Recovering and purifying resveratrol produced by microbial fermentation

Abstract

The present invention relates to a process for recovering and purifying resveratrol produced by microbial fermentation. More particularly, the present invention relates to a process for recovering and purifying resveratrol produced by yeast fermentation.

Claims

1. A process for recovering and purifying resveratrol from a microbial fermentation broth said process comprising: (a) increasing the pH of the fermentation broth to about 11; (b) separating and removing the host microbes such that a substantially microbe free liquid remains; (c) decreasing the pH of the substantially microbe free liquid to a value which is no less than about 7 to about 8.5 such that crude resveratrol is precipitated; (d) separating the precipitated crude resveratrol; (e) dissolving the crude resveratrol recovered in step (c) in a purification solvent to form a crude resveratrol containing solution; (f) contacting the solution produced in step (e) with one or more absorbents; and (g) crystallizing the purified resveratrol from the purification solvent, said purified resveratrol having a lower concentration of impurities including bisnoryangonin than the precipitated crude resveratrol of step (c).

2. The process according to claim 1 where the one or more absorbents are selected from carbon, optionally activated carbon, ion exchange resins, and/or Al.sub.2O.sub.3.

3. The process according to claim 1 where step (f) comprises the steps of: i) contacting the solution produced in step (e) with one or more absorbents selected from ion exchange resins and/or Al.sub.2O.sub.3; and ii) contacting the solution produced in step i) with carbon, optionally activated carbon.

4. The process according to claim 1, wherein the host microbes are removed by ultrafiltration, pressure filtration, vacuum drum filtration, or cross-flow filtration.

5. The process according to claim 4, wherein the host microbes are removed by ultrafiltration using an ultrafilter having a molecular weight cut-off of about 100 kDa to about 5 kDa or less.

6. The process according to claim 1, wherein the process includes a concentration step before step (c).

7. The process according to claim 6, wherein the concentration step is carried out by reverse osmosis.

8. The process according to claim 1, wherein the reduction of pH in step (c) is achieved by the addition of sulphuric acid or hydrochloric acid.

9. The process according to claim 1, wherein the purification solvent is selected from methanol, ethanol, propanol, acetone, ethyl acetate, and tetrahydrofuran.

10. The process according to claim 9, wherein the purification solvent is ethanol.

11. The process according to claim 10, wherein the resveratrol concentration of the solution formed in step (e) is in the region of from about 30 to about 50 g/L.

12. The process according to claim 3, wherein the amount of carbon to which the solution formed in step (e) or step i) is contacted will be from about 1.5 kg to about 2 kg per kg of resveratrol.

13. The process according to claim 3, wherein the solution formed in step (e) or step i) is contacted 3 times with 0.5 kg of carbon per kg of resveratrol.

14. The process according to claim 1, wherein crystallisation in step (g) is achieved by decreasing the concentration of the solvent solution to about 40% to about 15% by volume.

15. The process according to claim 1, wherein the precipitated crude resveratrol obtained in step (d) is subjected to pre-crystallisation before being passed to step (e), the pre-crystallisation comprising: (x) dissolving the crude resveratrol recovered in step (d) in a first solvent; and (y) crystallizing the resveratrol out of the first solvent, said resveratrol having a lower concentration of dihydroresveratrol impurities than present in the precipitated crude resveratrol.

16. The process according to claim 15, wherein the first solvent is the same or different to the purification solvent.

17. The process according to claim 16, wherein the first solvent is selected from methanol, ethanol, propanol, acetone, ethyl acetate, and tetrahydrofuran.

18. The process according to claim 17, wherein the first solvent is ethanol.

19. The process according to claim 18, wherein the ethanol concentration is about 65% to about 80% by volume.

20. The process according to claim 18, wherein the ethanol is mixed at a temperature of about 50° C. to about 80° C.

21. The process according to claim 15, wherein crystallisation in step (y) is achieved by decreasing the concentration of the solvent solution to about 40% to about 15% by volume.

22. The process according to claim 15, wherein steps (x) and (y) are repeated.

23. A composition comprising at least about 70 wt % resveratrol, about 10 to about 5000 mg/kg dihydroresveratrol, and about 10 to about 5000 mg/kg pinosylvin, wherein the composition further comprises bisnoryangonin at a maximum concentration of about 5000 mg/kg as measured by dry weight ratio to resveratrol and wherein the composition is free of emodin.

