ES2330602B1 - METHOD OF PRODUCTION OF PHYTOENE AND / OR PHYTOFLUENE, OR CAROTENOID MIXTURES WITH HIGH CONTENT IN THE SAME - Google Patents

Abstract

Method of production of phytoeno and / or phytofluene, or mixtures of carotenoids with high content in them. The present invention describes a biotechnological method that makes it possible to obtain, in a simple, effective and large-scale way, stabilized preparations of phytoeno and / or phytophane, or mixtures of carotenoids with high content thereof, by using new mutated strains of Paracoccus sp . (FA1 and FA3) superproducers of phytoene and / or phytofluene.

Description

Production method of phytoen and / or phytofluene, or mixtures of carotenoids with high content therein.

Field of the Invention

The present invention describes a biotechnological method that makes it possible to obtain, in a simple, effective and large-scale manner, stabilized preparations of phytoene and / or phytofluene, or mixtures of carotenoids with high content thereof, by using mutated strains of Paracoccus sp . Phytoenne and / or phytofluene superproducers. Therefore, the present invention is capable of being applied in various fields of life sciences, such as in the food, pharmaceutical or cosmetic fields.

State of the art

Carotenoids are pigments of nature Isoprenoid synthesized by certain bacteria, fungi and organisms photosynthetic They can be classified into two types:

(i)

Carotenes: they are pure hydrocarbons among which find compounds such as β-carotene, α-carotene, γ-carotene, lycopene, phytoen or phytofluene; Y

(ii)

Xanthophylls: these are molecules that contain oxygen in different forms (hydroxy, epoxy groups, etc.), among which find compounds such as astaxanthin, zeaxanthin, capsantin, cantaxanthin, lutein, etc.

All these compounds play a role important in the human diet as antioxidants and as precursors of vitamin A. Therefore, due to its characteristics, Carotenoids are effective against cancer and other diseases. Due to its beneficial effects on health and its attractiveness colors, carotenoids have great commercial importance as dyes and food additives [Ninet L. and Renaut J. (1979) In: Peppler HJ., Perlman D. (eds). Microbial Technology, 2 nd. Edn, vol. 1 Academic Press, NY, pp. 529-544].

Several investigations support the theory that oxidative damage on DNA, proteins and lipids is directly related to life expectancy, and that natural antioxidants such as ascorbate, tocopherol and carotenoids are important compounds in the prevention of these oxidations [Stadtman ER (1992) Science 257: 1220-1222; Sohal et al . (1993) Proceedings of the National Academic of Sciences USA 90: 7255-7256]. Particularly, in the case of phytoen, beneficial health effects have been described as it is a compound with antitumor activity [Mathews-Roth MM (1982) Oncology 39: 33-37].

The production of carotenoids by biosynthesis Microbial is a classic example of competition between processes Chemical and biological. Biotechnological processes show, among others, the advantage of allowing to obtain in a simple way the carotenoids of more complex structure, as well as isomers conformational that only exist naturally.

Despite the structural and functional diversity of carotenoids and xanthophylls, their biosynthetic pathway is similar in different organisms, with phytoene being the precursor molecule in all cases [Arrach N. et al . (2001) Proceedings of the National Academic of Sciences USA 98: 1687-1692; Velayos A. et al . (2000) European Journal of Biochemistry 267: 5509-5519; Lee & Schmidt-Dannert (2002) Appl. Microbiol Biotechnol 60: 1-11; Sandmann G (2003) Chem. Biol. 10: 478-479; Umeno et al . (2005) Microbiol. Mol. Biol. Rev. 69: 51-78].

Biosynthesis (Scheme 1) begins with the isomerization of isopentenyl pyrophosphate (IPP, C 5) to dimethylalkyl pyrophosphate (DMAPP, C 5), catalyzed by the enzyme IPP isomerase. Next, a DMAPP molecule is condensed with an IPP molecule to generate geranyl pyrophosphate (GPP, C 10), which is condensed again with an IPP molecule to generate farnesyl pyrophosphate (FPP, C 15); both reactions are catalyzed by the enzyme FPP synthase. A new condensation of an FPP molecule with an IPP molecule results in geranylgeranyl pyrophosphate (GGPP, C20); This reaction is catalyzed by the enzyme GGPP synthase (encoded by the crtE gene). The condensation of two GGPP molecules catalyzed by the enzyme phytoen synthase (encoded by the crtB gene) generates phytoen (C 40), a precursor molecule of the biosynthetic pathway of carotenoids. The enzyme phytoene desaturase (encoded by the crtI gene) introduces four double bonds in the phytoen molecule to synthesize phytofluene, zeta-carotene, neurosporene and lycopene (C 40) consecutively. Finally, the lycopene cyclase enzyme (encoded by the crtY gene) is responsible for converting the acyclic ends of the lycopene molecule into β rings to sequentially form γ-carotene and β-carotene (C 40).

