city. The solidified growth medium here refers to a growth medium added with a solidified substance, such solidified cedar such as gel, agarose, or agar. A preferred solidified growth medium of the present invention is a yeast extract glucose medium plus agar (YEP-agar). The best case is to expose the evacuated creature to every millisecond at about 35 degrees Celsius? The citrate buffer contains about 25 ug of NTG for about 30 minutes. Another aspect of the present invention selects better mutant algae withdrawing organisms. Strategies for better selection and identification of mutant microorganisms that can produce zeaxanthin include selection by color and selection by its ability to resist metabolic inhibitors (that is, its ability to grow in metabolic inhibitors). Metabolic inhibitors include but are not limited to inhibitors of the isoprenoid pathway, inhibitors of carotenoid biosynthesis, and free radical generators. Metabolic inhibitors can be used alone or in combination. The selected Tie organisms (that is, the Tie organisms selected according to the selection strategy of the present invention) are grown in an effective medium to determine the quality of zeaxanthin that the Tie organisms can produce. Zeaxanthin produced by selected strains can be determined by different methods, including but not limited to functional and chromatographic verification methods. In the present invention, the preferred method for determining the production of the biological zeaxanthin is reverse phase high pressure liquid chromatography (HPLC). —......................... According to an embodiment of the invention, the mutant biopsy is in a solidified growth medium (eg YEP -Qi fat culture medium), and choose according to color. The use of color to select mutant withdrawal organisms is a useful method to identify the withdrawal metabolites that can produce zeaxanthin. Thorough organisms that can make zeaxanthin usually show olive lines, yellow, red yellow, orange, or red. These colors here are referred to as the colors representing zeaxanthin production. The algae organisms that normally make zeaxanthin also show a background edge color because of the chlorophyll production. In the present invention, the microorganisms selected according to the color are referred to as the color selected by the color. The preferred colors (in terms of the selected organisms selected by the color) include yellow, red yellow, orange, and red. In a preferred embodiment, the mutated withdrawal organism is grown in solid growth medium (for example, grown at about 35 degrees Celsius for about 5 to 7 days, and the manufacturer of zeaxanthin is selected according to the appearance color. According to the present invention In one embodiment, the mutant microorganism is pseudo-selected as the producer of zeaxanthin based on its ability to grow in metabolic inhibitors. Metabolic inhibitors are preferably selected from isoprenoid pathway inhibitors, carotenoid biosynthesis inhibitors, It is selected from the group of free radical generators. These inhibitors can be used alone or in combination. Thorough organisms capable of producing zeaxanthin can also be selected based on the common criteria of color and ability to grow in metabolic inhibitors. In the example, the mutant withdrawal biopsies were selected as zeaxanthin producers according to their ability to grow in a medium containing an isoprenoid pathway inhibitor. The isoprenoid pathway inhibitor is a compound that inhibits one more step in the isoprenoid synthesis pathway , Including steps to inhibit the pathway of sterol synthesis. Such inhibitors include, but are not limited to, angiobacteria & -9-i, please read the precautions before filling in this Stapling wire 3 vx ^ / ί • β ο C '

66 AB V. Description of the invention (8) Please. Read the back and face matters first and then fill out this page, anti-microtoxin A, tangerine microtome, mevinolin, saponin, amphotericin B, basic acid salt farnesyl compound, azasqualenes, allyl_ Derivatives, thiocarbamates, pyrimidines, azoles, triazoles, organic lines, and mixtures thereof. The most useful isoprenoid inhibitor in the present invention is mitostatin because it can bind to membranous ergosterol and significantly interfere with the cell membrane. In one embodiment, mutant mutant organisms are selected to produce zeaxanthin in a solid growth medium (eg, YEP agar medium), which contains at least about 1 gram of chrysanthemum per milliliter, and most Fortunately, there are about 1 gram to 20 gram of anemectin per milliliter. In a preferred embodiment, the mutated withdrawal biopsies are grown at about 35 degrees Celsius for about 10 days in a medium containing about 5 to 7 gram / ml of staurostatin. In the S-embodiment of the present invention, the mutant biopsy was selected as the zeaxanthin maker based on its ability to grow in a culture containing a compound that inhibits the carotenoid biosynthetic pathway. The inhibitor of the carotenoids biosynthetic pathway, Meng, inhibits one or more steps in the synthetic pathway of cucurbitacins. Carotenoid biosynthesis inhibitors include but are not limited to norflurazon, metflurazon, phenfuranone, diphenylamine, nicotinic acid, oxyfiuor'fen, nuorfen, yS-ion ketone, and mixtures thereof. The carotenoids biosynthesis inhibitor which is more useful in the present invention is norflurazon, which suppresses the desaturation reaction necessary for the production of carotenoid. In one yellow embodiment, the mutant Pseudobiopse was selected to make zeaxanthin on a solid growth medium (such as YEP-agar medium), which contains about 10 to 100 millicograms of norflurazon per ml at about 35 degrees Celsius. It grows at about 10 days. The preferred concentration of norflurazon in this medium is between about 60 milligrams / ml to 100 milligrams / ml. Printed by the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs In another embodiment of the present invention, the mutated biopsy is based on its ability to grow in a medium containing a compound capable of producing free radicals (ie, free radical generator) Selected as the producer of zeaxanthin. It is generally believed that because of their anti-Weiwei properties, glucosinolates can protect cells from free radical damage. However, the current inventors are not aware of the use of compounds capable of producing 抸 radicals to select biomolecules that can produce a large number of Hulu S-like elements (such as zeaxanthin). Free radical generators include, but are not limited to, & Class, pervaporates, ultraviolet rays, x-rays, gamma rays, odor, and mixtures thereof. Preferred free radical generators (such as electricians and pervaporates are easily absorbed by the organism and will not be mutagenized. The best and most suitable free radical generator in the present invention is hard iMb. One embodiment In this case, the mutated withdrawal organisms were selected for the manufacture of zeaxanthin based on their ability to grow on solid growth media, such as YEP-agar medium, containing approximately 20 to 100 It grows at about 35 degrees Celsius for about 10 days. The preferred hard electrode concentration is about 55 molecules. In an embodiment of the present invention, the parent algae bio-image is exposed to a suitable mutagen (preferably NTG ) In order to obtain the withdrawal of the exam. The bio-manufacturer