EP1786919A1 - Process for the production of lutein - Google Patents

Process for the production of lutein

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Publication number
EP1786919A1
EP1786919A1 EP05761115A EP05761115A EP1786919A1 EP 1786919 A1 EP1786919 A1 EP 1786919A1 EP 05761115 A EP05761115 A EP 05761115A EP 05761115 A EP05761115 A EP 05761115A EP 1786919 A1 EP1786919 A1 EP 1786919A1
Authority
EP
European Patent Office
Prior art keywords
lutein
microorganism
nutrient medium
process according
pbi
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP05761115A
Other languages
German (de)
French (fr)
Inventor
Suresh Joseph
Princy Thankam
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pharma Base SA
Original Assignee
Pharma Base SA
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Filing date
Publication date
Application filed by Pharma Base SA filed Critical Pharma Base SA
Publication of EP1786919A1 publication Critical patent/EP1786919A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes

Definitions

  • the present invention relates to a new microorganism according to claim 1, a process for the production of lutein using said new microorganism according to claim 2, a process for isolating lutein according to claim 8 and the use of the new microorganism for the production of lutein according to claim 10.
  • Lutein is a naturally occurring carotenoid which is for example found in dark green leafy vegetables such as spinach, in yellow or orange fruits such as mango, papaya, peaches and oranges, in prunes, acorn squash and corn. It is also present in egg yolk and in the human plasma.
  • Lutein is represented by the following structural formula:
  • lutein There are three asymmetric centres in lutein, i.e. in C-3, C-3 f and C- ⁇ ' of the carbocycles.
  • the absolute configuration in natural occurring lutein is 3R, 3' R.
  • Lutein is mainly used as food colorant. Apart from its use as food colorant, lutein is known to have strong antioxidant capabilities and is useful in reducing the incidence of cancer. It has also been found that lutein is useful for the prevention or treatment of macular degeneration in humans . Macular degeneration is a wide-spread, age-related condition which often leads to blindness. Since an effective cure of macular degeneration is highly- desirable, several strategies for its prevention and treatment using lutein have been studied.
  • lutein used for these therapeutic applications needs to have a particularly high purity.
  • lutein is obtained from marigold petals which are known to contain high levels of lutein.
  • US-A-20050038271 describes a method for extracting and isolating carotenoid esters from marigold petals as well as a method for saponifying said carotenoid esters to obtain a carotenoid comprising lutein.
  • US-A-20040258782 discloses a process for an extraction and purification of xanthophylls such as lutein from plant material such alfalfa.
  • lutein contained in these plants is esterified, it is subjected to a saponification process in order to obtain free lutein. Due to the saponification process, the product thus obtained is contaminated with the saponifying reagent.
  • microorganism has been deposited under the Budapest Treaty at the following depository authority:
  • the date of deposit is 1 April 2005.
  • the deposit number is DSM 17218.
  • Example 1 The isolation process for obtaining the microorganism is given in Example 1.
  • the microorganism of the present invention synthesizes lutein when cultured in a nutrient broth. Consequently, the invention also relates to a process for the production of lutein wherein a microorganism of the strain Microbacterium sp. PBI 006 deposited under deposit number DSM 17218 is cultured in a nutrient medium, thereby producing lutein.
  • the microorganism used is non- fastidious, it can be cultured in a low cost nutrient medium, such as trypticase soy broth or Zobell marine broth, both known to a person skilled in the art. It is non-pathogenic and, thus, allows the process to be carried out without any precautionary measures. Consequently, the process of the present invention is inexpensive and easy to carry out on a large scale.
  • the pH of the nutrient medium ranges from 6.1 to 9.1, more preferably from 7.0 to 8.1. At such a pH, the microorganism is growing rapidly showing a high pigmentation.
  • the growth and the pigmentation of the microorganim is particularly well if the nutrient medium comprises 6 wt% to 10 wt% of sodium chloride, more preferably about 8 wt% of sodium chloride.
  • the nutrient medium comprises about 17.0 g of casein enzyme hydrolysate, about 3.0 g of soy bean meal, about 5.0 g of sodium chloride, about 2.5 g of dipotassium phosphate and about 2.5 g of dextrose per liter and its pH is about 7.3.
  • the nutrient medium has a temperature of 3O 0 C to 37 0 C, also promoting the growth of the microorganism and, thus, the pigmentation.
  • the invention also relates to a process for isolating the lutein obtained by the above process of the present invention.
  • the nutrient medium comprising the microorganism is generally centrifuged at about 5000rpm in order to separate the cells of the microorganism from the nutrient medium.
  • the separated cells are then subjected to solvent extraction using a mixture of saline and THF as an extraction solvent.
  • the ratio of saline: THF in the mixture is about 1:3.
  • the present invention also relates to the use of the microorganism of the strain Microbacterium sp. PBI 006 deposited under deposit number DSM 17218 for the production of lutein.