In the case of Phycomyces blakesleeanus, the yellow color of my mycelium can be modified by mutation, resulting in strains with red, white or several yellow gradations. Red mutants accumulate lycopene, while whites lack carotenoid production or accumulate phytoeno [Metha BJ and Cerdá-Olmedo E. (1995) Applied Microbiology and Biotechnology 42: 836-838].

The use of Paracoccus sp. For the production of astaxanthin, adonixanthin, equinenone, 3-hydroxyequinenone, zeaxanthin,? -carotene, lycopene, or? -cryptoxanthin is contained in EP 635576, EP 1138208, EP 1229126, EP 1676925, or US 5935808.

Description of the invention Brief Description of the Invention

The present invention describes a biotechnological method, hereinafter the method of the invention , which allows to obtain, in a simple, effective and large-scale way, stabilized preparations of phytoene and / or phytofluene, or mixtures of carotenoids with high content thereof, by means of the use of new mutated strains of Paracoccus sp. (FA1 and FA3) superproducers of phytoene and / or phytofluene.

Thus, the technical problem solved by the The present invention is to obtain in a simple, effective and large scale stabilized preparations of phytoene and phytofluene, or mixtures of carotenoids with a high phytoen content and phytofluene, which can be applied directly in the fields Food, pharmaceutical and cosmetic.

As cited in the prior art, there are several patent documents that describe the use of Paracoccus sp. for the production of various carotenoids other than phytoene and phytofluene. However, none of them refers to the use of the strains of Paracoccus sp. mutates (FA1 and FA3) achieved in the present invention for the production of phytoene or phytofluene. In the light of the documents cited in the prior art, the present invention is considered to present: (i) novelty, since none of the documents of the prior art discloses the use of the Paracoccus sp . mutated (FA1 and FA3) for simple, effective and large-scale production of stabilized preparations of phytoene and / or phytofluene; and (ii) inventive activity, since an average expert in the field could not obviously combine the documents set forth in the prior art and solve the technical problem raised above.

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Description of the figures

Figure one

HPLC chromatograms of the carotenoids accumulated by the strain Paracoccus sp . FA1

A: Left: HPLC chromatogram showing a Major peak with RT 9.46 corresponding to phytoen. Right: Absorption spectrum obtained by diodearray of the peak with RT 9.46. The maximum absorption obtained (286 nm) is characteristic of phytoeno (see Table 1).

B: Left: HPLC chromatogram showing a Major peak with RT 8.23 corresponding to phytofluene. Right: Absorption spectrum obtained by diodearray of the peak with RT 8.23. The maximum absorption obtained (348 nm) is characteristic of phytofluene (see Table 1).

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Figure 2

HPLC chromatograms of the carotenoids accumulated by the strain Paracoccus sp . FA3

A: Left: HPLC chromatogram showing a Major peak with RT 9.52 corresponding to phytoen. Right: Absorption spectrum obtained by diodearray of the peak with RT 9.52. The maximum absorption obtained (286 nm) is characteristic of phytoeno (see Table 1).

B: Left: HPLC chromatogram showing a Major peak with RT 8.29 corresponding to phytofluene. Right: Absorption spectrum obtained by diodearray of the peak with RT 8.29. The maximum absorption obtained (348 nm) is characteristic of phytofluene (see Table 1).

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Figure 3

A: Production of phytoene and phytofluene by flask fermentation of the strain Paracoccus sp . FA1. Ordered: phytoene (\ blacklozenge) and phytofluene (\ Box) (mg / L). Abscissa: fermentation time (h). The data is represented in Table 5.

B: Production of phytoeno and phytofluene by flask fermentation of the strain Paracoccus sp . FA3. Ordered: phytoene (\ blacklozenge) and phytofluene (\ Box) (mg / L). Abscissa: fermentation time (h). The data is represented in Table 6.

