KR20050021993A - Process for producing carotenoids - Google Patents

Abstract

The present invention can produce carotenoids in the presence of an inhibitor of sterol biosynthesis from farnesyl pyrophosphate and biologically produce carotenoids using microorganisms belonging to the genus Xanthophyllomyces ( Paffia ). It is about.

Description

Production method of carotenoids {PROCESS FOR PRODUCING CAROTENOIDS}

The present invention is capable of producing carotenoids in the presence of an inhibitor for biosynthesis of sterols from (hereinafter referred to later, FPP) Parr nesil pyrophosphate and using microorganisms belonging to the genus Xanthomonas Philo My process (Xantophyllomyces) (wave PIA (Phaffia)) A method for biologically preparing carotenoids.

More than 600 different carotenoids have been described from carotenogenic organisms found in bacteria, yeasts, fungi and plants. Currently two of these, beta-carotene and astaxanthin, are commercially produced in microorganisms and used in the food and feed industry. These carotenoids are of industrial importance as natural pigments and as functional substances for human health due to their strong antioxidant properties. In addition, in commercial properties, the need for astaxanthin as a coloring reagent is increasing, especially in the fish farming industry, such as salmon farming, because astaxanthin provides fish with a distinctive orange-red coloration and contributes to consumer preference. .

Other carotenoids such as lycopene, zeaxanthin, canthaxanthin and beta-kryptoxanthin are also of industrial importance as natural pigments and antioxidants. Lycopene shows that monolithic oxygen can be effectively removed in the ex vivo experiments performed. Lycopene inhibits lipid peroxidation, and the serum levels of this carotenoid are inversely related to the risk of cancer in the pancreas and cervix. Fruits and vegetables such as tomatoes, pink grapes, the surface of red grapes, the watermelon and the red color of red guava are due to lycopene. Other edible sources include papaya and apricots. Zeaxanthin is a yellow colored carotenoid and is an oxidized hydroxy derivative of beta-carotene. Zeaxanthin is abundant in spinach and corn and many other plant species. It is a strong antioxidant and is also observed in the retina. It is widely believed that zeaxanthin acts as a filter, blocks harmful blue light from the eye and protects against spot degeneration associated with aging. Canthaxanthin is a red-colored cannotenoid and is found in many plants and animals. It is used in egg yolks, broilers and farmed trout and foods and in cosmetics that require many orange-reds. Canthaxanthin also functions as a monolithic oxygen scavenger and free radical inert. Beta-Cryptoxanthin is a yellow-colored carotenoid found in oranges, mangoes, papayas, pumpkins and many other fruits. Beta-Cryptoxanthin is recognized as a powerful antioxidant useful for preventing cancer.

Among the microorganisms capable of producing significant amounts of carotenoids, Phaffia rhodozyma is one of the best known carotenogenic strains. This yeast strain is one of some microorganisms commonly used to produce astaxanthin and provide astaxanthin in the food and feed industry. In a recent taxonomic study, the meteor generation of Papia Rhododima was identified and its telemorph state was named Xanthophyllomyces dendrorose (Golubev, Yeast 11: 101-110 (1995)). ). In the present technology, the widely recognized name Papia Rhododima is used.

Several studies have been conducted to increase the level of carotenoid production by microorganisms, for example Esherichia coli , Saccharomyces cerevisiae or Candida utilis . Construction of recombinant microorganisms genetically engineered to obtain the ability to produce significant amounts of several carotenoids using appropriate host strains such as (Misawa et al., J. Bacteriol. 172: 6704 (1990); Yamano et al. , Biosci.Biotech.Biochem. 58: 1112 (1994); Miura et al., Biotechnol.Bioeng. 58: 306 (1998)], for example to obtain over-producers of astaxanthin from papia rhodoma. Strain improvement (Johnson et al., Critical Reviews in Biotechnology 11: 297-326 (1991)) and process improvements to optimize the fermentation process for, for example, astaxanthin production by papia rhodoma Patent No. US 5,9 72,642) It is described in US Pat. No. 5,356,809 that the addition of antimycin or another inhibitor of the main respiratory chain to papia rhodoma cells enhances astaxanthin production. Obviously, there is still a need for a simple and efficient method of increasing the production yield of carotenoids.