  • microorganism of the present invention has been isolated as follows:
  • Marine agar plates (containing marine agar medium comprising 5.00 g of peptic digest of animal tissue, 1.00 g of yeast extract, 0.10 g of ferric citrate, 19.45 g of sodium chloride, 8.80 g of magnesium chloride, 0.55 g of potassium chloride, 0.16 g of sodium bicarbonate, 3.24 g of sodium sulphate, 1.80 g of calcium chloride, 0.55 g of potassium chloride, 0.08 g of potassium bromide, 0.022 g of boric acid, 0.004 g of sodium silicate, 0.0024 g of sodium fluorate, 0.0016 g of ammonium nitrate, 0.008 g of disodium phosphate and 15.00 g of agar per liter of medium) have been exposed to sea atmosphere in Vasai
  • the new microorganism of the present invention- can be cultured in a conventional nutrient medium, such as trypticase soy broth (comprising 17.0 g casein enzyme hydrolysate, 3.0 g of soy bean meal, 5.0 g of sodium chloride, 2.5 g of dipotassium phosphate and 2.5 g of dextrose per liter and having a pH of about 7.3). It has been characterised according to Bergey' s Manual of Systematic Bacteriology (1. edition, 1984). The results are given in Table 1. Table 1
  • the profile of the cellular fatty acids accords to the typical components for the genus Microbacterium and related organisms.
  • the microorganism of the present invention has been classified by the "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH” (DSMZ) as belonging to the genus Microbacterium.
  • the partial 16SrDNA sequences show a similarity of 99.7% to the species Microbacterium esteroaromaticum.
  • the microorganism of the present invention is positive for gelatine hydrolysis and non-motile. In addition, it growths at a sodium chloride concentration of 2%.
  • the physiological results differ in some characteristics from the characteristics of Microbacterium esteroaromaticum and has been classified as new microorganism by the DSMZ.
  • the finding that the microorganism is new has further been confirmed by DNA/DNA hybridisation against the type-strain of the species Microbacterium esteroaromaticum.
  • the process parameters pH, temperature, salinity, light and aeration were standardized by inoculating 5% wt/vol of microbial cells in 100 ml of marine agar medium, incubated for 36 hours at 25 0 C.
  • the pigmentation was visually estimated based on the intensity of the colour, the growth was examined spectrophotochemically and by determining the wet cell mass.
  • a temperature of 30 to 37 0 C is preferred in the process of the present invention.
  • the process of the present invention is preferably carried out under aeration and under light irradiation.
  • Example 3
  • the cell mass of the microorganism is isolated by low temperature centrifugation at 5000 rpm.
  • the intracellular lutein is then separated from the cells by solvent extraction using different ratios of saline: THF.
  • ratios of saline: THF of about 1:3.
  • 1% wt/vol inoculation was done in 100 ml of trypticase soy broth and incubated at standard atmosphere for 36 hours. After incubation, the cells were separated by centrifugation at 4000 rpm for 5 minutes. The separated cells were estimated for wet cell mass (corresponding to 10.62 grams/100 ml).
  • the cells were extracted for lutein using 15 ml of a mixture of saline and THF, the ratio of saline: THF being 1:3. They were sonicated for 10 minutes and centrifuged at 3000 rpm for 5 minutes. Then, the supernatant was separated and lyophilized.
  • the solvent cell mixture was sonicated and the sonicated mixture was centrifuged at 4000 rpm. After centrifugation, three layers were obtained: a bottom solid layer comprising cell debris without lutein, an intermediate saline layer and an upper solvent layer comprising the extracted lutein. The upper solvent layer was decanted carefully. The separated layer was lyophilized and the final purified mass of lutein was collected.
  • lutein For the spectrophotometric study, 2 ml of the cells were collected from a stationary phase culture and centrifuged to collect the wet cell. To the collected wet cell, 1.5 ml of THF is added and the lutein comprised is extracted. The extracted lutein is micro-filtered and spectrophotometrically scanned from 390 to 650 nm to estimate the absorption maxima.
  • Sample A is a standard sample of lutein (in MeOH)
  • sample B is the product lutein obtained in Example 3 (in MeOH)
  • sample C is a mixture of the standard sample of lutein and of the product lutein obtained Example 3 (in MeOH) .
  • Fig. 4A shows the HPLC spectrum of sample A
  • Fig. 4B shows the spectrum of sample B
  • Fig. 4C shows the spectrum of sample C.
  • the retention times and the peak heights for each sample is given in Table 6.
  • UV-Vis spectra of the samples corresponding to the peaks of Fig. s 4A, 4B and 4C are given in Fig.s 5A, 5B and 5C, respectively (the wavelength being in a range of 350 to 600 nm) .
  • the spectrophotometric study shows that the absorption maximum of the product obtained in Example 3 (448.476 nm) is similar to the lutein absorption maximum reported in Botanical Bulletin of Academic Sinica, Vol. 43, 2002 (448.447 nm) .
  • the spectrophotometric study also shows that the lutein obtained by the process of the present invention is pure all-E-lutein (3R, 3' R, 6' R). Table 6
  • the microorganism identified under I above was accompanied by
  • microorganism identified under I above was received by this International Depositary Autho ⁇ ty on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion)