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Figure 4

A: Production of phytoeno and phytofluene by fermentation in a fermenting tank of the Paracoccus sp. FA1. Ordered: phytoene (\ blacklozenge) and phytofluene (\ Box) (mg / L). Abscissa: fermentation time (h). The data is represented in Table 7.

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B: Production of phytoeno and phytofluene by fermentation in a fermenting tank of the Paracoccus sp. FA3. Ordered: phytoene (\ blacklozenge) and phytofluene (\ Box) (mg / L). Abscissa: fermentation time (h). The data is represented in Table 8.

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Detailed description of the invention

As mentioned above, the method of the invention makes it possible to obtain, in a simple, effective and large-scale manner, stabilized preparations of phytoene and / or phytofluene, or mixtures of carotenoids with high content thereof, by using mutated strains of Paracoccus sp. superproducers of phytoen and / or phytofluene (FA1 and FA3).

The method of the invention comprises:

(i)

Obtaining and selecting mutants superproducers of phytoene and phytofluene from a strain of Paracoccus sp. xanthophyll producer;

(ii)

development of improved fermentation conditions to achieve maximum production of phytoeno and / or phytofluene, Y

(iii)

extraction and purification of phytoen and phytofluene produced.

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Obtaining and selecting mutants of paracoccus sp. Obtaining

In the first place and with the purpose of obtaining strains of Paracoccus sp. Superproducers of phytoene and phytofluene, a mutagenic procedure of Paracoccus sp . using the mutagenic agents ethylmethanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (NTG). The suspensions of cells to be mutated were obtained from culture in liquid medium, washing with saline solution to remove remains of the medium. The mutation procedure with EMS consisted of the incubation of approximately 10 11 cells / mL in a solution of 3% EMS in 0.1 M sodium phosphate buffer pH 7.0 at room temperature for 30 to 180 minutes, achieving Mortality rates between 90 and 99.9%. The mutated cells were washed three times with saline. The NTG mutation procedure consisted of the incubation of approximately 10 11 cells / mL in a solution containing 250 µg / mL of NTG and 0.1 M sodium phosphate buffer pH 7.0 at room temperature for 90 minutes , obtaining mortality rates of around 99%. The mutated cells were washed three times with saline. With the mutated cells, Petri dishes were seeded with solid medium and incubated at 28 ° C for 4 days to obtain isolated colonies.

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Selection

The strategy used for the selection of superproductive strains of phytoene and phytofluene from a strain of Paracoccus sp. Xanthophyll producer was based on the color of the colony. To do this, the mutated cells of Paracoccus sp. they were seeded in Petri dishes and, once grown at 28 ° C, those colonies that had a cream color were selected (the parental strain has an orange color). After analyzing about 100,000 colonies, 2 cream-colored colonies called FA1 and FA3 producing phytoene and phytophenol were isolated.

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Development of improved fermentation conditions

The Paracoccus sp . FA1 and Paracoccus sp. FA3 were fermented in a flask in order to determine the production level of phytoen and phytofluene in liquid medium. The analysis of phytoene and phytofluene was determined by reverse phase HPLC liquid chromatography, demonstrating that with both strains it is possible to produce phytoene and phytofluene together with a mixture of carotenoids in which phytoen is the majority carotenoid. Also, the strains Paracoccus sp . FA1 and Paracoccus sp. FA3 were fermented in pre-industrial fermenters in order to determine the production level of phytoene and phytofluene. In this case, two different fermentation conditions developed: one based on the use of glucose as a carbon source and another based on the use of glycerol as a carbon source. In both cases, it is shown that the FA1 and FA3 strains produce phytoene and phyto-glune together with a mixture of carotenoids in which phytoene is the majority carotenoid.

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Extraction and purification of phytoeno and phytofluene

The extraction and purification of phytoen and Phytofluene was performed by centrifuging the broth of fermentation to obtain a wet biomass, which was extracted with isopropyl alcohol. The extract obtained was concentrated and purified by two successive gel chromatographic separations of silica

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Deposit of microorganisms

As set forth above, the mutants FA1 and FA3 of the strain of Paracoccus sp ., Useful for obtaining stabilized preparations of phytoene and / or phytofluene in a simple, effective and large manner have been obtained and selected in the present invention. scale. The data related to the deposit of biological material are the following:

International Deposit Authority: Spanish Type Crops Collection (CECT).

Management of the International Deposit Authority: University of Valencia; Research building; Burjassot Campus; 46100 Burjassot (Valencia).

