Background Art Natural food colorants are steadily gaining importance in food and pharmaceutical products. Due to their health promoting effects and safety in comparison to synthetic dyes, red beet color extracts are under extensive research to develop alternative methods for replacing synthetic red dyes. Reference my be made to Piatelli and Minale (1964, Phytochemistry. 3: 307-310) wherein they reported that the color of red beet is due to water soluble nitrogenous pigments called'Betalaines'.

Betalaines are presently found in restricted number of plants belonging to the order Centrospermae (Piatelli, 1976. in the Biochemistry of plants. Vol. 7: Conn. E. E. , Academic Press: New york, pp 557-575). They are used to colour a range of food products viz. Ice creams, sherbets, meat products.

Betalaines are fairly stable in the pH range of 4.0-7. 0 under light. Stability of betalaines is enhanced by acidic pH obtained by using ascorbic acid & citric acid combinations. (Von- Elbe 1975, Food Technology pp 42-46) The hue value of Betalaines is very high, often higher than the synthetic colorant FD&C Red No. 2 (Von-Elbe 1975, Food Technology pp 42-46).

Hairy root cultures which are produced upon infection with Agrobacterium rhizogenes are known to produce a variety of bioactive secondary metabolites which find importance in food and pharmaceutical industries (Toivenon, L. 1993. Biotech prog. 9.12-20.). Hairy roots have the ability to synthesize and accumulate compounds, which are generally synthesized in root of intact plant. Due to their hormonal autotrophy, high growth rate, genetic as well as biochemical stability, hairy root cultures have gained economic importance in recent years (Hamill et al 1986 Plant Cell Rep. 5 : 111-114). So there is a need to scale up the hairy root cultures for higher biomass production as well as metabolite productivity.

The advantage of its cultivation in bioreactors include 1) controlled production of phytochemicals independent of plant availability (Sajc et al 2000. Biochemical Engineering Journal. (4). 89-99. ) 2) Better production system resulting in higher yields and consistent

quality of the product (Fowler 1985. In: Neumann, K. H, Barz, W, Reinhardt, E (eds).

Primary and secondary metabolism of plant cell cultures. Springer, Berlin pp. 362-378.).

Hairy roots have been cultivated in different types of bioreactors viz. airlift (Taya et al.

1989. J Chemical Eng Japan 22: 84-89), Stirred tank (Hilton et al. 1988. Appl. Microbiol.

Biotechnol. 33.132-138.), Bubble column (Wilson et al. 1987. Fermentation studies of transformed root cultures. In: Moody GW, Baker PB (eds). Bioreactors and biotransfromations. Elsevier, London, 38-51). Evaluation of a fermentor design depends on comparison of the performance ratios at the same oxygen mass transfer rate (Hatch 1976.

Single cell protein II. Jun 2.46-68).

The main problem of the bioreactor usage is the root disorganization and callus formation leading to reduced production especially in stirred tank reactors where the baffles could cause wounding to hairy roots (Hilton et al. 1988. In: Robins RJ, Rhodes MJC, (eds).

Manipulating secondary metabolism in culture. Cambridge university press, Cambridge, U. K, 239-245.

The designs were later modified accordingly to minimize the problems mainly to enhance better oxygen mass transfer. Hilton and Rhodes (1990. Appl. Microbiol. Biotechnol. <BR> <BR> <P>33: 132-138. ) have used a modified stirred tank reactor where they have used a stainless steel mesh cage that separated the roots from stirrer and also acted as an immobilization <BR> <BR> matrix. Reference may be made to Kwok et al. (1995. Biotechnol Prog. , 11,429-435) wherein they have used a modified bubble column reactor to facilitate better oxygen transfer by providing spargers at different levels of reactor vessel. Reference may also be made to kanokwaree and Doran (1998. Biotechnol Prog, 14,479-486), wherein the oxygen mass transfer problem was minimized by using a polypropylene tubing as a supplementary aeration device and addition of FC-43 perflurocarbon emulsion to the medium in case of Atropa belladonna hairy root cultures. Reference may also be made to Mukundan et al <BR> <BR> (1998. Biotech Lett 20 (5). 469-474. ) wherein a 2L bubble column reactor has been used for the cultivation of hairy roots of Beta vulgaris.

