WO2007012204A1

WO2007012204A1 - Process for the production of zeaxanthin

Info

Publication number: WO2007012204A1
Application number: PCT/CH2005/000447
Authority: WO
Inventors: Suresh Joseph; Princy Thankam
Original assignee: Pharma Base S.A.
Priority date: 2005-07-28
Filing date: 2005-07-28
Publication date: 2007-02-01
Also published as: AR058015A1

Abstract

The invention relates to process for the production of zeaxanthin wherein a microorganism of the species Dietzia maris is cultured in a nutrient medium, thereby producing zeaxanthin.

Description

Process for the production of zeaxanthin

The present invention relates to a process for the production of zeaxanthin according to claim 1, a process for isolating zeaxanthin according to claim 9 and the use of a microorganism of the species Dietzia maris for the production of zeaxanthin according to claim 11.

Zeaxanthin (3, 3' -dihydroxy-β-carotene) is a yellow carotenoid pigment. It is represented by the following structural formula:

Zeaxanthin is for example found in corn, egg yolk, crustaceans, fishes, poultry and toadstools. It is used as food colorant for various types of food, such as margarine, dairy products, syrups, cookie doughs and certain types of meat preparations which are not subjected to harsh cooking.

Apart from its use as colorant, zeaxanthin can specifically be used for preventing or treating macular degeneration in humans. Macular degeneration is a widespread, age-related condition often leading to blindness. Since an effective cure of macular degeneration is highly desirable, several strategies for its prevention and treatment have been studied.

Although the presence of zeaxanthin in the macula and its effects on the prevention of macular degeneration has been recognized, zeaxanthin has not been used for this purpose on a large scale due to its high costs.

Zeaxanthin is conventionally obtained from marigold by a complicated process. Efforts for obtaining zeaxanthin in a large quantity by microbial synthesis have been made.

Zeaxanthin is known to be synthesized biologically by very few bacterial species of the genus Flavobacter under certain process conditions.

US Patent No. 3,841,967 discloses a process for the preparation of zeaxanthin by culture of a zeaxanthin- producing microorganism of the genus Flavobacter in a defined nutrient medium.

US Patent No. 3,891,504 discloses a process for the preparation of zeaxanthin containing cell-mass, comprising cultivating a microorganism of a certain species of the genus Flavobacter.

US Patent No. 3,951,742 describes a process for the preparation of zeaxanthin which comprises culturing a microorganism of the genus Flavobacter producing this pigment in a nutrient medium to which pyridoxine is added.

US Patent No. 4,026,949 discloses the production of optically active intermediates used in the production of carotenoids, such as zeaxanthin; one of the bacteria used in this process is Flavobacterium dehydrogenans . US Patent No. 5,308,759 discloses a process for the production of zeaxanthin comprising the step of culturing a strain of Flavobacterium multivorum in a nutrient medium.

The above mentioned processes of the state of the art have not been thoroughly satisfying since the yield of zeaxanthin produced thereby is generally rather low.

It is therefore an object of the present invention to provide an inexpensive process for producing zeaxanthin in high yields.

The problem is solved by a process for the production of zeaxanthin according to claim 1 wherein a microorganism of the species Dietzia maris is cultured in a nutrient medium, thereby producing zeaxanthin.

By the process of the present invention, higher yields of free zeaxanthin and higher yields of zeaxanthin-containing cells are obtained compared to the processes of the state of the art mentioned above.

The high yields of zeaxanthin as achieved by the process of the present invention lead to a vast simplification of the processing of the zeaxanthin-containing fermentation broth obtained after culturing.

The microorganism of the species Dietzia maris used in the process of the present invention has been deposited at the following depositary authority:

DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen; Mascheroder Weg Ib; D-38124 Braunschweig.

The date of deposit is 1 April 2005. The deposit number is DSM 17219.

The characterization of the microorganism according to Bergey' s Manual of Systematic Bacteriology is given in Table 1. The isolation process for obtaining the microorganism is given in Example 1.

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 microorganism 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 17.0 g of 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 litre 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 (by a shaker) and the irradiation of light (by an ordinary lamp) . Thus, the process is preferably carried out under aeration and the irradiation of light.

