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WO2003080819A1 - Procede d'extraction de betalaines a partir de cultures de racines velues de beta vulgaris - Google Patents

Procede d'extraction de betalaines a partir de cultures de racines velues de beta vulgaris Download PDF

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Publication number
WO2003080819A1
WO2003080819A1 PCT/IB2002/001156 IB0201156W WO03080819A1 WO 2003080819 A1 WO2003080819 A1 WO 2003080819A1 IB 0201156 W IB0201156 W IB 0201156W WO 03080819 A1 WO03080819 A1 WO 03080819A1
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WO
WIPO (PCT)
Prior art keywords
betalaines
pigment
effluxing
hairy
hairy roots
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.)
Ceased
Application number
PCT/IB2002/001156
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English (en)
Inventor
Rudrappa Thimmaraju
Gokare Aswathanarayana Ravishankar
Neelwarne Bhagyalakshmi
Bhamidi Suresh
Mandayam Ungara Narayan
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Council of Scientific and Industrial Research CSIR
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Council of Scientific and Industrial Research CSIR
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Publication date
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Priority to AU2002247924A priority Critical patent/AU2002247924A1/en
Priority to PCT/IB2002/001156 priority patent/WO2003080819A1/fr
Publication of WO2003080819A1 publication Critical patent/WO2003080819A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin

Definitions

  • the present invention relates to an improved process for effluxing betalaines from Beta vulga ⁇ s hairy root cultures for continuous production of natural red beet pigment.
  • 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 z.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 (Non-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.
  • the present invention aims to develop an efficient method of recovery of beet color (betalaines) from hairy root cultures of beet root.
  • 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 Reto 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.
  • 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
  • the surfactant is selected from the group comprising Tween-80, Triton - X and cetyl trimethyl ammonium bromide .
  • the medium used comprises of Murashige and Skoog's salts with sucrose as carbon source in the range of 1.5-5%).
  • concentration of cetyl trimethyl ammonium bromide used was used in the range of 0.001-0.01%) for effluxing red beet pigment
  • cetyl trimethyl ammonium bromide used does not affect the viability during an effluxing period of 45 hours.
  • the stability of betalaines is ensured in the range of surfactant concentration adapted and the period of treatment.
  • 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.
  • 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).
  • 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.
  • 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.
  • vessel with culture medium was autoclaved at 15 lb pressure at 121 C temperature for 15 min to avoid contamination.
  • 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.
  • the effluxed betalaines were concentrated under vacuum to get food grade colour.
  • EXAMPLE 1 lOOmg 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 0.44 ⁇ M) 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%o,0.05%, 0.15%o.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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. he 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.
  • 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 "
  • 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.
  • 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..
  • Cetyl trimethyl ammonium bromide treated hairy roots can be reused for continuous culture of biomass for production of betalaines in a bioreactor.
  • the process can be scaled up in a large scale bioreactor for higher biomass growth and metabolite production .
  • the betalaines so obtained can be concentrated under vacuum and can be used for food and other applications as colorants.

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  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

La présente invention concerne un procédé amélioré d'extraction in situ de bêtalaïnes à partir de racines velues de Beta vulgaris. Selon ce procédé, des racines velues de betterave sont cultivées dans un milieu liquide nutritif dans des conditions d'asepsie et le pigment est extrait dans ce milieu. A l'issue de cette opération, les racines velues conservent leur viabilité, le pigment pouvant ainsi être produit en continu.
PCT/IB2002/001156 2002-03-26 2002-03-26 Procede d'extraction de betalaines a partir de cultures de racines velues de beta vulgaris Ceased WO2003080819A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2002247924A AU2002247924A1 (en) 2002-03-26 2002-03-26 Process for effluxing betalaines from beta vulgaris hairy root cultures
PCT/IB2002/001156 WO2003080819A1 (fr) 2002-03-26 2002-03-26 Procede d'extraction de betalaines a partir de cultures de racines velues de beta vulgaris

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PCT/IB2002/001156 WO2003080819A1 (fr) 2002-03-26 2002-03-26 Procede d'extraction de betalaines a partir de cultures de racines velues de beta vulgaris

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105671077A (zh) * 2016-04-21 2016-06-15 哈尔滨工业大学 一种利用发根农杆菌高效诱导食用甜菜产生毛状根的方法
US11299700B1 (en) 2021-02-19 2022-04-12 Acequia Biotechnology, Llc Bioreactor containers and methods of growing hairy roots using the same
WO2022198106A1 (fr) * 2021-03-18 2022-09-22 Calyxt, Inc. Production de bétalaïnes à l'aide de matrices de cellules végétales

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993000424A1 (fr) * 1991-06-21 1993-01-07 Worcester Polytechnic Institute Procede pour extraire des quantites accrues de metabolites secondaires de plantes avec perte limitee de la viabilite de la plante

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993000424A1 (fr) * 1991-06-21 1993-01-07 Worcester Polytechnic Institute Procede pour extraire des quantites accrues de metabolites secondaires de plantes avec perte limitee de la viabilite de la plante

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DILORIO A A ET AL: "GROWTH OF TRANSFORMED ROOTS IN A NUTRIENT MIST BIOREACTOR REACTOR PERFORMANCE AND EVALUATION", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 37, no. 4, 1992, pages 457 - 462, XP008008932, ISSN: 0175-7598 *
SUTTON D L ET AL: "EFFECT OF DIQUAT AND SEVERAL SURFACTANTS ON MEMBRANE PERMEABILITY IN RED BEET-D ROOT TISSUE", BOTANICAL GAZETTE, vol. 132, no. 4, 1971, pages 299 - 304, XP008008939, ISSN: 0006-8071 *
TAYA M ET AL: "PRODUCTION AND RELEASE OF PIGMENTS BY CULTURE OF TRANSFORMED HAIRY ROOT OF RED BEET", JOURNAL OF FERMENTATION AND BIOENGINEERING, vol. 73, no. 1, 1992, pages 31 - 36, XP008008924, ISSN: 0922-338X *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105671077A (zh) * 2016-04-21 2016-06-15 哈尔滨工业大学 一种利用发根农杆菌高效诱导食用甜菜产生毛状根的方法
US11299700B1 (en) 2021-02-19 2022-04-12 Acequia Biotechnology, Llc Bioreactor containers and methods of growing hairy roots using the same
WO2022198106A1 (fr) * 2021-03-18 2022-09-22 Calyxt, Inc. Production de bétalaïnes à l'aide de matrices de cellules végétales

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