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  • Engineering & Computer Science (AREA)
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  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
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  • Health & Medical Sciences (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract

The present invention relates to a microorganism of the strain Microbacterium sp. PBI 006 deposited under deposit number DSM 17218 and a process for the production of lutein wherein this microorganism is cultured in a nutrient medium, thereby producing lutein.

Description

Process for the production of lutein
The present invention relates to a new microorganism according to claim 1, a process for the production of lutein using said new microorganism according to claim 2, a process for isolating lutein according to claim 8 and the use of the new microorganism for the production of lutein according to claim 10.
Lutein is a naturally occurring carotenoid which is for example found in dark green leafy vegetables such as spinach, in yellow or orange fruits such as mango, papaya, peaches and oranges, in prunes, acorn squash and corn. It is also present in egg yolk and in the human plasma.
Lutein is represented by the following structural formula:
There are three asymmetric centres in lutein, i.e. in C-3, C-3f and C-β' of the carbocycles. The absolute configuration in natural occurring lutein is 3R, 3' R.
Lutein is mainly used as food colorant. Apart from its use as food colorant, lutein is known to have strong antioxidant capabilities and is useful in reducing the incidence of cancer. It has also been found that lutein is useful for the prevention or treatment of macular degeneration in humans . Macular degeneration is a wide-spread, age-related condition which often leads to blindness. Since an effective cure of macular degeneration is highly- desirable, several strategies for its prevention and treatment using lutein have been studied.
However, lutein used for these therapeutic applications needs to have a particularly high purity.
Conventionally, lutein is obtained from marigold petals which are known to contain high levels of lutein.
US-A-20050038271 describes a method for extracting and isolating carotenoid esters from marigold petals as well as a method for saponifying said carotenoid esters to obtain a carotenoid comprising lutein.
Extraction of lutein from other plants have been described in the state of the art.
US-A-20040258782 discloses a process for an extraction and purification of xanthophylls such as lutein from plant material such alfalfa.
As lutein contained in these plants is esterified, it is subjected to a saponification process in order to obtain free lutein. Due to the saponification process, the product thus obtained is contaminated with the saponifying reagent.
The chemical synthesis of lutein from commercially available starting materials is an extremely complex multi-step process. Due to its high complexity, no economical way of synthesizing lutein has been provided yet .
It is an object of the present invention to provide an economical process for producing lutein in highly pure form.
The problem is solved by the new microorganim of the strain Microbacterium sp. PBI 006.
The microorganism has been deposited under the Budapest Treaty at the following depository authority:
DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; Mascheroder Weg Ib; D-38124 Braunschweig .
The date of deposit is 1 April 2005. The deposit number is DSM 17218.
The isolation process for obtaining the microorganism is given in Example 1.
The characterization of the microorganism according to Bergey' s Manual of Systematic Bacteriology is given in Table 1.
It has surprisingly been found that the microorganism of the present invention synthesizes lutein when cultured in a nutrient broth. Consequently, the invention also relates to a process for the production of lutein wherein a microorganism of the strain Microbacterium sp. PBI 006 deposited under deposit number DSM 17218 is cultured in a nutrient medium, thereby producing lutein.