who can make zeaxanthin chooses the mutation from the mutated organisms based on the color and its selection? This paper scale uses the Chinese National Standard (CNS) A 4 specifications (210x297 Long) -10-Λ 6 Η 6 〇 & Γχδ V. Description of the invention (9) Please read the notes on it before filling this page isoprenoid pathway inhibitor, carotenoid biosynthesis inhibitor • and / or Grows in a free radical generator The selected microorganisms that can produce at least about 0.35 milligrams of zeaxanthin from the replacement of each gram of stem cells are pseudo-isolated and cultivated in an effective medium. The above mutation and selection procedures can be repeated repeatedly to obtain more Removal of zeaxanthin. Preferably, the selected strain should produce at least about 0.7 mg of zeaxanthin per gram of stem cell weight, and more preferably at least about 1.75 mg of zeaxanthin per gram of stem cell weight. And it is best to make at least about 2.8 mg zeaxanthin per gram of stem cell weight. Furthermore, the zeaxanthin produced by a certain biodegradation should account for at least about 5% of the total carotenoids production, compared with Preferably it is at least about 10¾, more preferably at least about 25SS, and most preferably at least about 40% of the total carotene production is zeaxanthin. The better organisms of the present invention include the withdrawal of Spongiococcura strain and its mutants, The mutant pseudo can produce at least about 0.35 milligrams of zeaxanthin per gram of stem cell weight. A better algae removal organism in the present invention is Spongiococcim ATCC No. 74108 (HZ 1236/274), which is divided The specialty is that it can produce about 3.5 mg of zeaxanthin per gram of stem cell weight. Another classification feature is that the total carotenoids produced by ATC: No. 74108 (HZ 1236/274) are about 40¾ to 50% is zeaxanthin. 3 Spongiococcum excentricum HZ1236 / 274 was deposited in the American Variety Cultivation and Storage (ATCC) on September 27, 1991. The address is 12301 ParHawn Drive, Rockville, Maryland, USA, and its number is ATCC 74108. Another preferred algae withdrawing agent of the present invention is Spongiococcura excentricum ATCC N0.74109 (HZ1236 / 437), and its classification is characterized by its ability to produce about 3 mg of zeaxanthin per gram of stem cell weight. Another category is proud that about 40% of the total carotenoids produced by ATCC No. 74109 (HZ1236 / 437) are zeaxanthin. Spongiococcum exceirtricum HZ1236 / 437 was also deposited with ATCC on September 27, 1991, and its MJ Staff and Consumers Cooperative Association's central printing number was ATCC 74109. Another preferred algae withdrawing agent of the present invention is Spongiococcum excentricum ATCC Ho. 74107 (HZ1236 / 538), and its special classification is that it can produce 3 mg of zeaxanthin per gram of stem cell weight. Another classification feature is that ATCC No. 74107 (HZ1236 / 538) makes about 40% of the total carotenoids manufactured by Zeaxanthin.

Spongiococcum excentricum HZ1236 / 538 was also deposited with ATCC on September 27, 1991, and its stomach number was ATCC 74107. ATCC Ho.74108 (HZ1236 / 274), ATCC No.74109 (HZ1236 / 437), ft ATCC No.74107 (HZ1236 / 538) are based on the Budapest Treaty of internationally recognized biological deposits for the purpose of patent procedures This paper scale is deposited with the Chinese National Standards (CNS) Grade 4 (210x297 g)-11-Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs V. Invention Instructions (10) and deposited. After the patent is approved, all regulations restricting the public ’s access to the deposited materials are definitely cancelled. Each test item should be stored for at least five years after the ATCC recently received a request to submit a sample of the stored organism, and under any circumstances should be stored for at least 30 years from the date of storage. Anyone who is familiar with this skill can understand that the present invention includes a thorough organism for the production of zeaxanthin, and that the proud biological image can be obtained through the method of transmission engineering. For example, the scope of the present invention includes the use of zeaxanthin biosynthetic pathway-encoded cormorants (which are obtained from the removal of algae such as Spongiococcmn that can produce zeaxanthin) to transform organisms. This gene may be a natural DNA sequence or a sequence obtained through the mutation source and the selection technique described herein. On the contrary, algae withdrawing organisms (such as Spongiococcum) can be converted by zeaxanthin biosynthesis pathway genes (this | because Ye is isolated from algae or other suitable organisms.) Manufacturing zeaxanthin The method of xanthin, which is to cultivate algae organisms capable of producing jade xanthin in an effective medium and recover zeaxanthin from it. According to the present invention, the algae bio-system capable of producing zeaxanthin is cultured in an effective medium (here, the culture I refers to any medium that can promote the production of zeaxanthin): preferably the effective medium can promote the growth of algae . The zeaxanthin-producing algae organisms in this Committee can also be cultivated by traditional fermentation methods, including but not limited to it, batch supply, and continuous fermentation methods. In an embodiment of the present invention, algae organisms (preferably Spongiococcura strains) are pseudo-cultivated in a continuous mounting fermentation process, and fresh fermentation medium is continuously or intermittently added during the process and used fermentation liquid is removed . During the fattening process, the changing factors including temperature, gas content, sulfide content of two gasification, pH value, and the addition rate of sulfonate are all controlled by Ϊ, so that the most zeaxanthin can be produced at the same time. Locally limit the time required for successful fermentation. Appropriate conditions and procedures are similar to pending US Patent Application No. 07 / 524,140 (Bailey et al., Filed on May 15, 1990). Bailey et. Al described a high-yield tandem fermentation method for the production of carotenoids including Spongiococcum ATCC No. 40335. The invention includes cultivating algae organisms capable of producing zeaxanthin, while adding fresh fermentation medium to dilute the hair solution or effective medium. The fresh fermentation medium is generally an aqueous solution, and the aqueous solution also includes a source (the amount of the source is sufficient to limit the growth rate of the organism). Specifically, the fresh fermentation medium may be water, or water with nutrients such as glucose and other substances added. (Please read the intentions of carrying goods first and then write this page) i

