FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)

1. Title of the invention.

A PROCESS FOR ENHANCING ASTAXANTHIN CONTENT OF CULTURES OF GENUS PHAFFIA THROUGH MUTAGENESIS

2. Applicant(s)
(a) NAME :
(b) NATIONALITY
(c) ADDRESS :

ALEMBIC LIMITED
An Indian Company.
Alembic Campus, Alembic Road, Vadodara - 390 003, Gujarat, India.

3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed:

Field of invention
The Invention pertains to a process for enhancing Astaxanthin content of cultures of genus Phaffia through mutagenesis.
Background of invention
During an expedition in 1967 for the purpose of studying yeast florae associated with the trees on the major Japanese Islands and in the Pacific North west, a peculiar Astaxanthin synthesizing organism was isolated from slime fluxes of various broad-leafed trees. The organism now known as Phaffia rhodozyma, was determined to have the capabilities of producing carotenoid pigments and fermenting several sugars.
Astaxanthin (3, 3'-dihydroxy-b, b'-carotene-4, 4'-dione) is an abundant carotenoid pigment in some marine animals such as Salmonids and Crustaceans. Since, these animals cannot synthesize Astaxanthin, de novo; this carotenoid has been added to the feed of the aquacuhivated salmonid, trout, and prawn to improve their color quality. Moreover, this carotenoid possesses a higher antioxidant activity than tocopherol and Beta carotene. Therefore, Astaxanthin has attained a commercial interest not only as a pigmentation source for fresh aquaculture but also as a powerful antioxidant reagent.
Due to lack of dietary Astaxanthin, fish raised on fish farms or in hatcheries are generally pale and lack the skin and flesh colors characteristic of fish grown in their natural environment. Whether or not the carotenoids are nutritionally important in the animal or human diet has not been determined, but pigments do make certain foods attractive. That is, since the color of a food is frequently an indicator of its quality, there is a strong consumer preference for fish having natural coloration, even though nutritionally the pale farm produced fish may be identical to those grown in their natural environment.


Astaxanthin has been produced mainly by chemical synthetic method on an industrial scale. In recent years, however, the use of chemical synthetic compounds as food additives is being strictly regulated. Since, a few microorganisms are capable of synthesizing Astaxanthin, the establishment of a natural Astaxanthin source by these microorganisms is now required.
In past, numerous studies have been carried out utilizing carotenoid containing crustaceans or crustacean processing wastes in salmonid diets. The pale color of fish produced on fish farms or in hatcheries is improved when the fish is fed a diet supplemented by large quantities of dried, ground up exoskeletal crustacean remains.
In an article in Journal of food Science, Volume 47 (1982), entitled, "Extraction of Astaxanthin pigment from Crawfish Waste Using Soy oil Process", various extraction techniques are described. However, the cost of natural isolates of Astaxanthin, especially from krill and crawfish shells, can cost anywhere from $5,000 to $15000 per kilogram. Obviously, a less source dependent and more economical process for production of Astaxanthin is needed.
The red yeast, Phaffia rhodozyma, has received great attention from industry as a natural source of Astaxanthin since it was isolated from the sap, and red color was identified as Astaxanthin. The potential advantages of Phaffia rhodozyma as a source of carotenoid pigments for aquaculture are that it is a natural product rich in essential nutrients (eg. Proteins, lipids, and B-vitamins) and that it contains Astaxanthin (Johnson, villa and Lewes, 1980 Aquaculture 20 :123-134) However, natural isolates of Phaffia rhodozyma produces very little Astaxanthin (typically 100-300 ppm) that they are not practical or economical pigment source for aquaculture. If Phaffia strains are to be used as an economically feasible feed additive for coloring aquatic animals, or any other potential foodstuff, then Astaxanthin over producing strains must be developed.