By the process of the present invention, high yields of lutein are obtained directly in a non-esterified form. Thus, no treatment using a saponifying agent is required and a product of higher purity is obtained compared to the process of isolating lutein from plant material such as marigold petals.
Due to the fact that the microorganism used is non- fastidious, it can be cultured in a low cost nutrient medium, such as trypticase soy broth or Zobell marine broth, both known to a person skilled in the art. It is non-pathogenic and, thus, allows the process to be carried out without any precautionary measures. Consequently, the process of the present invention is inexpensive and easy to carry out on a large scale.
In a preferred embodiment, the pH of the nutrient medium ranges from 6.1 to 9.1, more preferably from 7.0 to 8.1. At such a pH, the microorganism is growing rapidly showing a high pigmentation.
It has been found that the growth and the pigmentation of the microorganim is particularly well if the nutrient medium comprises 6 wt% to 10 wt% of sodium chloride, more preferably about 8 wt% of sodium chloride.
According to a preferred embodiment, the nutrient medium comprises about 17.0 g of casein enzyme hydrolysate, about 3.0 g of soy bean meal, about 5.0 g of sodium chloride, about 2.5 g of dipotassium phosphate and about 2.5 g of dextrose per liter and its pH is about 7.3.
It is further preferred that the nutrient medium has a temperature of 3O0C to 370C, also promoting the growth of the microorganism and, thus, the pigmentation.
Further studies have shown that growth and pigmentation of the microorganism is enhanced if the process is carried out under aeration and the irradiation of light.
The invention also relates to a process for isolating the lutein obtained by the above process of the present invention.
In this isolation process, the nutrient medium comprising the microorganism is generally centrifuged at about 5000rpm in order to separate the cells of the microorganism from the nutrient medium.
The separated cells are then subjected to solvent extraction using a mixture of saline and THF as an extraction solvent.
Preferably, the ratio of saline: THF in the mixture is about 1:3.
The present invention also relates to the use of the microorganism of the strain Microbacterium sp. PBI 006 deposited under deposit number DSM 17218 for the production of lutein.
Example 1
Isolation of microorganism of the strain Microbacter±wn sp. PBI 006 deposited under deposit number DSM 17218
The microorganism of the present invention has been isolated as follows:
Marine agar plates (containing marine agar medium comprising 5.00 g of peptic digest of animal tissue, 1.00 g of yeast extract, 0.10 g of ferric citrate, 19.45 g of sodium chloride, 8.80 g of magnesium chloride, 0.55 g of potassium chloride, 0.16 g of sodium bicarbonate, 3.24 g of sodium sulphate, 1.80 g of calcium chloride, 0.55 g of potassium chloride, 0.08 g of potassium bromide, 0.022 g of boric acid, 0.004 g of sodium silicate, 0.0024 g of sodium fluorate, 0.0016 g of ammonium nitrate, 0.008 g of disodium phosphate and 15.00 g of agar per liter of medium) have been exposed to sea atmosphere in Vasai
(Mumbai, India) . The marine agar plates have then been incubated at standard growth conditions known to a skilled person. The color of the grown colonies was analyzed. The colonies showing an orange colour were further examined for their carotenoid content by extraction of the carotenoid, followed by spectrophotometric scan. The corresponding UV-Vis spectrum is given in Fig. 1 (the wavelength being in a range of 400 to 800 nm) . The two peaks at 444.0 nm (0.5722 P) and 470.0 nm (0.4962 P) shown in the UV-Vis spectrum correspond to the carotenoid lutein. The finding that the isolated strain produces lutein has also been confirmed by IR spectroscopy (Fig. 2), mass spectroscopy (Fig. 3) and TLC (not shown) .
The new microorganism of the present invention- can be cultured in a conventional nutrient medium, such as trypticase soy broth (comprising 17.0 g casein enzyme hydrolysate, 3.0 g of soy bean meal, 5.0 g of sodium chloride, 2.5 g of dipotassium phosphate and 2.5 g of dextrose per liter and having a pH of about 7.3). It has been characterised according to Bergey' s Manual of Systematic Bacteriology (1. edition, 1984). The results are given in Table 1. Table 1