Lf (〇 & Vlb A 6 B 6 Printed by Beigong Consumer Cooperative of Central Bureau of Standards, Ministry of Economic Affairs V. Invention description (11) The dilution of the fermentation broth can be achieved by adding fresh fermentation medium to increase the total liquid volume, or a better method Poorly add fresh fermentation medium and remove the spent fermentation broth to maintain a consistent amount of fermentation broth. In the latter case, the dilution rate (D) is equal to the flow of fresh fermentation medium into the fermentation tank and the used fermentation broth out of the tank The unit of volumetric flow rate divided by the amount of fermentation broth D is the countdown time, which is represented by hJ unless otherwise indicated here. Dilution is equal to the hourly liquid passing through the container in the container, and is the average residence time Countdown. Both the addition of fresh fermentation medium and the removal of used fermentation broth can be carried out by secondary entanglement or intermittent softness, but the success is better. Add; | Fresh fermentation medium and removal of used fermentation broth can be carried out simultaneously or at different times. However, it is preferably carried out at the same time. In the case of maintaining a relatively stable amount of fermentation broth to promote growth and culture, dilution is generally effective between about 0.03 hrH and 0.13. The "growth rate" is represented by the "u" symbol, and refers to the large number of cells formed by each cell mass in the fermentation broth per basket time. Unless otherwise indicated, the unit of growth rate is hrM, which is stable In the state, the dilution rate is equal to the growth rate (D = u) in the present profit entanglement fermentation method. The dilution rate (D) is pseudo-dominated by the maximum growth rate (umax) of a specific withdrawn organism. One can use the entanglement culture The dilution rate is set to any level less than umaj (. In order to obtain the maximum amount, D is preferably between about 25% to 95% umax, more preferably about 90% uraax. This system will change when close to u It is unstable. It will cause erosion when D is greater than U. However, it should be noted that when adding fresh fermentation medium, the nutrient level in the fermentation broth must be maintained at a concentration sufficient to support the growth and / or manufacture of corn quality. These nutrients can be added to the fresh fermentation medium or can be added independently. Specifically, these nutrients include sources of nitrogen, nitrogen, phosphates, sulfates, and other trace metals that can be absorbed by magnesium, iron, etc. Sources include but are not limited to For sugars and their polymers, including starch, dextrin, sucrose, maltose, lactose. Glucose, mannose, sorbose, arabiose, xylose, fructose, cellobiose, and molasses, fatty acids; and poly 6, such as glycerin. The preferred sources of saccharin in the invention include monosaccharides, disaccharides, and trisaccharides. The best succulent sources are grapes. Sources of nitrogen absorbable include, but are not limited to, inorganic nitrides such as ammonium salts and animals, vegetables, and / or organic Source substances, such as protein hydrolysates, microbial hydrolysates, soybean meal, fish meal, meat meal, meat extract, (^ _, tryptoie, corn dipping solution, yeast extract, amino acid. Good nitrogen sources include yeast extract, and sulfuric acid is effective The culture medium may contain other compounds, such as vitamins, growth promoters, or color-forming promoters. These chemicals can be present in the source of nitrate, krypton, or minerals in the effective medium, or they can be added to the medium. C An embodiment of the present invention includes restricting the sources of fermentation in the fermentation while performing fermentation. A restricted growth rate yesterday η First read the notes on the back of ii and then fill out this page Μ It ax order book size using Chinese National Standards (CHS) A 4 specifications (210x297 male dragon) _ 13 _ ύ OBvio Α6 Β6 Economy Printed by the Central Committee of the Ministry of Public Security and Employee Consumer Cooperatives V. Description of invention (12) Degree. In this way, the fermentation time can be extended indefinitely, and the output can be increased. This aspect of the invention includes limiting the rate of addition of master sources. Better yet, all the other nutrients necessary for growth are much more than the source of the seeds. We 3 want to be limited by the doctrine, but generally believe that by restricting the availability of sources, the production of toxins can be greatly reduced, because the organism will be forced to give priority to the growth of existing lotus. We can monitor the addition rate of sulfonate sources and the supply rate of master sources is to achieve our desired limit i long rate. The limited growth rate is pseudo between 25% uiiax and 95% umax, preferably about 90% u max. In a preferred embodiment of the present invention, the fermentation broth or effective medium is diluted with fresh fermentation medium, and the supply rate of sulfonate source is generally limited to a high level. The method preferably includes three stages: (1) the transplantation stage; (2) the supply group growth stage; and (3) limiting the source of the seeds, and the continuous growth and cultivation stage. The transplantation phase includes the provision of actively growing biological implants in the fermentation tank. The growth phase of the supply group includes propagating the organisms in the fermentation tank, continuously adding the culture medium and keeping the source concentration less than 5 g plant liters. Generally speaking, the source of sulphur is between about 1 gram / liter and about 5 gram liters in the supply group stage. During the transition from the supply group stage to the stable continuous mulching stage, the continuous supply rate of fresh fermentation medium (which contains sulfonate sources) * is gradually increasing. When a satisfactory steady-state cell concentration is reached, the continuous fermentation stage begins. A preferred source of sulfon is a sugar, such as glucose. The glucose is consumed to a minimum concentration before switching to the continuous ore-growing stage that restricts M glucose. The concentration is as low as possible, generally about 1 g / L. . The fermentation process is switched to the tangled growth and culture stage where glucose is restricted. Lien Myanmar ’s supply includes supplying glucose and other nutrients that promote R production (ie nutrients such as krypton, potassium, phosphate, iron, sulfate, citrate, magnesium, and other trace elements, etc.) in fermentation culture. 4 base, but its initial dilution rate is low. The dilution rate then gradually increased to reach the growth rate of the organism almost the same as the highest growth rate in the group growth stage f, and a satisfactory steady state dilution rate was obtained. D is preferably about 90% umax. The addition rate of fresh fermentation medium is the same as the removal rate of used fermentation broth. Controlling temperature, acid age, gas, and sulphur dioxide pseudo-cultivation can produce zeaxanthin withdrawing organisms to obtain other important parameters of high growth rate and high p beige quality yield. In a preferred method, the cultivation or fermentation is carried out under the following conditions: the temperature is between 30 degrees Celsius and 38 degrees Celsius, the pH value is about 5.5 to 6.5, the dissolved oxygen content is at least about 10%, and the sulfur dioxide partial pressure is: j is less than about 0.04 atra. The best cultivation progress is as follows: the temperature is about 36 degrees Celsius, the pH value is about 6.0, the dissolved oxygen content is greater than about 10%, and the partial pressure of the two gasification sulfonic acid is less than about 0.04 atm. After the algae are removed from the organism to produce zeaxanthin at a satisfactory number, the recovery of zeaxanthin can be recovered by bio-mass containing zeaxanthin from the zeaxanthin-producing organisms. Here, the zeaxanthin-containing biomass pseudo-refers to the zeaxanthin-containing microorganism recovered from the fermentation medium, and the recovery is after the withdrawal of the organism to produce zeaxanthin in the cell. Read the backfill items first and then fill in%