One aspect of the invention is a biological method for the production of carotenoids comprising culturing microorganisms capable of producing carotenoids in aquatic nutrient media under aerobic conditions in the presence of an inhibitor of sterol biosynthesis from FPP.

It is more preferable to use highly carotenogenic microorganisms, such as those belonging to the genus Xanthophyllomyces (Papia). Thus, a preferred example of the microorganism of the present invention is a microorganism belonging to the genus Xanthophyllomyces (Papia). Preferred strains of Xanthophyllomyces (Papia) of the present invention are Xanthophyll Myces (Papia) ATCC 96594 from the American Type Culture Collection (20110-2209 Manassas University Boulevard 10801, VA) In accordance with the Budapest Treaty, reassigned to accession number ATCC 74438 dated April 8, 1998).

Accordingly, the present invention is particularly capable of producing carotenoids and belonging to the genus Xanthophyllomyces (Papia) in the presence of a substrate for producing carotenoids in aquatic nutrient medium under aerobic conditions and an inhibitor of sterol biosynthesis from FPP. It relates to a method for producing a biological carotenoid comprising culturing and isolating the carotenoid generated from the cells of the microorganism or from the culture.

In particular, the biological method of the present invention is more preferred for inhibiting the first reaction of the sterol pathway, which is catalyzed by squalene synthetase.

Squalene synthase inhibitors are reported at lower intracellular sterol levels. Squalene synthase is an enzyme that catalyzes the first performed step of sterol biosynthesis. Two molecules of FPP condense to form squalene.

Thus, a further aspect of the invention is a method of biologically producing a carotenoid comprising culturing a microorganism capable of producing the carotenoid in the presence of an inhibitor against squalene synthetase.

Several classes of squalene synthetase inhibitors have been reported in Miller et al., Current Pharmaceutical Design 2: 1-40 (1996). Approximately three groups of squalene synthase inhibitors have been reported as ammonium ion-based squalene synthetase inhibitors, phosphorus-containing FPP analogs and carboxylate-based inhibitors. Any kind of squalene synthetase inhibitor is preferred for the present invention. One preferred example of such a squalene synthetase inhibitor is an ammonium ion-based squalene synthetase inhibitor.

One preferred example of an ammonium squalene synthetase inhibitor is a phenoxypropylamine type squalene synthetase inhibitor (Brown et al., Journal of Medicinal Chemistry 38: 4157-4160 (1995)). Many phenoxypropylamine type squalene synthetase inhibitors, for example [3- (3-allyl-biphenyl-4-yloxy) -propyl] -isopropyl-amine, N-isopropyl-3- (4 -Acetamido-2-allylphenoxy) propylamine, N-methyl-N-isopropyl-3- (4-acetamido-2-allylphenoxy) propylamine, N-cyclopentyl-3- (4 -Acetamido-2-allylphenoxy) propylamine, N-cyclobutyl-3- (4-acetamido-2-allylphenoxy) propylamine, N-isopropyl-3- (2-allyl-4 -Butylamidophenoxy) propylamine, N-isopropyl-3- (4-acetamido-2-chloro-phenoxy) propylamine, N-isopropyl-3- (4-acetamido-2 -Propylphenoxy) propylamine and N-isopropyl-3- (4-acetamido-2-allylphenoxy) -1-methylpropylamine and their biologically acceptable salts are known (Brown et al. Literature).

Preferred squalene synthetase inhibitors used in this example are [3- (3-allyl-biphenyl-4-yloxy) -propyl] -isopropyl-amine, N-isopropyl-3- (4-acetamido -2-allylphenoxy) propylamine and N-methyl-N-isopropyl-3- (4-acetamido-2-allylphenoxy) propylamine.

Carotenoids are usually suitable macronutrients and micronutrients for cells, such as hydrocarbon sources for cell growth and as substrates for preparing carotenoids, molasses, saccharose or glucose, nitrogen sources such as corn soaks, yeast extracts, diammonium sulfate, Prepared by culturing a carotenogenous microorganism in a medium comprising ammonium phosphate, ammonium hydroxide or urea, a person, such as ammonium phosphate and phosphoric acid and added micronutrients or mineral salts such as magnesium sulfate, zinc sulfate and biotin or desthiobiotin do.