The profile of the cellular fatty acids accords to the typical components for the genus Microbacterium and related organisms. The microorganism of the present invention has been classified by the "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH" (DSMZ) as belonging to the genus Microbacterium. The partial 16SrDNA sequences show a similarity of 99.7% to the species Microbacterium esteroaromaticum. However, the microorganism of the present invention is positive for gelatine hydrolysis and non-motile. In addition, it growths at a sodium chloride concentration of 2%. Thus, the physiological results differ in some characteristics from the characteristics of Microbacterium esteroaromaticum and has been classified as new microorganism by the DSMZ. The finding that the microorganism is new has further been confirmed by DNA/DNA hybridisation against the type-strain of the species Microbacterium esteroaromaticum.
Example 2
Process parameters of preferred embodiments
Studies on the influence of certain process parameters have been carried out.
The process parameters pH, temperature, salinity, light and aeration were standardized by inoculating 5% wt/vol of microbial cells in 100 ml of marine agar medium, incubated for 36 hours at 250C. The pigmentation was visually estimated based on the intensity of the colour, the growth was examined spectrophotochemically and by determining the wet cell mass.
a) pH
The growth and the pigmentation of strain Microbacterium sp. PBI 006 at varying pH in the nutrient medium has been studied. The results are given in Table 2. The term "grade" means the growth of Microbacterium sp. PBI 006 determined on the basis of the visual color intensity of the culture. "+" means "weak" and "++" means "strong". Table 2
As can be seen from Table 2, a pH of 7.0 to is preferred in the process of the present invention.
b) Temperature
The growth of strain Microbacterium sp. PBI 006 and the production of lutein at varying temperature has been studied. The results are given in Table 3.
Table 3
As can be seen from Table 3, a temperature of 30 to 370C is preferred in the process of the present invention.
c) Salinity
In a further test, the growth of strain Microbacterium sp. PBI 006 and the production of lutein has been studied at varying concentrations of sodium chloride in the nutrient broth. The sodium chloride concentration is given in weight-% based on the weight of the nutrient medium. The results are given in Table 4.
Table 4
As can be seen from Table 4, a concentration of sodium chloride of about 8 weight-% is preferred in the process of the present invention. d) Light and aeration
In a further test, the growth of strain Microbacterium sp. PBI 006 and the production of lutein has been studied with and without aeration and with and without light irradiation. Aeration is carried out in a shaker using a baffled flask at 180 to 200 rpm. The light source is an ordinary laboratory lamp. The results are given in Table 5.
Table 5
Condition Growth
with aeration and slight growth and pigmentation without light
without aeration and slight growth and pigmentation with light
without aeration and slight growth and pigmentation without light
with aeration and with more growth and more light pigmentation
As can be seen from Table 5, the process of the present invention is preferably carried out under aeration and under light irradiation. Example 3
Isolation of lutein
After completion of the cultivation of the microorganism, the cell mass of the microorganism is isolated by low temperature centrifugation at 5000 rpm. The intracellular lutein is then separated from the cells by solvent extraction using different ratios of saline: THF. The best results were obtained with ratios of saline: THF of about 1:3. 1% wt/vol inoculation was done in 100 ml of trypticase soy broth and incubated at standard atmosphere for 36 hours. After incubation, the cells were separated by centrifugation at 4000 rpm for 5 minutes. The separated cells were estimated for wet cell mass (corresponding to 10.62 grams/100 ml). The cells were extracted for lutein using 15 ml of a mixture of saline and THF, the ratio of saline: THF being 1:3. They were sonicated for 10 minutes and centrifuged at 3000 rpm for 5 minutes. Then, the supernatant was separated and lyophilized.