Shi '丨 J The size of the bound paper used in the Chinese National Standards (CNS) Grade A (210x297 mm) -14-Printed by the Beigong Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 208713 A 6 __ B6_ V. Description of the invention (13 ) Λ In an embodiment of the present invention, zeaxanthin-containing algae are recovered as a feed additive to increase the color of the food animal. The method of increasing coloration here refers to the use of zeaxanthin to apply yellow or reddish yellow to animal meat, skin, other parts of the body, and / or egg yolk, or to apply a similar color to the composition (e.g. except for the food animals mentioned above) Other foods) or cosmetics. If food animals convert zeaxanthin to astaxanthin, the animal may show a red color. Edible animals mean feeding animals as food. Such animals include but are not limited to poultry, fish and crustaceans. The better zeaxanthin-containing biogroup according to the present invention is Spongiococcum, because of the zeaxanthin bioavailability that is possessed by Spongiococcum-based zeaxanthin-producing organisms. The zeaxanthin-containing Spongiococcum bio mass can be used directly as a feeding supplement to increase the coloration of food animals. The zeaxanthin-containing Spongiococcuin biomass used as a feeding additive can be separated from the fermentation medium and recovered from the zeaxanthin-containing organism. Better separation techniques include but are not limited to centrifugation and filtration. In a preferred embodiment, the zeaxanthin-containing Spongiococcum biocluster is recovered by the following method: centrifugation is used to separate the zeaxanthin-containing biocide; an aqueous solution is used to rinse the biocluster at least once to remove at least a portion of the chlorophyll Contaminants, thereby reducing bad thread color; spray dry the biomass to obtain bioplaque powder. The biomass powder preferably has a moisture content of about 3% to 5%. If the algae organisms that make zeaxanthin have indigestible cell walls, recovering the zeaxanthin-containing biomass from the organisms requires an additional cell lysis step before spraying the biomass. Such dissolution can be achieved by those skilled in the art using physical, chemical, or promoting methods known to them. In this embodiment, the total carotenoids content of the zeaxanthin biocluster powder is at least about 5 milligrams per gram of biological garden, preferably at least about 7 milligrams. According to the present invention, zeaxanthin accounts for at least about 5% of the total carotenoids content, g is preferably at least about 10%, more preferably at least about 25%, and most preferably at least about 40%. We can also use standard techniques to purify and isolate zeaxanthin from algae that can make zeaxanthin. Standard methods for purifying zeaxanthin from biological sources include the use of organic solvents to extract zeaxanthin from zeaxanthin-producing organisms and purifying zeaxanthin from other contaminants. In a preferred embodiment of the present invention, a standard purification method can be used to recover zeaxanthin from zeaxanthin-producing organisms to obtain very pure zeaxanthin with a purity greater than about 90%. Fortunately, it is at least about 97%, and preferably close to 100%. Very pure zeaxanthin can be used to formulate zeaxanthin-containing compositions and used to enhance the coloration of foods and cosmetics, and at the same time to reduce the damage caused by active gas species and light-damaging molecules. Reactive oxygen species here refers to molecules that vaporize other molecules and often cause damage to cells or tissues. Active oxygen species include photosensitizers, single-line gas, and gas radicals. The paper size here is the Chinese 8 standard (CNS) A4 specifications (210x297 male dragon) _ 15 _ (please read the precautions on the back before filling in this page) to install < line.

A 6 ^ _B6 _ V. Description of the invention (14) Light-loss molecules refer to substances (such as light) that degrade or inactivate photosensitive compounds and cause damage to animals and plants (including damage to cells and organs). An effective amount of zeaxanthin means that it can effectively reduce the damage caused by reactive oxygen species and optical loss molecules. For example, zeaxanthin can be used in mammals to prevent or treat certain cancers and alleviate intracellular and extracellular organ damage or organ damage caused by reactive oxygen species. _ Compositions containing zeaxanthin can be taken internally (including but not limited to oral, itching, intravenous, subcutaneous, intraperitoneal) or topical (including but not limited to topical application). For example, zeaxanthin-containing compositions can be added to anti-wake oils and emulsions to mitigate the damage caused by the active gas species. An effective amount of a zeaxanthin-containing composition can also be added to light-sensitive and / or gas-sensitive compounds to stabilize and mitigate damage caused by light or oxygen. „(Please read the precautions on the back before filling out this page) Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs to enhance coloring The method of improving the color. The effective amount of zeaxanthin is to provide a satisfactory yellow to red-yellow color in the amount of animal meat, skin, other body parts, and / or egg yolk. If the animal can use corn Xanthin is converted to astaxanthin, and the animal may appear red. According to the present invention, algae are pseudo-cultured to produce zeaxanthin and zeaxanthin-containing biomass powder is pseudo-recovered as described above. The zeaxanthin powder can be formulated into a zeaxanthin-containing composition for application to food animals. A preferred zeaxanthin-containing composition includes mixing zeaxanthin-containing biomass powder with animal feed. In one embodiment of the present invention In the process, the Spongiococcim microorganism that can produce zeaxanthin is pseudo-cultured and the recovery of zeaxanthin-containing biopowder powder is based on the following methods: Microbe microbes to obtain zeaxanthin-containing biomagnesia, ② rinse the obtained biomass, and ③ spray dry the biomass. The recovered zeaxanthin-containing biomass powder can be directly applied to, including but not limited to Poultry, fish, and crustaceans, etc. to enhance the burnt meat, skin, and other body parts of these animals, and / or egg yolk. In a preferred embodiment, the powder of the biomass containing Spongiococcura zeaxanthin is pseudo-mixed For poultry feed. The amount of powder added to the poultry feed is based on the total lutein content of the powder. Preferred zeaxanthin-containing compositions include about 10 to 40 grams of lutein per ton of feed. The best combination Wuxi contains about 40 grams of lutein per ton of feed. The following experimental results are for the purpose of understanding and not to limit the scope of the present invention. Special attention should be paid to the parameters (such as concentration, Time, temperature, and other reaction conditions) are examples of the present invention rather than limiting Λ. The appropriate range of such parameters is known to the general technician. 