Object of the invention
Primary object of the present invention is to develop a process for the production of yeast having enhanced astaxanthin content, comprising culturing a microorganism of genus Phaffia in a nutrient medium containing at least one of yeast extract, soypeptone, Corn Steep Liquor (CSL) and salts.
Another object of the present invention is to provide a process for the production of yeast having enhanced astaxanthin content, comprising subjecting a microorganism of genus Phaffia to mutation using physical and chemical mutagens.
Another object of the present invention is to develop a process for improving astaxanthin content of progeny derived from naturally occurring P. rhodozyma such as ATCC-74219 deposited with ATCC.
Another object of the present invention is to develop a process for preparation of Phaffia strain P. rhodozyma AL-8/32 comprising steps of:
(i) subjecting ATCC 74219 to UV mutation to give AL-6/29
(ii) subjecting AL-6/29 to NTG mutation to give Phaffia strain P. rhodozyma AL- 8/32
Another object of the present invention is to provide economical process for in vivo production of the pigment astaxanthin.
Another object of the present invention is to provide a process for obtaining cultures of the yeast Phaffia strain P. rhodozyma AL-8/32 having increased astaxanthin content.
Another object of the present invention is to develop a process for improving astaxanthin content of mutated strains of the yeast Phaffia strain P. rhodozyma AL-8/32.

Still a further object of the present invention is to provide a fermentation method of astaxanthin using Phaffia strain P. rhodozyma AL-8/32 comprising a step of growing a strain in a nutrient medium and using fermentation medium containing Sucrose, soyapeptone, yeast extract and salts.
Yet another object of the present invention is to provide a fermentation medium comprising of Sucrose, soyapeptone, yeast extract and salts wherein the astaxanthin produced in the harvested yeast cells is not less than 8000ppm of the dried yeast biomass and the biomass is at least 5g/l of total yeast solids.
A further object of the present invention is to provide a fermentation medium comprising of Sucrose, Corn steep Liquor and salts wherein the astaxanthin produced in the harvested yeast cells is not less than 5000ppm of the dried yeast biomass at a high biomass content of at least 15g/l of total yeast solids.
Still further object of the present invention to provide novel strains of Phaffia rhodozyma which produces greater than 6000ppm Astaxanthin, when grown under suitable conditions
Another object of the present invention is to provide Phaffia strain P. rhodozyma AL-8/32 characterized by high astaxanthin content.
Summary of the invention
In one embodiment of the invention, the present invention provides various methods of mutagenesis and methods for selecting Astaxanthin overproducing strains of Phaffia rhodozyma (Yeast genetics and molecular biology, Classical mutagenesis techniques).
Another object of the present invention is to develop a process for preparation of Phaffia strain P. rhodozyma AL-8/32 comprising steps of:


(i) subjecting ATCC 74219 to UV mutation to give AL-6/29
(ii) subjecting AL-6/29 to NTG mutation to give Phaffia strain P. rhodozyma AL-
8/32
In another embodiment of the invention, the present invention provides a fermentation method of astaxanthin using Phaffia strain P. rhodozyma AL-8/32 comprising a step of growing a strain in a nutrient medium and using fermentation medium containing Sucrose, soyapeptone, yeast extract and salts.
In another embodiment of the invention, the present invention provides a defined fermentation medium comprising of Sucrose, soyapeptone, yeast extract and salts wherein the astaxanthin produced in the harvested yeast cells is not less than 8000 ppm of the dried yeast biomass and the biomass is at least 5g/l of total yeast solids.
Yet in other embodiment of the invention, the present invention provides a fermentation medium comprising of Sucrose, Corn steep Liquor and salts wherein the astaxanthin produced in the harvested yeast cells is not less than 5000 ppm of the dried yeast biomass at a high biomass content of at least 15g/l of total yeast solids.
Still in further embodiment, the present invention provides novel strains of Phaffia strain P. rhodozyma AL-8/32 which produces greater than 6000ppm Astaxanthin, when grown under suitable conditions. These Phaffia strain P. rhodozyma AL-8/32 can produce Astaxanthin on a commercial production scale for the aquaculture and food industries.
Brief description of the drawings
FIG. 1 is a flow chart showing preferred sequences of mutant isolations in P. rhodozyma.