The solvent cell mixture was sonicated and the sonicated mixture was centrifuged at 4000 rpm. After centrifugation, three layers were obtained: a bottom solid layer comprising cell debris without lutein, an intermediate saline layer and an upper solvent layer comprising the extracted lutein. The upper solvent layer was decanted carefully. The separated layer was lyophilized and the final purified mass of lutein was collected.
HPLC-, IR-, spectrophotometric and mass spectrometric studies have confirmed that the product is lutein.
For the spectrophotometric study, 2 ml of the cells were collected from a stationary phase culture and centrifuged to collect the wet cell. To the collected wet cell, 1.5 ml of THF is added and the lutein comprised is extracted. The extracted lutein is micro-filtered and spectrophotometrically scanned from 390 to 650 nm to estimate the absorption maxima.
In addition, the three discrete samples A, B and C have been analyzed. Sample A is a standard sample of lutein (in MeOH) , sample B is the product lutein obtained in Example 3 (in MeOH) and sample C is a mixture of the standard sample of lutein and of the product lutein obtained Example 3 (in MeOH) .
HPLC study:
The HPLC studies were carried out using a PDA detector. Fig. 4A shows the HPLC spectrum of sample A, Fig. 4B shows the spectrum of sample B and Fig. 4C shows the spectrum of sample C. The retention times and the peak heights for each sample is given in Table 6.
UV-Vis spectra of the samples corresponding to the peaks of Fig. s 4A, 4B and 4C are given in Fig.s 5A, 5B and 5C, respectively (the wavelength being in a range of 350 to 600 nm) .
The spectrophotometric study shows that the absorption maximum of the product obtained in Example 3 (448.476 nm) is similar to the lutein absorption maximum reported in Botanical Bulletin of Academic Sinica, Vol. 43, 2002 (448.447 nm) . The spectrophotometric study also shows that the lutein obtained by the process of the present invention is pure all-E-lutein (3R, 3' R, 6' R). Table 6
The results confirm that the product obtained by the process of the present invention is lutein.
Annex to Fig. 2
LUTINA-2.SP 3551 4000.00 450.00 28.03 84.98 4.00 %T 8 0.30
B.No. PBI006
REF 4000 82.64 2000 84.95 600
3649.08 82.06 3392.16 76.93 2928.19 28.03 2855.12 37.08 2728.85 78.36
1730.41 57.52 1654.34 79.92 1601.07 74.58 1580.65 77.01 1513.57 74.34
1459.19 47.08 1377.12 57.69 1270.57 58.89 1204.05 71.27 1168.36 70.60
1121.66 65.55 1071.89 64.67 1039.26 70.27 1017.50 70.91 960.46 76.33
871.64 79.20 813.12 76.99 743.60 72.79 723.47 75.41 703.63 79.13
668.36 83.63 651.88 83.63 545.51 83.38 490.97 82.95 477.70 82.57
Annex to Fig. 4A
λrea Percent Report
Sorted By Signal
Multiplier 1.0000
Dilution 1.0000
Use Multiplier & Dilution Factor with STDs
Signal 1 : DAD1 A, Sig=450 ,4 Ref= 360,100
Peak RetTime Type Width Area Height Area
No. [min] [min] [mAU*s] [mAU] %
1 1.280 BP 0.1175 77.12190 8.86038 1.2289
2 1.571 W 0.1050 6001.64990 864.01160 95.6313
3 1.718 W 0.1102 138.36694 17.50082 2.2048
4 1.989 VB 0.1782 58.67996 4.38175 0.9350
Totals: 6275.81871 894.75455
Results obtained with enhanced integrator
Annex to Fig. 4B
Area Percent Report
Sorted By Signal
Multiplier 1.0000
Dilution 1.0000
Use Multiplier & Dilution Factor with STDs
Signal 1: DAD1 A, Sig=450,4 Ref= 360,100
Peak RetTime Type Width Area Height Area
No. [min] [min] [mAU*s] [mAU] %
1 0.822 BP 0.3541 243.10497 9.64494 3.6380
2 1.318 W 0.1132 154.71983 19.35742 2.3153
3 1.579 W 0.1541 6131.33398 549.80933 91.7534
4 2.218 W 0.1451 153.24924 15.53183 2.2933
Totals: 6682.40802 594.34352
Results obtained with enhanced integrator
Annex to Fig. 4C
Area Percent Report
Sorted By Signal
Multiplier 1.0000
Dilution 1.0000
Use Multiplier & Dilution Factor with STDs
Signal 1: DAD1 A, Sig=450,4 Ref= 360,100
Peak RetTime Type Width Area Height Area
