PRODUCTION OF BIOCOMPOUNDS
The present invention relates to the isolation of compounds such as carotenoids and flavonoids, in particular the highly oxidised carotenoid astaxanthin (astaxanthin, 3,3'-5dihydroxy- β,β-carotene-4, 4'-dione), and the flavonoid anthocyanin, and a marine bacterium belonging to the genus Pseudoalteromonas sp. which is capable of producing the aforementioned compounds.
Astaxanthin is a highly oxidised carotenoid commonly found in marine and animal tissue, such as the carapaces of lobsters, crabs and shrimps, the integuments of red sea bream, the muscle of salmonids, and the ovaries and gonads of fish and shellfish such as some species of sea urchins. Astaxanthin is known to have a number of biological functions including being a pre-cursor to Vitamin A production, acting as a scavenger/quencher of active oxygen species, as an anti-tumour promoter and as an enhancer of in vitro antibody production. In addition to these biological functions, astaxanthin is widely used in the aquaculture industry as an agent for pigmenting cultured fish and shellfish. Dietary pigments such as astaxanthin and canthaxanthin are included in fish feed to aesthetically improve the colouring of farmed fish. This is necessary, as in nature these animals are unable to synthesise the pigments for themselves and must obtain them from their diet. These pigments account for 10% of the overall costs of feed manufacture with synthetic pigments typically costing in excess of $2500 (£1500) a kilo.
Natural astaxanthin can be synthesised in nature by the aquatic alga Haematococcus pluvialis and the yeast Phaffia rhodozyma and currently the only commercially available source of natural astaxanthin is produced using the aquatic micro-alga Haematococcus pluvialis. Astaxanthin or pigment analogues can also be produced synthetically. However the petrochemical reactions used in such syntheses are often environmentally damaging. Moreover, synthetic analogues of astaxanthin, which are similar to their natural counterparts, may be not as effective as the natural pigment due to differences in the uptake of the pigments.
Anthocyanins are natural colourants belonging to a group of compounds called the flavonoids. Flavonoids are widely distributed amongst flowers, fruit and vegetables. In addition to their colourful characteristics, anthocyanins possess powerful antioxidant properties (equal to
or greater than vitamin E analogues, (Wang H., Cao G.H., Prior R.L, Oxygen radical absorbing capacity of anthocyanins J AGR FOOD CHEM 45 (2): 304-309 FEB 1997 (Hong et al. 1997)) and this may account for the beneficial health protection against cardiovascular and other diseases, such as cancer, provided by eating fruit and vegetables high in these pigments. In addition, anthocyanins isolated from wild blueberries have also been implicated in providing health benefits as far reaching as slowing down the ageing process (particularly loss of memory) and preventing cancer by blocking the actions of certain enzymes involved in the promotion stage of cancer (Prior R.L, Cao G., Hoang H., et al. In vivo antioxidant capacity in human subjects following consumption of prune juice, prunes or blueberries).
An object of the present invention is to provide an alternate source of natural astaxanthin (bio-pigments) derived from marine bacteria, marine bacteria being able to utilise cheap raw materials and showing high productivity through fermentation. A further object of the present invention is to provide novel compounds which display anti-bacterial/anti-microbial activity.
Accordingly, the present invention provides a method for the isolation of a red- pigmented compound, such as a carotenoid, preferably astaxanthin, and/or a flavonoid, such as anthocyanin, from the marine bacterium belonging to the genus Pseudoalteromonas sp., wherein the method comprises the steps:
(a) culturing said bacterium in a suitable culture medium under conditions and for a time sufficient to produce quantities of the red-pigmented compound(s);
(b) subjecting the bacterium to purification procedures so as to purify said red pigmented compound(s); and
(c) isolating said red-pigmented compound(s).
The colour of the compound is defined as red but may vary from a light red or pink colour to a dark red or purple colour. For example, the fraction that comprises the carotenoid may be characterised by an orange colour and that which displays anti-microbial activity is characterised by a dark red/purple colour, which is speculated to be a result of the presence of flavonoids, in particular, anthocyanins.
Preferably, the procedure for isolating the carotenoids from the bacterium includes column chromatography. Preferably, the carotenoid is isolated using HPLC on a reverse-phase (RP) column. Alternatively, the procedure for isolating flavonoids from the bacterium includes flash column chromatography.
Preferably, the marine bacterium belonging to the genus Pseudoalteromonas sp. is strain ASTA-1 as deposited in accordance with the requirements under the Budapest treaty at the NCIMB on 7th March 2001 under accession number NCIMB 41083 or a mutant or variant thereof having the property of producing a red-pigmented carotenoid and/or flavonoid, such as astaxanthin and/or anthocyanin respectively.