24. The composition according to claim 23, wherein the composition has about 2 wt % or less water.

25. The composition according to claim 23, wherein the compound has a yellowness index of about 40 to about 5 or less.

26. The composition according to claim 23, wherein the compound has a whiteness index of about −50 or less to about 100.

27. The composition according to claim 23, wherein the composition is dissolved or suspended in a solvent.

Description

(1) The present invention will now be described, by way of example, with reference to the following examples and figures in which:

(2) FIG. 1 is NMR data confirming that bisnoryangonin is an impurity in resveratrol;

(3) FIG. 2 is a summary of key yield and purity data for the results of Example 1;

(4) FIG. 3 is a summary of key yield and purity data for the results of Example 2;

(5) FIG. 4 is a summary of the experiments described in Example 3; and

(6) FIG. 5 is a graph illustrating the testing of bulk separation at pH11, repeated for various fermentation batches.

EXAMPLE 1

Full Downstream Process Tested at 1 L Broth Scale. Yeast Removed by Micron-Range Pressure Filter

(7) 1000 mL of broth was taken from the product of a fermentation using resveratrol-producing yeast strain EFSC4687 available from Evolve SA. The sample contained 21.6 g of trans-resveratrol. The pH of the sample was adjusted to 11 by addition of 63 mL of 30 wt % NaOH, dissolving the solid resveratrol into the liquid phase while leaving the yeast in the solid phase. The resulting yeast suspension was mixed for 1 h, and then centrifuged with a batch centrifuge for 20 min at 4400 rev/min, to deposit the yeast. 890 mL of liquid were decanted off of the yeast. 400 mL of deionized water were added and the yeast was resuspended. The suspension was then separated with the same batch centrifuge technique, and the liquid was decanted and added to the liquid that was initially decanted from the yeast. The total liquid volume recovered was 1285 mL.

(8) The pH of the liquid was then adjusted to 7.0 over 1 hour, by the addition of 37% HCl, causing the resveratrol to precipitate as a crude solid. The resulting suspension was filtered on a laboratory pressure filter with a filter paper rated for retention of particles>0.2 micron. The crude resveratrol solids were washed with 200 mL of deionized water. The resulting wet crude resveratrol solids had a mass of 87.8 g. Moisture content was measured as 50.8 wt % using a laboratory moisture analyzer. A sample of the solids was dissolved in ethanolic solution for analysis by HPLC, and was found to contain 45 g resveratrol per 100 g dry matter. So the estimated yield of resveratrol was 19.5 g, or 90.3% of the resveratrol contained in the broth at end of fermentation.

(9) The 87.8 g of crude wet resveratrol solids were mixed with 1080 mL of 70 vol % ethanol and heated to 55° C. for 3 hours, dissolving the solid resveratrol into ethanolic solution. The crude solids did not dissolve entirely into ethanol solution, and the remaining solids were filtered out with a vacuum filter, using a filter paper rated with a 12-25 micron exclusion size. The residual solids were washed with 220 mL of 70 vol % ethanol. This produced 1260 mL of ethanolic filtrate containing dissolved resveratrol. 2270 mL of deionized water were added to this filtrate during 2 to 4 hours, which decreased the ethanol content of the liquor to ˜25 vol % and caused crystallization of the resveratrol contained in the liquor. The material was stirred overnight and cooled to 5° C. to 10° C. for 3 hours to increase the crystallization yield. The resulting crude resveratrol solids were filtered out of the crystallization liquor using a vacuum filter with 12-25 micron filter paper. The solids were washed with 200 mL of deionized water, yielding 67.9 g of wet semi-crude solids. A sample of the semi-crude material was analyzed and it was found to contain 69.5 wt % H.sub.2O, with 71.8 g trans-resveratrol per 100 g of dry matter. So the estimated yield of trans-resveratrol was 14.9 g, or 76.1% of the resveratrol in the crude solids generated by acid precipitation.