Detailed description of the invention
Phaffia rhodozyma, the only species in this genus, reproduces exclusively by budding, and an examination of this mode of vegetative reproduction by scanning electron microscope reveals that the yeast buds repeatedly from the same site which leads to the formation of the multilayered cell wall. Attempts to find sexual cycle of Phaffia have all failed. (Eric A Johson, Int. Microbiology (2003), Phaffia rhodozyma: A colorful odyssey).
While the carotenoids of most plant and animal sources can be readily extracted with water miscible solvents, the yeasts are well known for the tenacious attachment to their pigments. Astaxanthin is firmly attached in the yeast cell and can not be extracted by lipid solvents unless the structure of the yeast cell is first altered.
Carotenoids and tetraterpenes and their basic pathway of synthesis is similar to that of their terpenoids. Acetyl CoA is the initial precursor and the first specific terpenoid compound is Mevalonic acid. Isopentenyl pyrophosphate is the fundamental precursor from which the carotenoids are derived.
It was found that Astaxanthin biosynthesis occurred maximally during the exponential growth phase. (Kim, Jeong Hwan, Cheol Won Yun, et al Journal of Microbial Biotechnology, (2006), Effect of culture conditions on Astaxanthin formation in red yeast Xanthophyllomyces dendrorhous Mutant JH1). The pigment yield in the growth medium was found not to be solely dependent on cell concentration but was influenced by the culture conditions. The optimal pH for Astaxanthin production was found to be in the range of 4.0 to 6.0. More preferably in the range of 4.5 to 6.0. AT the other pH tested, however, the concentration of Astaxanthin by Phaffia rhodozyma remained relatively constant.


The temperature of cultivation was found to influence the growth rate of Phaffia rhodozyma. The cells grown under light intensity appeared to have redder hue. It was found that a low glucose concentration and high air supply promoted efficient Astaxanthin production by phaffia rhodozyma (Yu-Ichi Yamane,Katsuya Higashida et al Applied and Enviromental Microbiology, Nov. 1997, Effect of oxygen and glucose on primary metabolism and Astaxanthin production by Phaffia rhodozyma in Batch and fed batch cultures.
In another aspect of this invention, it relates to a process for improving astaxanthin content of mutated strains of the yeast Phaffia strain P. rhodozyma undescribed microorganism, has been designated AL-8/32 in the culture collection of Alembic Ltd. India and a culture thereof has been placed on permanent deposit with the International Depositary Authority (IDA) Microbial Type Culture Collection and Gene Bank (MTCC) Institute of Microbial Technology (IMTECH) Council of Scientific and Industrial Research (CSIR) Sector-39A Chandigarh-160 036 on December 18, 2007 and has been assigned accession No. MTCC 5395.
With the objective of isolating a novel genetic mutant of Phaffia rhodozyma capable
of increased Astaxanthin production, Phaffia rhodozyma was subjected to
mutagenesis.
Survived mutant isolates from performed mutagenesis were selected visually having
darker reddish tinge and subjected to testing at shake flask level
Mutagenesis
Astaxanthin overproducing yeast strains can be obtained by consecutive mutagenesis followed by suitable selection of mutant isolates which demonstrate superior Astaxanthin production. Preferably the yeast is of the genus Phaffia, and more preferably of the species Phaffia rhodozyma. Starting wild type yeast cell strains


include those on deposit with various culture collections throughout the world eg. ATCC and CBS.
With the aim of obtaining Astaxanthin overproducing strains, a mutagenic method is developed. Mutagenic agents EMS, NTG and UV are commonly used.
UV Mutation UV mutagenesis
Parent culture isolate ATCC 74219 was inoculated in growth medium (YM medium) and the cells were harvested in their log phase. Culture suspension was centrifuged at 7000 rpm and washed twice with Citrate Buffer (pH 5.5). The cells were re-suspended in growth medium and minimal O.D. in the range of 0.2 to 0.6 was checked at 660nm with the aid of a Spectrophotometer. Cells were then exposed to U.V. light for 1-2 hours to obtain a kill rate of 90-99 %. Exposed cells were placed under dark conditions for 3-4 hours and re-incubated for 12 to 36 hours. Incubated cells were then plated on solid growth medium.
NTG and EMS mutagenesis
Parent culture isolate ATCC 74319 was inoculated in growth medium (YM medium) and the cells are harvested in their log phase. Culture suspension was centrifuged at 7000 rpm and washed twice with citrate buffer (pH 5.5). The cells were re-suspended in growth medium and exposed to NTG mutagen in concentration of 10 to 50 (Ag/ml, to obtain a kill rate of 90-99 %. Exposed cells were plated on solid growth medium.
Screening of High Astaxanthin Producing Mutants Initial screening on plates and in shake flasks
After the incubation period, those yeast colonies that appear better pigmented to the human eye than the mutagenized parent strain are selected and sub cultured onto the culture slants. After additional incubation period of 8-10 days under 21°C, the