Suitably, the bacterium may be cultured in media comprising sterilised seawater and tryptone. Another suitable media is 3% NaCI 1% peptone and 0.1% yeast extract source in purified seawater (natural).
Preferably, the bacteria may be cultured in media comprising sterilised seawater, tryptone, glucose, and Fe"'so4.
Typically, the bacterium may be cultured at 22°C to 28°C for 3 - 6 days.
More typically, the bacterium is cultured at 24°C for 5 days.
The carotenoid and/or flavanoid may be harvested from the cells of the marine bacterium grown in culture, and/or alternatively from the media in which the cells are grown.
Preferably, the carotenoid is astaxanthin.
Preferably, the flavonoid is anthocyanin.
Preferably, production of the carotenoid by the marine bacterium will occur at levels above basal (See table 1).
Table 1 : Level of crude pigment production (g) from 2.5L culture broth produced by Pseudoalteromonas sp. Strain ASTA-1 NCIMB 41083.
Studies have shown that production levels of astaxanthin from Haematococcus pluvialis is around 10 - 30g per kg dry biomass which is 1000 - 3000 times higher than what is found in salmon tissue. The present invention from the marine bacterium (see table 1) produces higher expression levels of astaxanthin typically 5-8 fold higher than from Haematococcus pluvialis. The present invention therefore provides a method of producing at least 50g of astaxanthin per kilogram of dry bacterial biomass, such as at least 100g per kilogram of dry bacterial biomass.
The present invention also provides a marine bacterium belonging to the genus Pseudoalteromonas sp. that produces carotenoids, such as astaxanthin, and flavonoids, such as anthocyanin. Desirably, said bacterium is isolated from other bacterial species and is in a purified form. Advantageously astaxanthin appears to be the only carotenoid compound produced by the strain ASTA-1 (accession no. NCIMB 41083) which is beneficial in terms of its purification and biomanufacture.
The present invention further provides the use of a marine bacterium belonging to the genus Pseudoalteromonas sp for the production of carotenoids, such as astaxanthin, and flavonoids, such as anthocyanin. Conveniently said marine bacterium is cultured in vitro to permit easy harvest of the required compounds.
The extracted astaxanthin produced by the marine bacterium and isolated from the cells and/or supernatant may be used in variety of ways. The astaxanthin either in its isolated form or whole cells containing the pigment may be added to, for example, fish feed pellets for aquaculture or added to animal feed where pigmentation is necessary for the feed strategy and
healthcare (pharmacological) of that animal as an environmentally friendly substitute to synthetic derivatives. Astaxanthin may also be formulated as a freeze-dried sample that has been incorporated into a lipid/gelatine capsule (liposome). The liposome matrix may be manufactured through the sonication of phospholipids (oil) containing the pigmented cells.
Alternatively liposomes can be incorporated into the complete marine bacterial cell (ASTA-1). The astaxanthin produced by the marine bacterium Pseudoalteromonas sp. may alternatively be used as an environmentally friendly substitute for use in a variety of industrial and consumer markets including paints and coatings, plastics, spin dried fibres, construction materials, paper, ceramics, opto-electronic devices, elastomers, inks, textiles, glass, food and pharmaceuticals, for example as an additive colouring agent, and cosmetics.
Alternatively the astaxanthin produced may be used to promote a pharmacological or physiological effect in animals and humans or as an anti-oxidant for use in the treatment and prevention of disease.
A fraction from an extract of the marine bacterium and which has been subjected to column chromatography is generally characterised by a dark red/purple colour. This fraction is referred to herein as the "anthocyanin fraction".
The anthocyanin fraction has been demonstrated to successfully display anti-microbial and/or anti-bacterial activity against Methicillin Resistant Staphylococcus Aureus (MRSA). Therefore, the present invention also provides the anthocyanin fraction of an extract of a marine bacterium belonging to the genus Pseudoalteromonas sp. for use as an anti-biotic and/or antimicrobial agent. The marine bacterium belonging to the genus Pseudoalteromonas sp. may be strain ASTA-1 NCIMB 41083. Anthocyanin may be used as a contact ink in the food packaging industry. Contact inks are understood to be inks which come into direct contact with a food product within its packaging and such inks should desirably be non-toxic and/or physiologically acceptable.