No. [min] [min] [mAU*s] [mAU] %
1 0.815 BP 0.3188 96.51286 3.84494 1.5525
2 1.296 W 0.1034 64.16727 9.19554 1.0322
3 1.570 W 0.1103 5452.21143 754.65997 87.7065
4 1.724 VB 0.1281 603.53418 61.71065 9.7087
Totals: 6216.42573 829.41109
Results obtained with enhanced integrator
INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL
(PCT RuIe 13Ms)
A. The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 3 , tine 6 .
B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | |
Name of depositary institution DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
Address of depositary institution (including postal code and country)
Mascheroder Weg 1 b D-38124 Braunschweig
Date of deposit Accession Number
1 April 2005 DSM 17218
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet
Identification reference given by the depositor: PBI 006 (see acknowledgement of receipt of the DSMZ) The microorganism is viable (see viability statement of the DSMZ)
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)
The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., "Accession Number of Deposit")
Form PCT/RO/134 (Julyl998; reprint January 2004) BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE D Deutsc5he MX.
Sam m lung von
Mi kro organism en ^ und Zβllkulturen GmbH • ^y
INTERNATIONAL FORM
Pharma Base S A Churerstr 166 8808 Pfaffikon/SZ RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7 1 by the SWITZERLAND INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page
I IDENTIFICATION OF THE MICROORGANISM
Identification reference given by the DEPOSITOR Accession number given by the INTERNATIONAL DEPOSfTARY AUTHORITY
PBI 006
DSM 17218
π SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I above was accompanied by
( X ) a scientific description
( jς) a proposed taxonomic designation
(Mark with a cross where applicable)
m RECEIPT AND ACCEPTANCE
This International Depositary Authoπty accepts the microorganism identified under I above, which was received by it on 2005-04-01 (Date of the original deposit)1
IV RECEIPT OF REQUEST FOR CONVERSION
The microorganism identified under I above was received by this International Depositary Authoπty on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion)
V INTERNATIONAL DEPOSITARY AUTHORITY
Name DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of ρerson(s) having the power to represent the
MHCROORGANISMEN UND ZELLKULTUREN GmbH Internationa! Depositary Authoπty or of authorized officials)
Address Mascheroder Weg Ib D-38124 Braunschweig
Date 2005-04-05
' Where Rule 6 4 (d) applies, such date is the date on which the status of international depositary authoπty was acquired Form DSMZ-BP/4 (sole page) 12/2001 BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Db X
FOR THE PURPOSES OF PATENT PROCEDURE Deutsche M Sαmmlung von
Mikroorgαnismen ^^ ^ und Zellkulturβn GmbH ***
INTERNATIONAL FORM
Pharma Base S A Churerstr 166 8808 Pfaffikon/SZ
VIABILITY STATEMENT SWITZERLAND issued pursuant to Rule 10 2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page
L DEPOSITOR π IDENTIFICATION OF THE MICROORGANISM
Name Pharma Base S A Accession number given by the Churerstr 166 INTERNATIONAL DEPOSITARY AUTHORITY Address 8808 Pfaffikon/SZ
DSM 17218 SWITZERLAND
Date of the deposit or the transfer1
2005-04-01
HI VIABILITY STATEMENT
The viability of the microorganism identified under π above was tested on 2005-04-01 On that date, the said microorganism was
Cx)1 viable
( y no longer viable
IV CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED4
V INTERNAT[ONALDEPOStTARY AUTHORITY
Name DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of person(s) having the power to represent the
MKROORGAMSMEN UND ZELLKULTUREN GmbH International Depositary Authority or of authorized official(s)
Address Mascheroder Weg Ib
D-38124 Braunschweig
Date 2005-04-05
Indicate the date of original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer)
In the cases referred to m Rule 10 2(a) (iι) and (in), refer to the moat recent viability test
Mark with a cross the applicable box
Fill in if the information has been requested and if the results of the test were negative
Form DSMZ-BP/9 (sole page) 12/2001