装 * This paper scale is in use 8 sample quasi-secretaries 5) A 4 specifications (2〗 0 father 297 male dragons) -16- Printed by the employee consumer cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs A 6 _B6__ V. Description of invention (15)] Example 1 Zeaxanthin manufacturer Isolation of S. excentricum HZ1236 Spongiococcum excentrlcum ATCC 40335 is a yeast extract / ^ J3. (G / l yeast extract, 5.0g / l and 20g / l glucose) medium grown in a vibrating fermentation flask at a temperature of 35 ° C Degree, grown to a visual concentration (OD) between about 1.0 and 2.0 at 620 nm. Cells obtained by centrifugation from 10 ml of culture broth are sterile citrate buffer (0.1M sodium citrate, pH 5.5 ) 〇These cells are resuspended in 10 ml of citrate buffer. 250 mL of 1 gram / microliter NTG was added to the suspended cells to obtain the final HTG physique 25 gram / mL. (These conditions generally kill 65% to 80% of the cells.) The cells mutate by shaking at 3 degrees Celsius in the dark for 30 minutes. After the mutation occurred, the cell suspension was separated by centrifugation, and the cell pellet was washed twice with citrate buffer. The mutated cells were resuspended in 10 ml of YEP and cultured at 35 degrees Celsius. Overnight, the cell culture solution was diluted with citrate buffer to a certain concentration, and at this concentration, approximately 50 strains of bacteria will grow well on a 100 mm Petri dish. The dilute number of the diluted culture solution was pseudo-spread on a petri dish containing a solid YEP medium (YEP medium containing 20 g / L agar) and grown at 35 degrees Celsius for 5 to 7 days. After growth, the flora was visually evaluated for tb by color, and transferred to a freshly solidified YEP Petri dish containing 5 掞 g / ml skimmer to re-grow for 10 days to select the manufacturer of zeaxanthin. The olive-green algae (represented by Spongiococcum excentricura HZ1236) was found to have grown on a solidified YEP petri dish containing 5 μg / ml mitostatin and was isolated. When grown in a shake flask with YEP medium, the S. (short for Spongiococcum) excentricum HZ1236 line was found to have approximately 15% zeaxanthin in the total Huluo Si production. This strain can generally produce zeaxanthin and lutein, and the ratio (Z: L) of zeaxanthin to lutein is about 0.15. Relatively speaking, the parent S. excentricum ATCC No. 40335 generally produces 2 to 4% zeaxanthin in the total humectin production, and the Z: L ratio is 0.03 to 0.06. Example 2 Isolation of zeaxanthin manufacturer S. excentricum HZ1236 / 274 S. excentricum HZ1236 was exposed to NTG under the conditions described in Example 1. The mutated flora was grown as described in Example 1. The difference is that the flora grown in the solidified YEP Petri dish was pseudo-transferred to a system containing 7 g / ml (please read the precautions on the back before filling this page). Order ···· Line · The paper size is free to use the Chinese National Standard (CNS) A 4 specifications (210 father 297 male dragon) -17-208713 printed by the Central Standard of the Ministry of Economic Affairs Employee Consumer Cooperatives A 6 B6 V. Description of invention ( 16) Renew growth of bacteriocin in a freshly solidified YEP petri dish for 10 days to select the organisms that can produce high-yield zeaxanthin. The pongiococcum excentricum HZ1236 / 274 is a yellow-green flora and was found to grow in a solidified YEP petri dish containing 7 gram / ml of Anisomycetes, and was isolated. When grown under 2 liters of fermentation broth P under the conditions described in Example 4, S. excentricura HZ1236 / 274 was found to produce approximately 3 mg and 5 mg of E-bexanthin per gram of stem cells. In general, about 50% of the total carotenoids production of this strain is zeaxanthin. S. exhntricum Z1236 / 274 was found to produce zeaxanthin and lutein at a Z: L ratio of about 1.1. Example 3 Isolation of zeaxanthin manufacturer S. excentricum HZ1236 / 538 S. excentricum HZ1236 was exposed to NTG again as in Example 1. The mutated bacterial group grows as described in Example 1. The difference is that the strains grown on the solidified YEP petri dish are pseudo-transferred in a fresh solidified YEP g petri dish containing 55 Μ hardness to grow for 10 days to select the energy system. Removal of high-yield zeaxanthin: S. excentricum HZ1236 / J8 pseudo-orange-green flora was found to grow on a solid YEP Petri dish containing 55 mol) | 1 and was isolated when it was in 2 liters When grown in the fermentation broth as described in Example 4, S. excent "icin» HZ1236 / 538 was found to produce approximately 3 mg of zeaxanthin per gram of stem cell weight. The total yield of carotenoids of this strain is approximately 40 % Yong corn I. S. excentricum HZ1236 / 538 was found to be able to produce zeaxanthin and I flavin at a Z: L ratio of about 0.6. Example 4: Preparation of inoculum and culture medium Spongiococcum excentricura HZ1236 / 274 strains were prepared. Initially, a yeast 3: extract / ^ (2 X YEP) inoculum culture medium was prepared. The culture medium included 6.0 g / + Difco yeast extract in 960 ml of deionized water, and 10 g / L Bacto-^. The mixture is autoclaved at 121 degrees Celsius: 5 minutes. After high ® disinfection , Add 40 ml of 50% glucose solution. A single S. excentiMcum flora is prepared for transplantation in a 1 liter shaking flask containing 200 ml of the aforementioned inoculum medium. The flask is pseudo-incubated at 400 rpin (per minute) at 35 degrees Celsius ± Rotational speed) Cultivated in a rotary shaker. The flask was cultivated to a visual concentration of 7 g 10 (absorbed at 620 nm). This paper scale uses the Chinese B standard (CNS) A 4 specifications (210x297 mm) _ 18 _ (Please read the precautions on the back before filling in this page) Pack-Λ6 13 6 V. Description of the invention (17) A fermentation medium is prepared, the medium includes 3.88 g / l potassium dihydrogen phosphate, 1.50 g / l Ambers yeast extract, 2.75 g / L of sulfuric acid, and 0.20 g / L of citric acid in 9 L of deionized water. The solution was autoclaved at 121 degrees Celsius for 60 minutes. Then the autoclaved solution was added with 5 g / L 1 liter of sugar, 5.0 ml / litre of trace Jinmao 8 solution, 4.5 ml / litre magnesium sulfate, and 0.1 g / litre ferric sulfate. The trace amount of Jinluo 8 solution includes 200 ml of 6.0 litre deionized water Concentrated hydrochloric acid (97%), 114 g calcium chloride CaCUHiO, 12.3 g sulfur Manganese, 0.314 g copper sulfate, 1.6 g cobaltous vapor, 9.12 g boric acid, 17.65 g zinc sulfate, 0.48 g sodium molybdenateJHiO, 0.8 g vanadium sulfate, 0.4 g nickel sulfate, 0.4 g sodium selenite , And 2.5 grams of sodium citrate. After all solids are added and dissolved, the final amount (with deionized water) becomes 0 liters.