Preferred conditions for the cultivation are a pH in the range of 4 to 8, a temperature in the range of 15 to 26 ° C. and 24 to 500 hours. More preferred conditions for incubation are pH in the range of 5-7, temperature in the range of 18-22 ° C. and 48-350 hours.

In culture, aerobic and agitation usually leads to carotenoid production.

In the present invention, an inhibitor for sterol biosynthesis from FPP is added to the medium. Suitably, the concentration of the inhibitor varies depending on the species of the inhibitor and the microorganisms used for the carotenoid production, for example, at a concentration that provides a reduction of less than 50% of cell growth under carotenoid production conditions. More preferred concentrations of inhibitors may be in a range of concentrations providing less than 30% reduction in cell growth.

In the present invention, inhibitors for sterol biosynthesis from FPP can be added to the medium during any period of culture.

The carotenoids produced by culturing the carotenogenic microorganisms using the method of the present invention can be isolated from the medium or from cells of the microorganism when secreted into the medium and, if necessary, by methods known in the art. Isolation and Analysis, Britton et al., Birkh user Verlag, Basel (1995)] may be isolated from other carotenoids that may be present if one particular carotenoid is required.

The carotenoids produced according to the invention can be used in a method for the production of food or feed. Those skilled in the art are familiar with these methods. Such compound foods or feeds may also comprise additives or ingredients commonly used for this purpose and known in the art.

The following examples also illustrate the invention but do not limit the scope of the invention.

The squalene synthetase inhibitor used in the examples is [3- (3-allyl-biphenyl-4-yloxy) -propyl] -isopropyl-amine, which is one of the penoxyflophyllamine type inhibitors. This compound was synthesized according to the method described by Brown et al., Supra. That is, biphenyl-4-ol is reacted with allyl bromide and potassium carbonate in butan-2-one and thermally rearranged to produce 3-allyl-biphenyl-4-ol (product code H7751, Sigma, USA). It was. Reacting 3-allyl-biphenyl-4-ol with 1,3-dibromopropane and potassium carbonate in butan-2-one followed by isopropylamine in 2-propanol [3- (3-allyl-biphenyl 4-yloxy) -propyl] -isopropyl-amine was obtained.

Example 1

Effect of Squalene Synthase Inhibitor Addition on the Cell Growth of Papia Rhodonma

Papia Rhododima ATCC 96594 was inoculated in YPD medium (DIFCO, Detroit, USA, 10 mL) and shaken for 2 days at 20 ° C. 0.5 ml of culture was added with 30 g / L glucose and [3- (3-allyl-biphenyl-4-yloxy) -propyl] -isopropyl-amine 0, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 and 20.0 μg. Fresh YPD medium (10 ml in tubes) containing / ml each was inoculated and shaken for 5 days at 20 ° C.

Aliquots of cultures were often collected during the cultivation and measured using an UV-1200 photometer (Shimadzu Corp., Kyoto, Japan) at an optical density (OD) of 660 nm to analyze cell growth. The results are shown in Table 1 below.

Cell growth was ineffective when the concentration of squalene synthase inhibitor was 2.0 μg / ml or less. An inhibition of about 23% of cell growth was observed at day 2 at 5.0 μg / ml of inhibitor.

Example 2

Effect of Squalene Synthase Inhibitor Addition on Astaxanthin Production by Papia Rhododima

Papia Rhododima ATCC 96594 was inoculated in YPD medium as in Example 1 and shaken at 20 ° C. for 2 days. 2.5 ml of culture contained 22 g / l glucose and 0, 0.5, 1.0, 2.0 and 5.0 μg / ml [3- (3-allyl-biphenyl-4-yloxy) -propyl] -isopropyl-amine, respectively Fresh YPD medium (50 mL in flask) was inoculated and shaken for 7 days at 20 ° C. On day 2 of culture, 50 g / l of glucose was added to the culture. The addition of inhibitors in the middle of the culture was also tested. Aliquots of the culture were recovered on days 4 and 7 of the culture and the optical density (by using the same method as described in Example 1) and astaxanthin content in the culture were measured at 660 nm.