Number and date of deposit of the strain Paracoccus sp. FA1: CECT 7383; deposited on March 4, 2008.

Number and date of deposit of the strain Paracoccus sp. FA3: CECT 7384; deposited on March 4, 2008.

The Examples of embodiment, which are intended to illustrate the present invention without limiting it.

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Example 1 Obtaining mutants of Paracoccus sp.

First, a mutagenic procedure of a strain of Paracoccus sp . Producer of xanthophylls, for which we analyzed: (i) different types of mutagenic agents, (ii) concentration of the mutagen, (iii) concentration of cells, (iv) incubation pH and (v) treatment time. Thus, ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) were selected as mutagenic agents.

The suspensions of cells to be mutated are obtained from cultures in a liquid medium, described in EP 1229126, whose composition is as follows: corn steep liquor 30.0 g / L, sucrose 30.0 g / L, KH 2 PO 4 0.54 g / L, K 2 HPO 4 2.78 g / L, MgSO 4 • 7 H 2 O 12.0 g / L, CaCl 2 • 2H 2 O 0.1 g / L, and FeSO 4 • 7 H 2 O 0.3 g / L, adjusted to a final pH of 7.2 with NaOH. Once grown the cells were washed twice in saline (9 g / L NaCl) and were adjusted the concentration of cells in the suspension to approximately 10 11 cells / mL.

The mutation procedure with EMS consisted of in the incubation of approximately 10 11 cells / mL in a 3% EMS solution in 0.1 M sodium phosphate buffer pH 7.0 a room temperature for 30 to 180 minutes, getting rates of mortality of around 90-99.9%. The cells mutates were washed three times with saline, centrifuging at 3,000 r.p.m. and 15 ° C for 5 minutes.

The NTG mutation procedure consisted of in the incubation of approximately 10 11 cells / mL in a solution containing 250 µg / mL of NTG and sodium phosphate buffer 0.1 M pH 7.0 at room temperature for 90 minutes, getting mortality rates of around 99%. The cells mutates were washed three times with saline, centrifuging at 3,000 r.p.m. and 15 ° C for 5 minutes.

Plates were seeded with the mutated cells Petri containing a solid medium with the following composition per liter: corn steep liquor 30.0 g / L, sucrose 30.0 g / L, KH 2 PO 4 0.54 g / L, K 2 HPO 4 2.78 g / L, MgSO 4 • 7H 2 O 12.0 g / L, CaCl 2 • 2 O 0.1 g / L, FeSO4 \ 7H2O 0.3 g / L, and 15 g / L agar, adjusted a final pH of 7.2 with NaOH. The seeded plates were incubated at 28 ° C for 4 days to obtain isolated colonies.

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Example 2 Selection of mutants of Paracoccus sp . producers of phytoeno and phytofluene from a strain of Paracoccus sp . xanthophyll producer

The strain selection strategy of Paracoccus sp . producers of phytoeno and phytofluene from a strain of Paracoccus sp . Xanthophyll producer was based on the color of the colony. The selection of said mutants was made from cells mutated with EMS and NTG as described in Example 1. The mutated cells were seeded in the solid medium described in Example 1 and incubated at 28 ° C for 5 days. After this time, those colonies that had cream color were selected instead of the typical orange color of the parental strain Paracoccus sp . Xanthophyll producer. In this way, from approximately 100,000 colonies analyzed, 12 cream-colored colonies were isolated, which could be producers of phytoene and phytofluene. In order to determine the carotenoids produced by these mutants, an extraction of the biomass obtained directly from the solid medium was performed with a mixture of tetrahydrofuran: methanol: hexane (20: 1: 40) (v / v) and the extract was analyzed by HPLC liquid chromatography with the following methods: (I) For the analysis of β-carotene, lycopene, phytoene and phytofluene, a Hypersil ODS 5 µm column (100 x 4.6 mm) was used with a mobile phase of methanol: acetonitrile : chloroform (47: 47: 6) (v / v) at a flow of 1 mL / min. (II) For the analysis of xanthophylls a 100 NH2 5 µm (250 x 4.6 mm) nucleosyl column with a mobile phase of hexane: ethyl acetate (1: 1) (v / v) was used at a flow of 1 mL / min A diodearray detector was used to adapt to the maximum absorption wavelengths of the different carotenoids, which are shown in Table 1.