The pH of the fermentation medium is about 4.8. Adjust the pH to 6.0 with ammonium hydroxide solution (14%) before transplantation. The conditions before transplantation are as follows: temperature = 36 degrees Celsius 1 degree; pH = 6.0 ± 0.2; stirring rate = 400 rpm; gas rate = 1 vvm; and dissolved gas = 100% saturated D. The graft is transferred to the fermentation vessel under sterile conditions. The final concentration of the graft in the container is 5%: the initial visual concentration is about 0.5. In the feeding group stage, the fresh fermentation medium is fed including 500 g / l of 1 sterilized sugar at 121 degrees Celsius for 1 minute, followed by the addition of 28.57 ml / l magnesium sulfate and 5.72 ml / l phosphoric acid. The feed stream for fresh fermentation medium in the Burmese stage includes There are 0.048 grams of dipotassium phosphate, 0.0025 grams of iron sulfate, 0.003 grams of anhydrous citric acid, 0.11 milliliters of 1 gram of magnesium sulfate, and 0.125 milliliters per gram of glucose. Trace metal 8 solution. Example 5 Printed by the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling in this page). Using S. excentricum HZ1236 / 274 to manufacture zeaxanthin. This example explains how to use the above method parameters to 1. Zeaxanthin is produced in the fermentation of experimental plants. A. Preparation of implants: Add 7 liters of water to a 14-liter fermentation vessel, which contains 31 grams of potassium dihydrogen phosphate, 3 grams of Amberex 695 yeast extract, 22 grams of ammonium sulfate, and 1.0 ml of MAZU204 anti-foaming agent. Monthly items in the container are sterilized by high-pressure steam at 121 degrees Celsius for 90 minutes. 500 ml of other nutrient solutions were aseptically filtered into the fermentation vessel with a toxin filter. The nutrient solution contains 160 grams of glucose, 40 milliliters of trace metal 8 solution, and 9 paper scales. Chinese National Standard (CNS) A 4 specifications (210X297 male dragon) -19-13 Λ 6 Β6 V. Description of the invention (18) Grams of magnesium sulfate, 0.8 grams of iron sulfate, and 1.6 grams of olive acid. The fermentation vessel was then filled with 500 ml of raw YEP and cultivated for 48 hours to grow Spongiococcum excentricum HZ1236 / 274 (YEP contains 9 g / l yeast § fetch, 15 g / l t and 10 g / l glucose). The aeration rate was maintained at 1.0 to 1.2 vvm during the process, and the agitation rate was maintained at 400 to 600 rpm. The pH value is controlled at 6.0 by adding anhydrous ammonia. _ When the residual glucose is reduced to about 5 g / L, the nutrient supply starts, which includes the following substances ^ Glucose 320 g / L potassium dihydrogen phosphate 62 g / L gland sulfate * 44 g / L trace metal 8 80 mL / L magnesium sulfate 17.8 g / L ferric sulfate 1.6 g / L citric acid 3.2 g / L The supply solution makes the residual glucose in the fermentation vessel between 2 to 5 g / L after about 24 hours of supply Accumulate to 15-20g / L, and used for transplantation test fermentation container B. Culture medium preparation and disinfection: add 190 liters of water (including Sigma corn dip 600g-3.0g / L and MAZU204 anti-foaming agent 10ml- -0.05ml / liter of the solution was sterilized at 121 degrees Celsius for 60 minutes 8 and then cooled to 36 degrees Celsius. The following substances were then added to 5 liters (final amount) of water, and all substances were added through Millidisk sterilizing filter in a self-free state In the fermentation vessel, the medium composition is added to the total final concentration of potassium hydroxide (87.5%) 348 g 1.83 g / l phosphoric acid (85%) 625 g 3.29 g / l sulfuric acid & 523 g 2.75 g / l magnesium sulfate 211 g 1.11 G / l trace gold 8 Solution 950ml 5.0ml / l Printed by the Staff Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) Add sterilized water to the fermentation container to adjust the volume to 200 liters, and use an anhydrous atmosphere The pH value is adjusted to 6.0. C. Fermentation stage of the supply group: In this stage, all the substances in the 14-liter transplant container are transferred aseptically to the 4-liter fermentation container. When the residual sugar in the fermentation container is reduced to 5g / l (when about 12 to 15 sheets of paper are used in the Chinese standard (CNS) A4 specifications (210x297 g *) _ A 6 B6 V. Invention description (19), within hours), then Start adding nutrients to the fermentation vessel. The two sterilized nutrient supply streams are aseptically added to the fermentation vessel at a ratio of 1 to 1, so that the residual glucose is maintained at 3.5 g / L during this fermentation supply stage. The supply flow is as follows: Supply solution Supply solution 1 M glucose (600 g / L) Ferric sulfate (0.6 g / L) Citric acid (1.2 g / L) Phosphoric acid (61 g / L) Potassium hydroxide (34 g / L) Trace metal 8 solution (96 milligrams) L / L) Ammonium nitrate (42 g / L) In the nutrition supply stage, the acid test value is controlled at 6.0 ± 0.2 by adding anhydrous ammonia, and the dissolved oxygen is maintained at more than 40% saturation. After about 15 hours of nutrition supply Afterwards, the biomass accumulates to 25 to 30 g / L and begins the entanglement phase of carotenoid production. D. Lian Fermentation Phase: The waiting period of this successive stage is that the addition rate of glucose pseudo-limits growth and After the satisfactory dilution rate is reached, Ε ie maintains a fixed dilution rate. In this example, the dilution rate is hr1. As in the supply group stage of fermentation, the nutrition supply includes two different sterilization supply streams, and the composition of these supply streams is as follows. However, the ratio of sugar supply to salt supply here is virtually kept at 1 to 3.9. Supply solution Supply solution 1

Cerelose (600 g / L) Ferric sulfate (0.6 g / L) Citric acid (12 g / L) (Please read the notes on the back before filling this page) Phosphoric acid (6.1 g / L) Potassium hydroxide (34 g / L) L) Magnesium sulfate (47 g / l) Printed trace metal 8 solution (9.6 ml / l) Ammonium nitrate (4.2 g / l) in the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs in this example using the comprehensive nutritional supply To a dilution rate of 0.045 hr_ 丨. This dilution rate was then maintained at this level during 80 hours of β-lianata. In this continuous Burmese fermentation stage, the carotenoids of the biomass averaged 0.53% < Ε beige matter accounted for 40% of the total carotenoids. Therefore, in this fermentation, s. Excentricum HZ1236 / 274 produces about 2.1 mg of zeaxanthin with an average weight of gram of stem cells. The cumulative production of the above lotus plant is 3.9 mg of xanthin per liter hour. This paper scale uses the China Sleepy Family Standard (CNS) Grade 4 specification (210X297 mm) _ 21-A 6 136 Printed by the Beigong Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs V. Description of invention (20) Example 6 Poultry house breeding test 'A poultry feeding test compared the color enhancement capabilities of the following items: S. excentricura HZ123 & / 274 containing zeaxanthin biolan powder (BioZea); S. excentricum ATCC No. 40335 containing xanthophyll bio-painting) ), And lutein extraction based on marigold (Chromophyll-Oro). S. excentricum HZ1236 / 274 and S. excentricura ATCC 40335 strains were cultivated