Till date, the most successful aspect of cultivation of hairy roots in bioreactors is the scale- up of Panax ginseng hairy root cultures in 20 tons bioreactor (Scheidegger 1990).

Initial methods of color recovery from plant sources involved grinding and extraction in a particular solvent of target pigments. This method is a fully destructive process where not only the entire source tissue is ground to recover the pigment, but also undesirable extractives may alter the quality and stability of the pigment of interest.

Therefore, recently several workers have tried specific recovery methods where cytosolic pigments get extracted in a particular solvent. Ethanol and methanol have been extensively used to recover the Betalaines from red beet tissue (Nillsson, 1970. Lantbruckshogskolens Anals 36: 179, Metivier et al 1980. J. Food Sci. 45: 1099-1100, Francis 1982 In : Anthocyanins as food colours, eds. Markakis, P. Academic Press, NY, pp. 182-200). This method though results in better and more specific extraction leading to clearer pigments than the earlier methods. Such solvents are not desirable in food products for safety reasons.

It is therefore, desirable for the products to be released from hairy roots into the culture medium so that continuous operation can be carried out. (Sim and Chang 1993. Biotech lett. 15 (2) 145-150.). In situ adsorption and extraction have been widely applied for the removal of inhibitory products in the field of biotechnology including the plant cell cultures. It is very important to see that the cell viability is not lost while extraction for a continuous process.

Most of the secondary metabolites obtained from hairy root cultures are generally located within cell vacuoles or cell walls, which results in inhibition of cell growth and further synthesis of products. Lowering the pH of the medium is known to efflux the vacuolar contents, including pigments into the exterior (Mukundan et al, 1998, Microbial Biotech.

50: 241-245).

Reference may be made to studies of Taya et al (1992. J. Fermentation and Bioengineering Vol. 27: 31-36) wherein leaching of only 20% of pigments from hairy root cultures of red beef was achieved by the limitation of oxygen supply for a time period of 48 hours.

Reference may also be made to the studies of Mukundan et al (1998, J. Fermentation and Bioengineering Vol. 27: 31-36) wherein, betalaines were made to release from hairy root cultures with the influence of pH. Most of the above methods involved either poor recovery or destruction of the tissue leading to non-repetitive use of biomass.

Therefore, the present invention aims to develop an efficient method of recovery of beet color (betalaines) from hairy root cultures of beet root.

Objects of the invention The main object of the present invention is to develop an efficient process for the efflux of betalaines which is suitable for food grade from hairy roots of Beta vulgaris into the medium where they can be used for further processed to get beet root colour concentrate.

Another object of the invention is to maintain the viability of the hairy roots for a continuous culture and production of betalaines.

Detailed descriptiomn of the invention: Accordingly, the invention provides an Improved process for production of betalaines from Beta vulgaris, said method comprising the steps of : a) culturing hairy root explants of Beta vulgaris infected with wild strain of agrobacterium rhizogenes in a nutrient medium comprising Murashige and Skoog's salts and supplemented with 1.5 to 5% sucrose, and sterilized in an autoclave for 15 min at 121°C at 15 lb per square inch, for about 3-7 days, b) adding a surfactant such as cetyl trimethyl ammonium bromide at a concentration of 0. 001-0. 1%, to the culture and culturing it for 3 to 10 days, c) effluxing red beet pigment from hairy root cultures and concentrating the red beet pigment obtained under vacuum using a flash evaporator till desirable concentration is reached or dehydrating using dextrose as carrier, d) reculturing the hairy roots after effluxing of betalaines for further growth and production of pigment.

To describe in detail, the invention provides an improved process for the effluxing of betalaines from Beta vulgaris wherein: a. cultuirng hairy root explants of Beta vulgaris infected with wild strain of agrobacterium rhizogenes in shake flasks and in a bubble column reactor using nutrient media comprising of Murashige and skoog's salts + 1.5 to 5% sucrose and sterilized in an autoclave for 15 min at 121 C at 15 lb per square in, b. inoculating hairy roots (in the range of 2-6 g fresh wt/L) grown for 3-6 days to obtain higher growth and metabolites in shake flask and bubble column bioreactor, c. supplying air in the range of 0.5 to 2 WM for better mass transfer and to avoid necrosis of tissue, d. obtaining biomass of hairy roots containing red beet pigment, followed by adding a surfactant in the concentration range of 0. 001-0. 1% to the culture medium during 15 to 20 days of culture, e. effluxing red beet pigment from hairy root cultures and concentrating the red beet pigment obtained under vacuum using a flash evaporator till desirable concentration is reached or dehydrating using dextrose as carrier f. reculturing the hairy roots after effluxing of betalaines (step e) for further growth and production of pigment.