Of the 12 initially isolated mutants, they selected 2, called FA1 and FA3, which accumulated phytoeno and phytofluene, phytoeno being the main carotenoid. The Figures 1 and 2 show the HPLC chromatograms of the carotenoids accumulated by both strains. The phytoen production level and Phytofluene from strains FA1 and FA3 was subsequently analyzed in liquid medium as described in Examples 3, 4 and 5.

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Example 3 Production procedure of phytoen and phytofluene by flask fermentation of the Paracoccus sp . FA1 and Paracoccus sp . FA3

The Paracoccus sp . FA1 and Paracoccus sp . FA3, selected as described in Examples 1 and 2, were fermented in a flask in order to determine the production level of phytoene and phytofluene in liquid medium. For this, an inoculum medium, described in EP 1229126, was prepared with the following composition per liter: corn steep liquor 30.0 g / L, sucrose 30.0 g / L, KH 2 PO 4 0, 54 g / L, K 2 HPO 4 2.78 g / L, MgSO 4 • 12.0 g / L, CaCl 2 • 2H 2 O 0.1 g / L, and FeSO 4 • 7 H 2 O 0.3 g / L, adjusted to a final pH of 7.2 with NaOH. Each 250 mL inoculum flask with 50 mL of medium was seeded with 100 µl of the frozen culture and incubated at 28 ° C, 250 rpm and 5 cm eccentricity for 48 hours.

The fermentation medium, described in EP 1229126, has the following composition per liter: corn steep liquor 30.0 g / L, glucose 30.0 g / L, KH 2 PO 4 1.5 g / L, Na 2 HPO 4 • 12H 2 O 3.8 g / L, MgSO 4 • 7H 2 O 3.0 g / L, CaCl 2 • 2 O 0.2 g / L, and FeSO4 \ 7H2 O 1.0 g / L, adjusted to a final pH of 7.2 with NaOH. The medium was distributed in 250 Erlenmeyer flasks mL at a rate of 20 mL per flask. The flasks with the medium of fermentation were seeded with the inoculum described above and were incubated at 28 ° C and 250 r.p.m. Once the fermentation is finished (5-6 days), a broth mixture of fermentation and isopropyl alcohol (1/1). Concentration and Purity of phytoene and phytofluene was determined by the use of HPLC liquid chromatography in reverse phase.

Production levels of phytoene, phytofluene and other carotenoids obtained with FA1 and FA3 strains are shown in Tables 2, 5 and 6. Figure 3 shows the evolution of the production of phytoen and phytofluene throughout the fermentation in flask

This Example clearly demonstrates that with both strains and the described procedure it is possible to produce phytoen and phytofluene together with a mixture of carotenoids in which phytoene It is the majority carotenoid.

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Example 4 Production procedure of phytoen and phyto-glucan by fermentation in fermentation tank of the Paracoccus sp . FA1 and Paracoccus sp . FA3 using a specific glucose addition program

The Paracoccus sp . FA1 and Paracoccus sp . FA3, selected as described in Examples 1, 2, and 3, were grown in pre-industrial fermenters for the purpose of determining the production level of phytoen and phytofluene. For this, a pre-inoculum medium was prepared with the composition described in Example 3 for the inoculum medium. The pre-inoculums were seeded in 500 mL flasks with 100 mL of medium and incubated at 28 ° C and 250 rpm with 5 cm eccentricity for 48 hours. Inoculants were subsequently seeded in 500 mL flasks with 100 mL of inoculum medium (Example 3) with the pre-inoculum culture, and incubated at 28 ° C and 250 rpm with 5 cm eccentricity for 30 hours.

With the inoculum culture they were sown 30 L fermenters with 16 liters of a fermentation medium with The following composition per liter: corn steep liquor 30.0 g / L, glucose 30.0 g / L, KH 2 PO 4 1.5 g / L, Na 2 HPO 4 • 12H 2 O 3.8 g / L, MgSO 4 • 7H 2 O 3.0 g / L, CaCl 2 • 2 O 0.2 g / L, FeSO 4 • 7 H 2 O 1.0 g / L, and PPG antifoam 1 mL / L. The pH was adjusted to 7.2 with ammonia. The fermentation was incubated. for 96-144 hours at a temperature of 28 ° C with variable agitation between 150 and 500 r.p.m. and an aeration of 0.25-1 v / v / m.

From 20 hours to 90 hours it remained a 70% glucose addition to maintain a range of 10 to 30 g / L of glucose in the fermentation broth.