Claims

Claims
1. Microorganism of the strain Microbacteriuia sp. PBI 006 deposited under deposit number DSM 17218.
2. A process for the production of lutein wherein the microorganism of claim 1 is cultured in a nutrient medium, thereby producing lutein.
3. The process according to claim 2 wherein the nutrient medium has a pH of about 7.0.
4. The process according to claim 2 or 3 wherein the nutrient medium comprises about 8 weight-% of sodium chloride .
5. The process according to claim 2 wherein the nutrient medium comprises about 17.0 g casein enzyme hydrolysate, about 3.0 g of soy bean meal, about 5.0 g of sodium chloride, about 2.5 g of dipotassium phosphate and about 2.5 g of dextrose per liter and the pH is about 7.3.
6. The process according to any of claims 2 to 5 wherein the nutrient medium has a temperature of 300C to
37°C.
7. The process according to any of claims 2 to 6 wherein the process is carried out under aeration and the irradiation of light.
8. A process for isolating lutein obtained by the process according to any of claims 2 to 7 comprising •
separating the cells of the microorganism from the nutrient medium and
extracting lutein from the separated cells using a mixture of saline and THF as extraction solvent.
9. The process of claim 8 wherein the ratio of saline: THF in the extraction solvent is about 1:3.
10. Use of the microorganism of the strain Microbacterium sp. PBI 006 deposited under deposit number DSM 17218 for the production of lutein.
EP05761115A 2005-07-28 2005-07-28 Process for the production of lutein Withdrawn EP1786919A1 (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CH2005/000448 WO2007012205A1 (en) 2005-07-28 2005-07-28 Process for the production of lutein

Publications (1)

Publication Number Publication Date
EP1786919A1 true EP1786919A1 (en) 2007-05-23

Family

ID=36072095

Family Applications (1)

Application Number Title Priority Date Filing Date
EP05761115A Withdrawn EP1786919A1 (en) 2005-07-28 2005-07-28 Process for the production of lutein

Country Status (4)

Country Link
EP (1) EP1786919A1 (en)
AR (1) AR054570A1 (en)
CA (1) CA2579669A1 (en)
WO (1) WO2007012205A1 (en)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2007012205A1 *

Also Published As

Publication number Publication date
WO2007012205A1 (en) 2007-02-01
AR054570A1 (en) 2007-06-27
CA2579669A1 (en) 2007-02-01

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