as described in Examples 4 and 5. The preparation system of BioZea is: ① centrifugation is used to separate zeaxanthin from the fermentation medium, and ② spray drying the biological enclosure to obtain zeaxanthin-containing biomass powder BioZea with a humidity of 3 to 5%. BioZea contains about 7 mg of lutein per gram of stem cell weight, of which about 50% is zeaxanthin. The method prepared by BioXan is similar, but the biomass is not rinsed before spray drying. BioXan contains about 7 mg of lutein per gram of stem cell weight, of which about 2 to 4% is zeaxanthin. ChromophyH-Oro was obtained from Laboratorios Bioquimex, S, A. (company name). In this experiment, the dosage of BioZea, BioXan, and ChromoPhyH-OR (standardized according to the amount of total lutein. A total of 640 12-day-old commercial breeding cocks (of Callus donesticus species) are divided into 10 test groups, each group Feed lutein from one of the following houses per ton (t) of poultry feed within 18 days: (1) None, (2) 10 g / ton BioZea, (3) 20 g / ton BioZea, (4) 40 ^ tons BioZea, (5) 10 g / ton BioXan, (6) 20 g / ton BioXan, (7) 40 g / ton BioXan, (8) 1 g / um Chromophyl Bu Oro, (9) 20 g / ton Chromophyll-Oro, (10) 40 g / Bll Chromophyil-Oro. The composition of poultry feed is shown in Table 1. Table 1 Composition of poultry feed and percentage of nutrient elements Milled Milo 61.15 Soybean material-47.5% 25.77 Fat: Mixed -3800 5.87 Meat and Aggregate -50% 5.13 The size of this paper is in accordance with Chinese National Standard (CNS) A 4 specifications (210x297 g) _ 22 (please read the precautions on the back before filling in this page) Install &Order; V. Description of the invention (21) Phosphate / defluorination 0.72 Limestone 0.44 Salt 0.33 DL-methionine 99% 0.25 Chick Vitamin Premix 0.15 Kefen Coban) 0.11 Trace Boy Metal Premix 0.05 L-Lysine HCL 98% 0.03 100.00 Protein 20.31 Fat 8.05 Cellulose 2.24 Calcium 0.90 Existing Phosphate 0.47 Calories / Lb 1464,64 Existing Lutein 0.00 Feed and water can be whatever you want The chicken is weighed at the beginning and the end of the test. At the end of the test, the leaf side of the chicken is evaluated by the Merlot colorimeter and the Rocker color fan test. The standard test system measures the color samples. The brightness, sharpness, and chromaticity. The results of the test are shown in Table 2. Table 2 Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page). The ranking of poultry coloring Handling US D, E. 1 40 g / ton BioZea 50.62 2 40 g / Chromophy 1 丨 -Or *. 45.54 3 40 g / L. BioXan 43.60 4 20 g / _ BioZea 41.13 This paper size is easy to use aa National Standard (CNS) A 4 specifications (210X297 mm) -23-A 6 B6 V. Description of invention (22) 5 20 g / s Chromophyll-Oro 38.15 6 20 g / t BioXan 36.47 7 10 g / t Chromophy1l-〇 ro 34.57 8 10 g / ton BioZea 34.37 9 10 g / ton BioXan 31.54 10 control 26.46 rank treatment Rock color fan 1 40 g / ton BioZea 5.11 2 40 g / ton BioXan 4.64 3 40 g / ton Chromophy1l-〇ro 4.49 4 20 g / ton BioZea 4.22 5 20 g / Ton Chromophyll-〇ro 3.75 6 20 g / ton BioXan 3.58 7 10 g / ton BioZea 3.35 8 10 g / ton Chromophy11-Oro 3.31 9 10 g / ton BioXan 3.00 10 Control 1.58 (please read the notes on the back before filling in this Page) The employee consumer cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs was printed in each treatment group, and no differences were found in the status of feathers and feces. As shown above, the chickens fed with BioZea showed much better color than the chickens fed with Chromophyll-Oro or BioXan, showing that BioZea is a better poultry color enhancer. Although several embodiments of the present invention have been described in detail, those skilled in the art can make various modifications to these embodiments, and these modifications are included in the patent application scope of the present invention. The paper size is in accordance with the Chinese National Standard (CNS) A 4 specifications (21〇Χ297 male dragon) -24-

Claims (1)

Sixth, patent application Fan Ji 1, a method of manufacturing zeaxanthin, including (a) cultivating a kind of algae that can produce at least about 0.35 milligrams of zeaxanthin per gram of stem cell weight in an effective cultivating base; And (b) recovery of zeaxanthin manufactured by Pak Bi. 2. The method according to item 1 of the patent application scope, in which the Spongiococcu · strain should be withdrawn. 3. The method according to item 1 of Shenzong's patent scope, wherein the cultivation includes continuous ore fermentation. 4. The method according to item 1 of the patent scope of the application, in which the cultivation step is performed at about 30 degrees Celsius to about 38 degrees Celsius, the acid value is between about 5.5 to 6.5, and the concentration of dissolved gas is at least about 10¾ And the partial pressure of the second gasification sulfonate is less than about 0.04 atm. 5. The method according to item 1 of the patent application garden, in which the recovery step includes separating the organism from the meal base to form a zeaxanthin-containing biological garden. 6. The method according to item 5 of the patent application, wherein the recovery step further comprises washing the biological garden containing zeaxanthin at least once to remove at least a part of the chlorophyll contaminants. 7. The method according to claim 1 of the patent scope, wherein the step of recovering includes extracting zeaxanthin from an organic solvent in the organism. 8. The method according to claim 1 of the patent scope, wherein at least about 5% of the total carotenoid production of the organism is corn yellow. 9. The method according to item 1 of the patent application scope, in which the withdrawn organism can produce at least about 1.75 mg of zeaxanthin per gram of stem cell weight. 10. The method according to item 1 of the patent application circle, wherein at least about 25% of the total carotenoid M production of the skimmer is zeaxanthin. Printed by the Central Standard of the Ministry of Economic Affairs and Employee Consumer Cooperatives (please read the notes on the back before filling in this page) 11. According to the method of item 1 of the patent application scope, the withdrawal of the organism can produce at least about 2.8 per gram of stem cell weight Milligrams of zeaxanthin. 12. The method according to item 1 of the patent application scope, wherein at least about 40% of the microbial entanglement vegan production is corn zeaxanthin. 13. A method of producing zeaxanthin, These include: (a〇 Mutating the parent microorganism of the Spongiococcun strain; (b) Selecting one of these mutant microorganisms that can produce at least about 0.35 milligrams of corn wood paper weight per gram of stem cells; 丨] Zhongmin Standard (CNS) 〒 4 specifications (210x297 male 81. 4. 5.000 (H) 8 ireο C six 'from the i special ten 丨 Fan Ji Huang Jiao biological; (c) cultivate the selected microorganism in an effective medium to Manufacturing zeaxanthin; and (d) recovering zeaxanthin produced by the selected organism. 14. The method according to item 13 of the patent application, wherein the mutation step includes exposing the maternal microorganism to nitrogen-A base- Nitrogen-nitrosoguanidine, ultraviolet rays, X rays, gamma rays, ethylaethane sulfonate, nitrous acid, or a mixture thereof. 