To analyze the content of astaxanthin, the carotenoids were extracted from the cells of Papia Rhododima by mixing the recovered cultures with a solvent mixture (ethyl alcohol, hexane and ethyl acetate) and vigorously mixing with glass beads. After extraction, the destroyed cells and free beads were removed by centrifugation and the resulting suspension was analyzed for astaxanthin content by HPLC. HPLC conditions used were as follows:

Reference samples of astaxanthin were obtained from Hoffmann La-Roche (Basel, Switzerland). The results are shown in Table 2 below.

Astaxanthin production was enhanced under all conditions tested. The best results were obtained when the inhibitor was added on day 2 of the culture, but the inhibitor was also effective for astaxanthin production from the beginning of the culture.

Claims (12)

A method for biological preparation of carotenoids comprising culturing microorganisms capable of producing carotenoids in aquatic nutrient media under aerobic conditions in the presence of an inhibitor of sterol biosynthesis from farnesyl pyrophosphate. It is possible to produce carotenoids in the presence of a substrate for producing carotenoids in aquatic nutrient medium under aerobic conditions and an inhibitor of sterol biosynthesis from farnesyl pyrophosphate and belong to the genus Xanthophyllomyces ( Paffia ). Culturing the microorganism and isolating the carotenoid generated from the cells of the microorganism or from the culture medium. The method of claim 2, The microorganism is Xanthophyllomyces dendrorhous ( Paffia rhodozyma ) ATCC 96594. The method according to claim 1 or 2, The inhibitor for the biosynthesis of sterols from farnesyl pyrophosphate is selected from the group consisting of squalene synthase inhibitors. The method of claim 4, wherein Wherein the squalene synthase inhibitor is selected from the group consisting of ammonium ion-based squalene synthetase inhibitors. The method of claim 5, wherein A method wherein the ammonium ion-based squalene synthase inhibitor is selected from the group consisting of phenoxypropylamine type squalene synthetase inhibitors. The method of claim 6, Phenoxypropylamine type squalene synthetase inhibitor [3- (3-allyl-biphenyl-4-yloxy) -propyl] -isopropyl-amine, N-isopropyl-3- (4-acetamido-2-allylphenoxy) propylamine, N-methyl-N-isopropyl-3- (4-acetamido-2-allylphenoxy) propylamine, N-cyclopentyl-3- (4-acetamido-2-allylphenoxy) propylamine, N-cyclobutyl-3- (4-acetamido-2-allylphenoxy) propylamine, N-isopropyl-3- (2-allyl-4-butyramidophenoxy) propylamine, N-isopropyl-3- (4-acetamido-2-chloro-phenoxy) propylamine, N-isopropyl-3- (4-acetamido-2-propylphenoxy) propylamine, N-isopropyl-3- (4-acetamido-2-allylphenoxy) -1-methylpropylamine and Their biologically acceptable salts Method selected from the group consisting of. The method of claim 7, wherein Phenoxypropylamine type squalene synthetase inhibitor [3- (3-allyl-biphenyl-4-yloxy) -propyl] -isopropyl-amine or a biologically acceptable salt thereof, N-isopropyl-3- (4-acetamido-2-allylphenoxy) propylamine or N-methyl-N-isopropyl-3- (4-acetamido-2-allylphenoxy) propylamine. The method according to claim 1 or 2, Wherein the concentration of inhibitor is within a range that provides a reduction of less than 50% of cell growth under carotenoid production conditions. The method of claim 9, Wherein the concentration of inhibitor is within a range that provides less than 30% reduction in cell growth under carotenoid production conditions. The method according to claim 1 or 2, Culturing is carried out for 24 to 500 hours at a temperature in the range from 15 to 26 ° C. at a pH in the range from 4 to 8. The method of claim 11, Incubating at a pH in the range of 5-7 for a temperature of 18-22 ° C. for 48-350 hours.