The assessment of the concentration and purity of phytoene and phytofluene at the end of the fermentation was performed as and as described in Example 2. Production levels of phytoeno, phytofluene and other carotenoids are shown in the Table 3.

This Example clearly demonstrates that with both strains and the described procedure it is possible to produce phytoen and phytofluene together with a mixture of carotenoids in which phytoene It is the majority carotenoid.

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Example 5 Production procedure of phytoen and phyto-glucan by fermentation in fermentation tank of the Paracoccus sp . FA1 and Paracoccus sp . FA3 using a specific glycerol addition program

The Paracoccus sp . FA1 and Paracoccus sp . FA3, selected as described in Examples 1, 2, and 3, were grown in pre-industrial fermenters for the purpose of determining the production level of phytoen and phytofluene. For this, a pre-inoculum medium was prepared with the composition described in Example 3 for the inoculum medium. The pre-inoculums were seeded in 500 mL flasks with 100 mL of medium and incubated at 28 ° C and 250 rpm with 5 cm eccentricity for 48 hours. Inoculants were subsequently seeded in 500 mL flasks with 100 mL of inoculum medium (Example 3) with the pre-inoculum culture, and incubated at 28 ° C and 250 rpm with 5 cm eccentricity for 30 hours.

With the inoculum culture they were sown 30 L fermenters with 16 liters of a fermentation medium with The following composition per liter: corn steep liquor 30 g / L, glycerol 30 g / L, KH 2 PO 4 1.5 g / L, Na 2 HPO 4 • 12H 2 O 3.8 g / L, MgSO 4 • 7H 2 O 3.0 g / L, CaCl 2 • 2 O 0.2 g / L, FeSO 4 • 7 H 2 O 1.0 g / L, and PPG antifoam 1 mL / L. The pH was adjusted to 7.2 with ammonia. The fermentation was incubated. for 96-144 hours at a temperature of 28 ° C with variable agitation between 150 and 500 r.p.m. and an aeration of 0.25-1 v / v / m.

From 20 hours to 90 hours it remained an addition of 66% glycerol to maintain a range of 10 to 30 g / L glycerol in the fermentation broth.

The assessment of the concentration and purity of phytoene and phytofluene at the end of the fermentation was performed as and as described in Example 2. Production levels of phytoeno, phytofluene and other carotenoids obtained are shown in the  Table 4

This Example clearly demonstrates that with both strains and the described procedure it is possible to produce phytoen and phytofluene together with a mixture of carotenoids in which phytoene It is the majority carotenoid.

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Example 6 Procedure for recovery and purification of phytoene and phytofluene from the fermentation broths of Paracoccus sp . FA1 and Paracoccus sp . FA3

The fermentation broth obtained as it is described in Examples 3, 4 and 5 was centrifuged, recovering the wet biomass, which was extracted with at least 2 volumes per weight of isopropyl alcohol, preferably 6 volumes by weight. The mixture was stirred for 1 hour at room temperature and It was then filtered through a Büchner funnel. The liquid obtained was concentrated under vacuum to dryness, obtaining an extract which was analyzed spectrophotometrically at 286 nm [Britton G., Liaaen-Jensen S. and Pfander H. (1995) Carotenoids. Volume 1B: Spectroscopy, p. 60, Birkhauser, Basel] showing the presence of 15-25% phytoeno and 0.5-1% phytofluene.

Then the first purification was performed chromatographic silica gel using at least 5 grams per each gram of extract to be purified, preferably 10 grams per each gram The column preparation was done by mixing gel  silica and dichloromethane. The concentrated extract, obtained as and as described above, it was dissolved in dichloromethane and loaded in the column. Fractions eluted after the process chromatography were analyzed spectrophotometrically and those that presented the UV / visible spectrum coinciding with the presence of phytoene and phytofluene evaporated to dryness. The product pre-purified obtained was analyzed spectrophotometrically showing the presence of 70-80% phytoeno and 1.0-2.0% phytofluene The aforementioned pre-purified product must be stored at -20ºC in an inert atmosphere to avoid degradation chemistry.

Finally the second purification was performed chromatographic silica gel using at least 10 grams of silica gel per gram of product pre-purified, preferably 20 grams per gram. The column preparation was done by mixing gel silica and cyclohexane or n-hexane. The product pre-purified, obtained as described previously, it was dissolved in cyclohexane or n-hexane  and loaded in the column. At the end of chromatography it is necessary increase polarity by adding ethyl acetate, eluting with mixtures of increasing polarity cyclohexane or n-hexane: ethyl acetate 98: 2, 97: 3, or 96: 4.