isolates are inoculated into the seed flasks, e.g. 20-50ml blend of glucose and yeast extract in 250 ml conical flask. Flasks are incubated for 40-48 hours at 18-21°C and 5-10 % of the seed is transferred to production medium, which is additionally incubated for 6-7 days at 18°C to 21°C with low intensity light. Flasks are harvested and Astaxanthin content is determined.
Pigment Extraction and Astaxanthin Analysis
Pigment Extraction was performed according to the procedures mentioned in reference (4) J.James Sedmak, Deepthi K Weerasinghe and Setsuko O Jolly, Biotechnology Techniques Vol 4, No.2 (1990), Extraction and Quantitation of Astaxanthin from Phaffia rhodozyma.) And carotenoid content was determined from the absorbency at 474nm. The total carotenoids are calculated using the extinction coefficient. (Based on determination using authentic synthetic Astaxanthin Sigma Aldrich)
The Astaxanthin content is then determined by HPLC. Samples for HPLC analysis is performed by using an eluent in Hexane: Acetone (80:20 volume basis). The column used is an Phenomenex, Luna 5 u Silica (2), 100 A 150 x 4.6 mm. (Biological Research, 37: 2004 p: 83-93)
Astaxanthin Concentration is then calculated as follows:-
Total Carotenoid mg/ml of the fermentation broth:-
Absorbance at 474 nm / 21 x dilution where 21 is the extinction coefficient
Astaxanthin in mg/g of yeast:-
Astaxanthin in ug of fermentation broth / gm dry yeast solids
In one embodiment of the invention, the present invention provides a defined fermentation medium comprising of Sucrose, soyapeptone, yeast extract and salts wherein the astaxanthin produced in the harvested yeast cells is not less than 8000 ppm of the dried yeast biomass and the biomass is at least 5g/l of total yeast solids.


Example 1
Development of Mutant strains and cultivation of the novel strain The data in Tables demonstrate that the phaffia strains of this invention grow to high level of cell solids and produce high levels of Astaxanthin. The Astaxanthin values in the tables are based on HPLC quantifications of Astaxanthin.
Shake flask fermentation
Loopful of slant culture is inoculated into the seed medium containing glucose and yeast extract. About 5 to 10% of actively growing culture at 21°C in 20 to 50ml growth medium is inoculated at 48hr into a 20 to 50 ml production medium preferably in 30ml production medium containing Erlenmeyer flask. A typical formulation of the set broth is:

Ingredient Amount (g)
Sugar 2 to 8
Yeast extract 0.1 to 0.8
Soypeptone 0.1 to 1.0
Potassium Hydrogen phthalate 1.0 to 3.0
Sodium Chloride 0.1 to 1.0
Magnesium sulphate 0.01 to 0.1
Calcium Chloride 0.005 to 0.01
Sodium dihydrogen Phosphate 0.001 to 0.005
Antifoam 1.0ml
Urea 0.001 to 0.005
Tap water 100 ml
Vitamin Mixture 0.2 - 0.5 ml
pH 5.0 to 5.5

Vitamin mixture constituted of

Biotin 0.05 to 0.1
Inositol 2.0 to 5.0
Thiamine 2.0 to 5.0
Calcium Pantothenate 0.5 to 1.0
Pyrodixine HCL 1.0 to 2.0
Water 1 litre
Shake flask fermentation is proceeded for 5 days at 18°C to 21°C under low intensity light. Results of such fermentations with AL- 8/32 are presented below:-
Astaxanthin production at shake flask level