In an embodiemnt, the surfactant is selected from the group comprising Tween-80, Triton- X and cetyl trimethyl ammonium bromide.

In an embodiment, the medium used, comprises of Murashige and Skoog's salts with sucrose as carbon source in the range of 1.5-5%.

In another embodiment, the concentration of cetyl trimethyl ammonium bromide used was used in the range of 0.001-0. 01% for effluxing red beet pigment In still another embodiment, the cetyl trimethyl ammonium bromide used does not affect the viability during an effluxing period of 45 hours.

In another embodiemnt, the stability of betalaines is ensured in the range of surfactant concentration adapted and the period of treatment.

In yet another embodiment, the betalaines effluxed are concentrated under vacuum using a flash evaporator to powder form using dextrose as a carrier for further use in food application.

In another embodiment, the hairy roots of beet root are cultured in nutrient liquid medium, aseptically and pigment is effluxed to the medium, while retaining the viability of the hairy roots, for continuous production of the pigment using shake flask or bioreactors, as the case may be, to obtain food grade colour upon concentration under vacuum or dehydrated to powder form using dextrose as carrier which is here in described with examples.

The present invention provides an improved method of in situ extraction of betalaines from hairy roots of Beta vulgaris grown in 150 mL Erlenmeyer flasks and 40 ml medium and in a 3L bubble column reactor where the hairy roots of Beta vulgaris were obtained upon infection of the explant using an isolate of Agrobacterium rhizogenes wild stain and were maintained by sub culturing in MS (Murashige and Skoog 1962. Physiol Plant, 15,473- 479 (1962). medium without any phytohormones and with the addition of 3% sucrose and the pH of the medium was adjusted to 5.8 before autoclaving and the flasks were kept on a shaker at 90 rpm and cultured in dark for 4 days and were inoculated in to 150 ml Erlenmeyer flasks and also into a bubble column reactor made up of coming glass (Length 22 cm, diameter 14 cm) of volume 3L with a working volume of 1.75 L. where the reactor lid was provided with openings for air inlet, outlet, inoculation port and for sample drawing and the compressed air through the PTFE filter (0. 2211m) which was sparged at a rate of 1.7VVM, which results in bubbling supplying oxygen to the hairy roots inoculated in a plastic autoclavable basket (Height 10.5 cm, diameter 8.5 cm) supported by a stainless

steel stand at 7 cm distance from the bottom of the reactor and Cetyl trimethyl ammonium bromide (Cl9H42BrN, M. W 364.46) which is a cationic surfactant was added at different concentrations i. e 0.001%, 0.002%, 0.005%, 0.01% which was procured from Merck kGa A, Darmstadt, Germany., Triton-X, procured from Hi-media, Mumbai, India at concentrations of 0. 01.-1. 5% W/V and Tween-80 procured from Hi-Media, Mumbai, India at concentrations of 0.01-0. 15% W/V were dissolved in a standard volume of distilled water to make up the concentration then filter sterilized in a laminar flow under aseptic condition and added to the hairy root culture flask and also to the reactor and after 6 hours of the surfactant treatments the medium was drawn periodically at 6,12, 24,36, 48 and 72 hours from the shake flasks as well as bioreactor (5mL) for evaluation of betalaine efflux and then after 48 hours of treatment the hairy roots were carefully taken from the reactor in laminar flow (200mg) and were kept on MS solid medium and hairy roots grew well and resulted in normal growth and metabolite production in the cultures fed with Cetyl trimethyl ammonium bromide implying that Cetyl trimethyl ammonium bromide was not inhibitory to hairy root cultures and later the betalaines were further concentrated under vacuum and used as colourant.

In an embodiment of the present invention, hairy roots of red beet root were obtained using the wild strain of Agrobacterium rhizogenes.

In yet another embodiment of the present invention, they were cultured on Murashige- Skoog basal medium in 150 mL Erlenmeyer flask and bubble column reactor.