Fractions eluted after the process chromatography were analyzed spectrophotometrically and those that presented the UV / visible spectrum coinciding with the presence of phytoene and phytofluene evaporated to dryness. The product obtained was analyzed spectrophotometrically showing the presence 80-90% phytoeno and 0.5-1.5% phytofluene

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Scheme one

Biosynthetic Route of carotenoids and xanthophylls

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one

TABLE 1 Maximum absorption wavelengths of different carotenoids

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2

TABLE 2 Production of phytoen, phytofluene and others carotenoids of the FA1 and FA3 strains in flask at 120 hours of fermentation. The production values of each carotenoid are express as a percentage of total carotenoids produced

3

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TABLE 3 Production of phytoen, phytofluene and others carotenoids of the FA1 and FA3 strains in a 30 L fermenter with glucose addition at 96 hours of fermentation. The values of Production of each carotenoid is expressed as a percentage of the total carotenoids produced

4

TABLE 4 Production of phytoen, phytofluene and others carotenoids of the FA1 and FA3 strains in a 30 L fermenter with addition of glycerol at 96 hours of fermentation. The values of Production of each carotenoid is expressed as a percentage of the total carotenoids produced

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5

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TABLE 5 Values (mg / L) of phytoene and phytofluene production obtained by the FA1 strain in flask. These values are represented in Figure 3A

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TABLE 6 Values (mg / L) of phytoene and phytofluene production obtained by the FA3 strain in flask. These values are represented in Figure 3B

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TABLE 7 Values (mg / L) of phytoene and phytofluene production obtained by strain FA1 in a fermenter tank with glucose. These values are represented in Figure 4A

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TABLE 8 Values (mg / L) of phytoene and phytofluene production obtained by strain FA3 in a fermenter tank with glucose. These values are represented in Figure 4B

9

Claims (16)

1. Method of obtaining, effectively and on a large scale, stabilized preparations of phytoene and / or phytofluene, or mixtures of carotenoids that contain a high percentage of phytoene and / or phytofluene, which comprises the fermentation of the mutant strains Paracoccus sp . FA1 and / or Paracoccus sp . FA3. 2. Method according to claim 1, characterized in that the fermentation of the mutant strains Paracoccus sp . FA1 and Paracoccus sp . FA3 is carried out in a fermenter using a specific glucose addition program. 3. Method according to claim 2, characterized in that the specific glucose addition program comprises a 70% glucose addition from 20 hours to 90 hours to maintain 10 to 30 g / l glucose in the fermentation broth . 4. Method according to claim 1, characterized in that the fermentation of the mutant strains Paracoccus sp . FA1 and Paracoccus sp . FA3 is carried out in a fermenter using a specific glycerol addition program. 5. Method according to claim 4, characterized in that the specific glycerol addition program comprises a 66% glycerol addition from 20 hours to 90 hours to keep 10 to 30 g / l glycerol in the fermentation broth . Method according to any one of claims 1 to 5, characterized in that the proportion of phytoen in the stabilized preparation obtained is at least 10% of the total carotenoids. 7. Method according to claim 6, characterized in that the proportion of phytoen in the stabilized preparation obtained is at least 90% of the total carotenoids. Method according to any of the preceding claims, characterized in that the level of phytoen production of the Paracoccus sp . FA1 is at least 866 mg / L. 9. Method according to any of claims 1 to 7, characterized in that the level of phytofluene production of the Paracoccus sp . FA1 is at least 7 mg / L. Method according to any one of claims 1 to 7, characterized in that the level of phytoen production of the Paracoccus sp . FA3 is at least 2,066 mg / L. Method according to any one of claims 1 to 7, characterized in that the phytofluene production level of the Paracoccus sp . FA3 is at least 19 mg / L. 12. Mutant strain of Paracoccus sp . FA1 characterized by being a producer of phytoene and / or phytofluene. 13. Mutant strain of Paracoccus sp . FA3 characterized by being a producer of phytoene and / or phytofluene. 14. Use of a mutated strain of Paracoccus sp . to produce phytoene and / or phytofluene. 15. Use according to claim 14, wherein the mutated strain of Paracoccus sp . used to produce phytoen and / or phytofluene is FA1. 16. Use according to claim 14, wherein the mutated strain of Paracoccus sp . used to produce phytoene and / or phytofluene is FA3.