Strain Hrs Biomass g/1 dry yeast solids Astaxanthin mg/g dys
AL 8/32 120 5.0 8000
10.0 6000
* dys - dry yeast solids
In other embodiment of the invention, the present invention provides a fermentation method of astaxanthin using Phaffia strain P. rhodozyma AL-8/32 comprising a step of growing a strain in a nutrient medium and using fermentation medium containing Sucrose, soyapeptone, yeast extract and salts.
Still in another embodiment of the invention, the present invention provides a fermentation medium comprising of Sucrose, Corn steep Liquor and salts wherein the astaxanthin produced in the harvested yeast cells is not less than 5000 ppm of the dried yeast biomass at a high biomass content of at least 15g/l of total yeast solids.

Example 2
Production medium for Astaxanthin production including Corn Steep Liquor (CSL)
Shake flask fermentation
Loopful of slant culture is inoculated into the seed medium containing glucose and yeast extract. About 5 to 10% of actively growing culture in 20 to 50 ml growth medium is inoculated into a 20 to 50 ml production medium preferably in 30ml production medium containing erlenmeyer flask. A typical formulation of the set broth is:

Ingredient Amount (g)
Corn Steep Liquor (CSL) 1.0 to 5.0
Sugar 2 to 8
Potassium Hydrogen phthalate 1.0 to 3.0
Sodium Chloride 0.1 to 1.0
Magnesium sulphate 0.01 to 0.1
Calcium Chloride 0.005 to 0.01
Sodium dihydrogen Phosphate 0.001 to 0.005
Antifoam 1.0 ml
Tap water 100 ml
Vitamin Mixture 0.2 - 0.5 ml
pH 5.0 to 5.5


Vitamin mixture constituted of

Biotin 0.05 to 0.1
Inositol 2.0 to 5.0
Thiamine 2.0 to 5.0
Calcium Pantothenate 0.5 to 1.0
Pyrodixine HCL 1.0 to 2.0
Water 1 litre
Shake flask fermentation is proceeded for 5 days. Results of such fermentations with AL- 8/32 are presented below:-
Astaxanthin production at shake flask level

Strain Hrs Biomass g/1 dry yeast solids Astaxanthin ug/g dys
AL- 8/32 120 15.6 5000
* dys - dry yeast solids


We claim:
1. A process for the production of yeast having enhanced astaxanthin content, comprising culturing a microorganism of genus Phaffia in a nutrient medium containing at least one of yeast extract, soypeptone, Corn Steep Liquor (CSL) and salts.
2. A process as in claim 1, wherein the astaxanthin in harvested yeast is 6000 ppm or more based on dry weight of yeast cells.
3. A process for the production of yeast having enhanced astaxanthin content, comprising subjecting a microorganism of genus Phaffia to mutation using physical and chemical mutagens.
4. A process for preparation of Phaffia strain P. rhodozyma AL-8/32 comprising steps of:
(i) subjecting ATCC 74219 to UV mutation to give AL-6/29
(ii) subjecting AL-6/29 to NTG mutation to give Phqffia strain P. rhodozyma AL-8/32
5. A fermentation method of astaxanthin using Phaffia strain P. rhodozyma AL-8/32 comprising a step of growing a strain in a nutrient medium and using fermentation medium containing Sucrose, soyapeptone, yeast extract and salts.
6. A defined fermentation medium comprising of Sucrose, soyapeptone, yeast extract and salts wherein the astaxanthin produced in the harvested yeast cells is not less than 8000 ppm of the dried yeast biomass and the biomass is at least 5g/l of total yeast solids.


7. A fermentation medium comprising of Sucrose, Corn steep Liquor and salts wherein the astaxanthin produced in the harvested yeast cells is not less than 5000 ppm of the dried yeast biomass at a high biomass content of at least 15g/l of total yeast solids.
8. Phqffia strain P. rhodozyma AL-8/32
9. A mutant strain of Phaffia rhodozyma of claim 1 which produces at least 6000mg of Astaxanthin per gm dry weight of Phaffia at 120 hours.

ABSTRACT
A process for production of Astaxanthin pigment and particularly process for enhancing Astaxanthin content of cultures of genus Phaffia through mutagenesis.