In yet another embodiment of the present invention, a bubble column reactor of 3L capacity with a working volume of 1.75 L was chosen for a better growth and metabolite production, vessel with culture medium was autoclaved at 15 lb pressure at 121 C temperature for 15 min to avoid contamination.

In yet another embodiment of the present invention, which may comprises of hairy root cultures of Beta vulgaris of 3-6 day old for inoculation into the Erlenmeyer flasks and also bioreactor for good growth and betalaine production.

In yet another embodiment of the present invention, which may comprises of the compressed air which was sparged at a rate of 0.5-2 WM range for better mass transfer resulting in good growth and metabolite production.

In yet another embodiment of the present invention, which may comprises of Cetyl trimethyl ammonium bromide at a concentration of 0.001%-0. 05% added on 17th day where the biomass is at the peak of its metabolite production as well as growth

In yet another embodiment of the present invention, after 48 hours of Cetyl trimethyl ammonium bromide treatment the hairy roots were carefully taken from the reactor in laminar flow (200mg) and were kept on MS solid medium. The hairy roots grew well and resulted in normal growth and metabolite production implying that Cetyl trimethyl ammonium bromide was not inhibitory to hairy root cultures.

In yet another embodiment of the present invention, the effluxed betalaines were concentrated under vacuum to get food grade colour.

The following examples are given by way of illustration of the present invention and these should not be considered to limit the scope of invention.

EXAMPLE 1 100mg hairy roots from 4 day old cultures were inoculated in to 40ml nutrient medium comprising of Murashige and Skoog's salts in a 150ml Erlenmeyer's flask. After 17 days, the cultures were treated with filter sterilized (with membrane filters of 0. 44M) Cetyl trimethyl ammonium bromide at concentrations of 0.0005%, 0.001%, 0.002%, 0.01%, Triton-X at concentrations of 0. 01%, 0.05%, 1%, 1.5% and Tween 80 at concentrations of 0.01%, 0.05%, 0.15%. The control culture did not receive these detergent treatments. The flasks were kept in static and agitated conditions under two illuminations of 1000 and 2000 lux and one set was kept in dark. The release of pigment was observed for various time periods. The quantity and quality of the pigment released was analyzed spectrophotometrically using three replicates. The pigment remaining in the hairy root biomass was extracted in acidified water (ascorbic acid water) of respective treatment by way of grinding, centrifuging at 5000 X g for 10 minutes, collecting the supernatant and quantifying the betalaine pigments by measuring OD at 480 and 540nm spectrophotometrically. Betalaine pigment remaining in the biomass was quantified.

The cultures treated as above with 0.001% to 0.01% of Cetyl trimethyl ammonium bromide Started leaching betalaines, with more than 50% of leaching in 24 hours and 80% leaching in 48 hours with excellent stability even after 48 hours at the concentration of 0.002%. In case of Tween-80 and Triton-X the leaching was not proper and also the colour degraded as soon as it got leached at the concentrations tested. Cetyl trimethyl ammonium bromide at low levels was efficient in effluxing the pigment as well as protecting the released pigments from immediate degradation. Thus, for beet pigment extraction from hairy roots using Cetyl trimethyl ammonium bromide was highly effective. Betalaines thus obtained were concentrated under vacuum to obtain food grade colour. Moreover, the hairy roots

after effluxing of pigment remained viable for continuous culture and production of betalaines.

EXAMPLE 2 The bubble column reactor of 3L volume with a working volume of 1.75 L was inoculated with 10 grams fresh weight of 4 day old hairy root cultures of Beta vulgaris. The medium used in the bioreactor was Murashige-Skoog medium and the pH was adjusted to 5.8 before autoclaving the bioreactor for 15 min at 121 C at 15 lb pressure. After 17 days of growth of hairy roots in the bubble column reactor Cetyl trimethyl ammonium bromide was added at a concentration of 0.01%. The betalaines started leaching into the medium after 6 hours of its addition and the colour of the medium turned brown in 24 hours showing the degradation of the betalaines. The roots turned brown showing necrosis and death. The roots were not able to grow when kept on fresh MS medium. The betalaine efflux with time is shown here Time period Betalaine content (hrs) (mg/L) 6 10. 2 12 22.4 24 28.0 48 9. 4 EXAMPLE 3 The bubble column reactor of 3L volume with a working volume of 1.75 L was inoculated with 10 grams fresh weight of 4 day old hairy root cultures of Beta vulgaris. The medium used in the bioreactor was Murashige-skoog medium and the pH was adjusted to 5.8 before autoclaving the bioreactor for 15 min at 121 C at 15 lb pressure. After 17 days of growth of hairy roots in the bubble column reactor Cetyl trimethyl ammonium bromide was added at a concentration of 0. 001%. The leaching of betalaines was very slow i. e after 12 hours and even after 48 hours also there was no increase in betalaine content compared to the one with 0.002% Cetyl trimethyl ammonium bromide.