The isolation, growth and production of the carotenoid astaxanthin and the flavonoid anthocyanin by the marine bacterium from the genus Pseudoalteromonas sp. Strain ASTA-1 NCIMB 41083 is described in the following figures and examples, in which:
Figure 1 is an HPLC chromatogram (480nm) (VARIAN STAR) of compound AQP001 and a synthetic standard of astaxanthin eluting with a reverse phase column with a 5 min run of methanol: water (95: 5), and a linear gradient of methanol: 29 tetrahydrofuran (7: 3) for 15 min and a final run of methanol (100%);
Instrument : Varian star #4 Sample Rate : 10.00 Hz
Channel : 1 = 470 nm Run Time : 19.983 min
*********** star Chromatography Software ********** version 4.0 *************** Chart Speed = 1.07 cm/min Attenuation = 58 Zero Offset = 3%
Start Time = 0.000 min End Time = 19.983 min Min/Tick = 1.00
Figure 2 is an HPLC chromatogram (480nm) (PHENOMENEX Luna silica column) of compound AQP001 (250 mg crude pigment extracted in methanol and re-dissolved in CHCL (20ml) - 10μL injection) eluting with a hexane and acetone. Mobile Phase 86% hexane 14% acetone at 1.2ml/min for 15 minutes;
Start Time : 0.00 min End Time : 15.00 min Low Point : -17.85 mv
Scale Factor : 0.0 Plot Offset : -18 mv Plot Scale : 357.0 mv
Figure 3 is a Standard Astaxanthin (8 ppm) HPLC chromatogram (480nm) (PHENOMENEX Luna silica column) eluting with a hexane and acetone. Mobile Phase 86% hexane 14% acetone at 1.2ml/min for 15 minutes;
Figure 4 H is an NMR spectrum of AQP001 dissolved in Chloroform-D recorded using an AMX400 spectrometer;
Figure 5 shows the UV-VIS spectrum of sample of AQP002 dissolved in chloroform showing adsorption peak data at 530nm using a Kontron UVIKON 932 spectrometer;
Figure 6 shows the UV-VIS spectrum of sample of AQP002 dissolved in chloroform showing adsorption peak data at 530nm using a Kontron UVIKON 932 spectrometer;
Figure 7 is an H NMR analysis of flavonoid sample AQP002 recorded using an AMX400 spectrometer; and
Figure 8 illustrates a structure analysis of anthocyanin compounds based on absorption spectrum of AQP002.
Example 1
Isolation of Pseudomonas sp. Strain ASTA-1 NCIMB 41083
Seawater was collected from several shallow water locations for locating bacteria. 100 μl samples of seawater were plated out onto a base medium (filtered sterilised seawater, 0.02% Tryptone, 0.02% Fe'"sθ4, 0.05% Glucose, Bacto Agar 1%, w/v, pH6.8). The agar plates were then incubated over 4 days at 28°C.
Colonies that displayed shades of yellow, red and orange were sub cultured and purified resulting in the isolation of particular strains. One of these strains that displayed a red shade was chosen (named ASTA-1) and has been deposited at NCIMB under accession number NCIMB 41083
Strain ASTA-1 exhibited a strong red colour on agar media and when cultured in a marine broth produced dark red pigments. Molecular analysis of the 16S rRNA gene of the strain showed it to be a new species of the genus Pseudoalteromonas.
Isolation of astaxanthin from the supernatant
Strain ASTA-1 was cultured in 2.5L of media (filter sterilised seawater, 0.2% tryptone, 0.05% glucose, 0.02% Fe'"so4, PH6.8) at 24°C for 5 Days. The culture was then centrifuged (3500 rpm, 30 min, 4°C) and the supernatant and cell pellet separated. 2.5L of the pigmented supernatant was extracted once with an equal volume of chloroform (CHCI3) and twice with an equal volume of ethyl acetate (EtOAc) to extract the pigments. The extract was dried over anhydrous sodium sulphate (NaS04) filtered and concentrated to dryness.
The extract was then re-dissolved in hexane, and chromatographed on silica gel 60 (Merck) with 30% Ethyl Acetate in hexane.
Astaxanthin was purified using HPLC on a reverse-phase (RP) column eluting with a 5 min run of methanol: water (95:5), and a linear gradient of methanol: tetrahydrofuran (7:3) for 15 min and a final run of methanol (100%). The purified astaxanthin was compared to analytical standards (See figure 3)
Isolation of astaxanthin from the cell biomass
From a 2.5L culture 4.5g of dry weight of ASTA-1 cells was collected and pigments extracted with a stabilised mixture (0.5g BHT / L), Methanol : Acetone mixture 7:3 v/v. (4 x 100ml methanol: acetone). The crude pigment supernatant was filtered using a Whatman No.3 filter to remove cell debris and concentrated to dryness under reduced pressure.