15. The method according to item 13 of the patent application, wherein the selection step includes the mutation Withdrawal organisms are exposed to a metabolic inhibitor, and the inhibitor is replaced from a group consisting of isoprenoid pathway inhibitors, carotenoid biosynthesis inhibitors, free radical generators, and mixtures thereof. 16 The method according to item 15 of the patent application scope, in which the isoprenoid pathway inhibitors include anabolic hormones, anti-microbolin A, somatobolin, nevilonin, sapolin, amphotericin B, chain acid fanesyl compounds, azasqua lenes, allyl蚞 Derivatives, thiocarbamate, pyridine, azole, triazole, orphol ines, or a mixture thereof. 17. The method according to item 15 of Shenzhao ’s patent scope, in which the carotenoids are biosynthesized Inhibitors include norflurazon, metflurazon, benzoan_, diphenylamine, niacin, oxyfluorfen, fluorfen, / 8-_sub_, or mixtures thereof. 18. The method according to item 15 of the patent application, in which the ^ The radical generator includes hard, other types, peroxides, ultraviolet rays, X-rays, gamma rays, Austrian gas, or a mixture thereof. 19. The method according to item 13 of the patent application letter, in which the selection step Including the isolation of a mutant coloring organism with zeaxanthin as the representative color. 20. The method according to item 13 of the patent application, in which the mother-colored microorganisms include microorganisms with a special withdrawal of the classification of SpongiococcuB excentricu · ATCC No. 40335 or its mutants. 21. According to the method of item 13 of the patent application, where at least about 5% of the selected biotic nodules carotenoids producers are zeaxanthin. 22. The method according to item 13 of the patent application scope, in which the withdrawal of the organism can produce at least about 1.75 mg of zeaxanthin per gram of stem cell weight. 23. According to the method of item 13 of the patent application, the production of carotenoids of the microorganisms produced by the S is at least about (Ji-T first read the notes on the back of the Ji and then fill out this book. Printed by the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. Line. The size of the paper is suitable; 丨] 屮 "« Home Standard (CNS) Specification 81. 4. 5,000 (Η) C7 __ D7 VI. Shenjing Patent Fan Garden 25% It is zeaxanthin. 24. According to the method of claim 13 of the patent application, wherein the selected organism can produce at least about 2.8 milligrams of zeaxanthin per gram of stem cell weight. 25. According to the patent application No. 13 The method of item 1, wherein at least about 40% of the total humubulin-like product produced by the biological organism is zeaxanthin. 26. The method according to item 13 of the patent application scope, wherein the cultivation includes continuous ore fermentation. 27. The method according to item 13 of the patent application scope, wherein the culturing step is performed at about 30 degrees Celsius to about 38 degrees Celsius, the acid ife value is between about 5.5 and 6.5, and the concentration of dissolved oxygen is at least about 10%, And the K-force of the second gasification sulfonate part is less than about 0.04 atm. 28. The method according to item 13 of the patent scope Wherein the recovery step includes separating the microorganism from the culture medium to form a biological painting containing zeaxanthin. 29. The method according to item 28 of the patent application scope, wherein the recovery step further includes washing the zeaxanthin-containing biological painting Biological garden at least once to remove at least a part of the chlorophyll contaminants. 30. The method according to item 13 of the patent application scope, wherein the recovery step includes extracting zeaxanthin from an organic solvent in the organism. 31. One capable of manufacturing The manufacturing method of zeaxanthin algae removal organisms includes: (a) mutating algae microorganisms; and 0> selecting one of the mutant organisms that can produce at least about 0.35 mg zeaxanthin per gram of stem cell weight. . 32. The method according to item 31 of the patent application scope, wherein the algae emblem is Spongiococcu. The Ministry of Economic Affairs, Central Bureau of Standards and Staff ’s Consumer Cooperation Du Duan 33, according to the method of item 31 of the patent application scope, wherein the mutant organisms include the classification characteristics of the strain Spongiococcu · excentricu · ATCC No. 40335 or its mutant snakehead Microbes. 34. The method according to item 31 of the patent application scope, wherein at least about 5% of the total Huhuan glucoside production of the extracted organism is zeaxanthin. 35. The method according to item 31 of the patent application scope, wherein the bio-energy produced by the plant produces at least about 1.75 mg of zeaxanthin per gram of stem cell weight. 36. The method in accordance with item 31 of the patent scope of the application, in which the total category of the selected withdrawal organisms Hu Huo "at least about 25% of the prime producers is zeaxanthin." 81. 4. 5.000 (H) (Please read the precautions on the back before writing this page) The size of this paper is suitable for W t «Μ standard (CNSVP4 specification (2 丨 0x25) 7) If) ⑽ 竹 3 C- ___ [) 7 37. The method according to item 31 of the patent application scope, wherein the selected withdrawn bioenergy produces at least about 2.8 mg of zeaxanthin per gram of stem cell weight. 38. The method according to item 31 of the patent application scope, wherein at least about 40% of the total carotenoid production of the selected mold organism is zeaxanthin. 39. The method according to item 31 of the patent application scope, in which the algae withdrawing organism is a Spongiococcum strain, which has the classification of Spongiococcum excentricun HZ1236 / 274 (ATCC 74108) and its mutants, among which The mutant can produce at least about 0.35 mg of zeaxanthin per gram of stem cell weight. 40. The method according to item 31 of the patent application scope, wherein the algae withdrawing organism is a Spongiococcum strain, which has the classification of Spongiococcum esccentricun HZ1236 / 437 (ATCC 74109) and its mutants, where such mutations The body can produce at least about 0.35 mg of zeaxanthin per gram of stem cell weight. 41. The method according to item 31 of the patent application scope, wherein the algae organism is a Spongiococcum strain, which has the classification of Spongiococcum excentricum HZ1236 / 538 (ATCC 74107) and its mutants, where such mutations The body can produce at least about 0.35 mg of zeaxanthin per gram of stem cell weight. 42. A zeaxanthin-containing composition comprising algae extracts capable of producing at least about 0.35 mg zeaxanthin per gram of stem cell weight, wherein zeaxanthin accounts for at least about 50% of the total carotenoid production of the extract 5%. 43. The zeaxanthin-containing composition according to item 42 of the patent application scope, in which the algae withdrawing organism is the Spongiococcum strain (-read the precautions on the back and then fill in this page) Employee Consumption Cooperation Duin of the Central Standards Bureau of the Ministry of Economic Affairs The size of the paper to be used is suitable for Sichuan, Shu, η, Jia Bingping (CNS), MM specification (2) 0> < 297 mm) 81. 4. 5,000 (H)