The betalaine efflux with time is shown here: Time period Betalaine content (hrs) (mg/L) 6 0. 26 12 1. 1 24 4.32 48 9. 89

At this concentration, Cetyl trimethyl ammonium bromide was less effective in the release of betalaines suggesting a higher concentration could be used for better efflux. The hairy roots showed good growth when they were subcultured on to a fresh medium after 48 hours of Cetyl trimethyl ammonium bromide treatment suggesting that the process could be used as a continuous operation in bioreactors. The betalaines in the effluxed medium was concentrated under vacuum to obtain food grade colour.

EXAMPLE 4 The bubble column reactor of 3L volume with a working volume of 1.75 L was inoculated with 10 grams fresh weight of 4 day old hairy root cultures of Beta vulgaris. The medium used in the bioreactor was Murashige-Skoog medium and the pH was adjusted to 5.8 before autoclaving the bioreactor for 15 min at 121 C at 15 lb pressure. After 17 days of growth of hairy roots in the bubble column reactor Cetyl trimethyl ammonium bromide was added at a concentration of 0.002% which was found to be the best concentration for both betalaine release as well as to get viable hairy roots for re use for continuous mode of operation where an initial biomass of 5.7 g/L was used and after 20 days a final biomass of 29.7g/L was obtaines showing a specific growth rate of 0.04 day' Time period Betalaine content (hrs) __Cmg/L) 6 1.86 12 4.36 24 9.4 36 12. 45 48 17.3 72 16. 5 The roots after the reactor run were extracted for betalaines which resulted in the production of 21.36 mg L~1. The colour of the betalaines was measured using a colour measuring system Minolta spectrophotometer CM-3500 d, Japan. The extracts were centrifuged for 15 min at 3000rpm. And the supernatant was taken for measurement of the colour. Time (hrs) Lightness Redness Yellowness 12 91. 69 5. 44 2. 23 24 80.09 17.6 7.06 48 70.91 26.13 11.43 72 65 20 8

It was observed that the betalaines were extracted into the medium without harming the hairy roots and the time for maximum extraction is 48 hours after Cetyl trimethyl ammonium bromide treatment and the concentration of Cetyl trimethyl ammonium bromide was also very critical i. e 0.002% for the proper efflux of betalaines without any damage to the hairy roots. The re growth of the hairy roots after Cetyl trimethyl ammonium bromide treatment gives very useful data for the use of this Cetyl trimethyl ammonium bromide in a continuous mode of operation in a larger scale for the scale up of the procedure for high biomass production and secondary metabolite recovery. The betalaine pigments were then concentrated under vacuum to obtain food grade colour.

The main advantages of the present investigation are: 1. In vitro culture of hairy roots obviates the use of whole red beet for betalaine production.

2. Hairy root cultures can be grown independent of environmental conditions and on a simple hormone free medium.

3. The hairy roots can be grown in a bioreactor where they are more amenable for scale- up in a large scale.

4. Development of a process for in situ extraction of betalaines from hairy root cultures of Beta vulgaris.

5. Use of Cetyl trimethyl ammonium bromide, which is a cationic non-toxic surfactant, effluxes betalaines without killing the hairy roots. Further retaining the stability of the pigment..

6. The Cetyl trimethyl ammonium bromide treated hairy roots can be reused for continuous culture of biomass for production of betalaines in a bioreactor.

7. The process can be scaled up in a large scale bioreactor for higher biomass growth and metabolite production.

8. The betalaines so obtained can be concentrated under vacuum and can be used for food and other applications as colorants.