The pigment extract was then re-dissolved in hexane and chromatographed on silica gel 60 (Merck) with 30% Ethyl Acetate in hexane.
Astaxanthin was purified from the crude pigments using HPLC on a reverse-phase (RP) column (VARIAN STAR). 10μL of pigment sample was eluted with a 5 min run of methanol: water (95: 5), and a linear gradient of methanol: tetrahydrofuran (7: 3) at a flow rate of 2ml / min for 15 min and a final run of methanol (100%) scanning at 480nm.
In addition, astaxanthin was purified from the crude pigments using normal-phase HPLC (NP-HPLC) using PHENOMENEX Luna silica column scanning at 480nm. 10μL of the crude pigment was eluted using a solvent mixture of hexane and acetone. The mobile phase consisted of an 86% hexane: 14% acetone mixture running at 1.2ml/min for a total run time of 15 minutes.
The purified astaxanthin was compared to analytical chemical standards (astaxanthin, Sigma) on HPLC and NMR (See figures 1-4) and shows similarity to these standards.
Example 2
Extraction of anthocyanins from the cell mass using silica flash chromatography.
1g of freeze dried bacterial cells (strain ASTA-1) was extracted with a stabilised mixture (0.5g BHT / L) Acetone : Methanol mixture 7:3 v/v. (4 x 50 ml methanol: acetone). The crude pigment supernatant was then filtered using a Whatman no.3 filter to remove cell debris and concentrated to dryness under reduced pressure.
Flash column chromatography (Whatman Sep-Pak 30 g) of the crude pigment eluting with a mobile phase of 90% Chloroform: 10% Methanol yielded a seven fractions that included a dark red/purple fraction.
Spectrographic analysis of this dark red/purple fraction using a Kontron UVIKON 932 UV-VIS spectrometer showed a peak adsorption value of 530nm when dissolved in chloroform, and, together with the NMR data, indicated that this compound belonged to the flavonoid group anthocyanins (See figures 5-8).
Antibiotic analysis of red/purple compound (Anthocyanin)
The crude extract was purified using silica gel gravity chromatography eluting with a chloroform : methanol (90:10) to yield a red/purple fraction which was tested for antibiotic activity using the radial diffusion assays against MRSA and Ps. aerugenosa (See table 2).
Table 2: Antibiotic activity using radial diffusion assays associated with red/purple colourants from Pseudoalteromonas sp. ASTA-1
Radial Diffusion Assay
The standard protocol for the Radial Diffusion Assay (RDA) is carried out as follows:
1. Place 10ml of the indicator microorganism (Methicillin Resistant Staphylococcus Aureus (MRSA)) into sterile polypropylene tubes and centrifuge (900 x g, 4°C, 10 min).
2. Decant supernatant and wash cells in saline.
3. Re-suspend cells in saline
4. Measure the O.D570 0.20 = 5 x 107 CFU ml"1 add 1 or 4 x 106 bacterial CFU to 11ml of an appropriate base agar (1/10th strength) (Nutrient agar for nosocomial pathogens).
5. Mix and pour into a square petri dish and allow to set.
6. Using a grid template, create as required no. of wells by removing gel plugs from the centre of each grid square with a sterile Pasteur pipette (aseptically cut off the tip first!).
7. Add 4 - 5μl of a known quantity of the test material (antibiotic istamycin) (mgml"1) to each individual well. Also include positive and negative controls, e.g. penicillin (50ng ml"1 5ppm sample) and distilled water (-Ve control).
8. Incubate plates 5°C for three hours.
9. Melt top agar (2 x strength NA), and pour onto base agar, allow to set and incubate at 37°C overnight (terrestrial bacteria).
RDA Gel stain RDA Gel De-stain
2mg Coomassie brilliant blue R250 Aqueous solution of 10% acetic acid and 2%
27ml methanol dimethyl sulfoxide (DMSO)
63ml dH20
15ml 37% formaldehyde
An alternative method for obtaining the biocompounds from within the cells involves subjecting the bacterial cells to a process of French Pressing and sonication to disrupt cell membranes and release cell contents including pigments. The biocompounds can then be extracted using a solvent based system and purified using a chromatographic technique.
It will be understood that the isolation of these compounds can be applied on an industrial scale, and which is within the capabilities of the skilled person. For example, fermentation of the bacterium can be scaled up to more than 1000L using industrial scale equipment, and the compounds extracted using similar techniques as described above i.e. using solvents and chromatography to purify compounds, but using larger scale (industrial) equipment.
Although the invention has been particularly shown and described with reference to a number of preferred embodiments, it will be understood by those skilled in the art that various changes in the foπm and details may be made therein without departing from the spirit and scope of the invention.