The invention also relates to a process for isolating the zeaxanthin produced.

In this isolation process, the nutrient medium comprising the microorganism is generally centrifuged at about 5000 rpm 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.

Example 1

Isolation of microorganism of -the species Dietz±a. marls

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 colour of the grown colonies was analysed. 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 446.0 nm (0.8146 P) and 472.0 nm (0.7398 P) shown in the UV-Vis spectrum correspond to the carotenoid zeaxanthin. The finding that the isolated strain produces zeaxanthin has also been confirmed by IR spectroscopy (Fig. 2), mass spectrometry (Fig. 3) and TLC (Thin Layer Chromatography; not shown) .

In addition, three discrete samples A, B and C have been analyzed. Sample A refers to zeaxanthin obtained by the process of the present invention, sample B refers to a mixture of zeaxanthin obtained by the present invention and a standard sample of zeaxanthin and sample C refers to a standard sample. The corresponding HPL chromatogram of samples A, B and C is given in Fig. 4A, 4B and 4C, respectively. UV-Vis spectra of the samples corresponding to the peaks of Fig. 4A and Fig. 4B are given in Fig. 5A, 5B and 5C, respectively (the wavelength being in a range of 350 to 600 nm) , showing that the sample corresponding to Fig. 5A is pure zeaxanthin. The results confirm that the isolated strain produces zeaxanthin.

A characterisation of the corresponding species is given in Table 1.

Table 1

The strain has been characterised according to Bergey' s Manual of Systematic Bacteriology (1. edition, 1984).

As can be seen from Table 1, the microorganism is gram- positive. It is in the shape of short rods occurring in V- forms . Primary tests showed that the microorganism is strictly anaerobic, KOH-, aminopeptidase- and oxidase negative and catalase-positive.

Based on the above testings, both the DSMZ and the NCCB

("The Netherlands Culture Collection of Bacteria") have identified the microorganism of the present invention as belonging to the species Dietzia maris (deposited under deposit No. DSM 17219) .

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 25°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) pH

The growth and the pigmentation of Dietzia maris at varying pH in the nutrient medium has been studied. The results are given in Table 2. The term "grade" means the growth of Dietzia maris 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 8.1 is preferred in the process of the present invention.

b) Temperature

The growth of Dietzia maris and the production of zeaxanthin 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 Dietzia maris and the production of zeaxanthin has been studied at varying amounts of sodium chloride in the nutrient broth. The amount 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, an amount 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 Dietzia maris and the production of zeaxanthin 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 preferrably carried out under aeration and under light irradiation.

Example 3

Isolation of zeaxanthin

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 zeaxanthin 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 pitching 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 11.985 grams/100 ml). The cells were extracted for zeaxanthin 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 6000 rpm. After centrifugation, three layers were obtained: a bottom solid layer comprising cell debris without zeaxanthin, the intermediate saline layer and the upper solvent layer comprising the extracted zeaxanthin. The upper solvent layer was decanted carefully. The separated layer was lyophilized and the final purified mass of zeaxanthin was collected.

The product has been identified spectrophotometrically (as described by Britton et al., GENERAL CAROTENOID METHOD - Methods in enzymology, Vol. Ill) by using sodium borate as being an oxocarotenoid. HPLC, IR, UV-Vis and mass spectrometry studies have shown that the oxocarotenoid is zeaxanthin (as shown in Fig.s 1 to 5) .

Annex to Fig. 2

ZEAXAN-2.SP 3551 4000.00 450.00 78.30 88.92 4.00 %T 8 0.10

B.No. PBI007

REF 4000 88.53 2000 87.56 600

3306.82 86.21 2964.65 81.33 2925.42 78.30 2878.63 83.78 2854.06 83. 08

2345.79 87.51 1730.45 86.51 1653.99 85.76 1559.21 86.49 1463.32 85. 22

1380.05 85.96 1261.04 86.38 1019.88 84.94 723.06 87.79 668.14 87. 77

508.86 88.25 490.90 87.75 477.83 87.72 462.69 88.45

Annex to Fig. 4A

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 1.065 BP 0.2271 299.67856 16.88381 18.3341

2 1.510 W 0.0941 1259.41589 188.04942 77.0501

3 1.870 VB 0.1822 64.02560 4.90879 3.9170

4 2.332 BP 0.1333 11.42184 1.31510 0.6988

Totals: 1634.54189 211.15712

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 0.851 BV 0.2738 206.68558 10.58397 6.7917

2 1.143 VP 0.0748 55.17566 10.58554 1.8131

3 1.583 VB 0.1346 2772.28516 334.92215 91.0968

4 2.401 VP 0.1263 ^• 9.08424 1.10086 0.2985

Totals: 3043.23063 357.19252

Results obtained with enhanced integrator

Annex to Fig. 4C

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 1.176 BV 0.0524 17.25638 4.98157 0.8896

2 1.278 VP 0.0989 50.49450 7.46965 2.6030

3 1.565 VB 0.1185 1872.11536 271.33560 96.5074

Totals: 1939.86623 283.78682

Results obtained with enhanced integrator

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13 to)

A. The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 3 , line ?3 .

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 17219

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet identification reference given by the depositor: PBI 007 (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")

BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

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 DEPOSITARY AUTHORITY:

PBI 007

DSM 17219

II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I. above was accompanied by:

( X ) a scientific description

( ^) a proposed taxonomic designation

(Mark with a cross where applicable).

in. RECEIPT AND ACCEPTANCE

This International Depositary Authority 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 Authority 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 person(s) having the power to represent the MKROORGANISMEN UND ZELLKULTUREN GmbH International Depositary Authority or of authorized officials):

Address: Mascheroder Weg Ib D-38124 Braunschweig

Date: 2005-04-05

¹ Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired. Form DSMZ-BP/4 (sole page) 12/2001 BUDAPEST TREATY ON THb LN 1 bKNA 1 IUNAL Deutechθ

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Sαmmlung von

FOR THE PURPOSES OF PATENT PROCEDURE Mikroorgαnismen und Zellkulturen GmbH •*'

INTERNATIONAL FORM

Pharma Base S. A. Churerstr. 166 8808 Pfaffikon/SZ

VIABILITY STATEMENT SWITZERLAND issued pursuant to Rule J 0.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

I. 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 17219 SWITZERLAND Date of the deposit or the transfer¹:

2005-04-01

III. VIABILITY STATEMENT

The viability of the microorganism identified under II above was tested on 2005-04-01 On that date, the said microorganism was

(_xy viable

( )³ no longer viable

IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED⁴

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature^) of person(s) having the power to represent the MKROORGANISMEN UND ZELLKULTUREN GmbH International Depositary Authority or of authorized official(s):

Address: Mascheroder Weg Ib

D-38124 Braunschweig

Date: 2005-04-05

¹ 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). ¹ In the cases referred to in Rule 10.2(a) (ii) and (iii), refer to the most recent viability test.

³ Mark with a cross the applicable box.

⁴ 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

1. A process for the production of zeaxanthin wherein a microorganism of the species Dietzia maris is cultured in a nutrient medium, thereby producing zeaxanthin.

2. The process according to claim 1 wherein the nutrient medium has a pH from 6.1 to 9.1.

3. The process according to claim 2 wherein the nutrient medium has a pH from 7.0 to 8.1.

4. The process according to any of the preceding claims wherein the nutrient medium comprises 6 wt% to 10 wt% of sodium chloride.

5. The process according to claim 4 wherein the nutrient medium comprises about 8 wt% of sodium chloride.

6. The process according to claim 1 wherein the nutrient medium comprises 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 litre and the pH is about 7.3.

7. The process according to any of the preceding claims wherein the nutrient medium has a temperature of 30⁰C to 37°C.

8. The process according to any of the preceding claims wherein the process is carried out under aeration and the irradiation of light.

9. A process for isolating zeaxanthin obtained by the process according to any of the preceding claims comprising

separating the cells of the microorganism from the nutrient medium and

extracting zeaxanthin from the separated cells using a mixture of saline and THF as extraction solvent.

10. The process of claim 9 wherein the ratio of saline: THF in the extraction solvent is about 1:3.

11. Use of a microorganism of the species Dietzia maris for the production of zeaxanthin.