(10) The 67.9 g of wet semi-crude solids were dissolved in 517 mL of 70 vol % ethanol and mixed for 2 hours at ambient temperature (approx. 20° C.). 6.2 g of activated carbon (CECA Acticarbone ENO-PC) were added and the mixture was stirred for 2 hours, to allow for adsorption of impurities including color bodies. The carbon was then removed by vacuum filtration and washed with 75 mL of 70 vol % ethanol. A second dose of 6.2 g of activated carbon was added to the resulting filtrate, and stirred for 2 hours, to adsorb more impurities and color inducing impurities. The carbon was again removed by filtration, this time with the aid of a Celite 512 filter aid precoat. Again the carbon was washed with 75 mL of 70 vol % ethanol, yielding a total of 516 mL of ethanolic filtrate. 930 mL of deionized water were added slowly over 2 to 4 hours, which decreased the ethanol content of the liquor to about 25 vol % and caused crystallization of the resveratrol contained in the liquor. The material was stirred overnight and cooled to 5° C. to 10° C. for 3 hours to increase the crystallization yield. The resulting resveratrol solids were filtered out of the crystallization liquor using a vacuum filter with 12 to 25 micron filter paper. The solids were washed with 200 mL of deionized water, then dried overnight in a vacuum oven. This yielded 12.2 g of dried product resveratrol, or 82.2% of the resveratrol contained in the semi-crude material. The product was analyzed and found to contain 99.7 g of resveratrol per 100 g of dried product. So the overall downstream process yield was calculated as 56% (12.2 g/21.6 g). The product contained 0.04% pinosylvin and 0.02% of cis-resveratrol. The product color was analyzed according to ASTM method E308, and the yellowness and whiteness indices were calculated according to ASTM E313. The Yellowness Index was 15 and the Whiteness Index was 41.

(11) The test parameters and results are summarized in Table 3 below and FIG. 2.

(12) TABLE-US-00003 TABLE 3 Op # Operation Lab parameters Lab example 1 Add base to Adjust pH to 11.0 1000 mL broth (21.6 g trans-resveratrol) dissolve Add ~0.06 L of 30 wt % NaOH per 63 mL of 30% NaOH resveratrol L of broth Mix 1 h Mix 1 h 2 Separate Batch centrifuge, 4400 rpm × 20 400 mL wash water yeast solids min. 1285 mL total supernatant + wash from liquid Separate supernatant (~0.89 L volume product supe per L broth). Wash solids w 0.4 L wash water per L of broth, add to first supernatant. 3 Neutralize Adjust pH to 7.0 37% HCl pH with ~0.007 L of 96% H.sub.2SO.sub.4 per L of H.sub.2SO.sub.4 supernatant 4 Separate Pressure filter, wash solids with Pressure filter 1360 mL of crude 0.15 L of water per L of slurry neutralized slurry resveratrol filtered +200 mL of wash water from −>1580 mL filtrate + 87.8 g wet suspension crude (50.8 wt % H.sub.2O, 43.2 g dry matter, 19.5 g trans-resv) 5 Dissolve Measure dry matter content, add 1080 mL of 70 vol % EtOH added wet crude 25 L of 70 vol % EtOH/kg crude to 87.8 g wet crude solid in (dry basis), mix 3 h at 55° C. ethanol 6 Filter out Vacuum filter, wash with 5 L of 70 Vacuum filter (12-25μ filter paper) undissolved vol % EtOH/kg crude (dry basis) Wash with 220 mL of 70 vol % solids EtOH 7 Slowly add Add 53 L water per kg crude (dry 1260 mL filtrate water, cool basis) to adjust ethanol content to Add 2270 mL H2O and hold to 25 vol %, dropwise for ~2-4 h crystallize (overnight). Cool to 5-10° C. for 3 h 8 Filter out Vacuum filter, wash with 5 L water/ Vacuum filter (12-25μ filter paper) semi-crude, kg crude Wash with 200 mL water water wash −>3560 mL filtrate 67.9 g wet semi-crude (69.5% H.sub.2O, 20.7 g dry matter, 14.9 g trans-resv) 9 Dissolve Measure dry matter content, add 517 mL of 70 vol % EtOH wet semi- 25 L of 70 vol % EtOH/kg crude crude in (dry basis), mix 2 h at ambient ethanol temperature 10 Add first Add 0.3 kg C/kg crude (dry 6.2 g Acticarbone ENO-PC dose of basis), mix 2 h at ambient carbon + temperature mix 11 Filter out Vacuum filter, wash with 3.6 L of Filter, wash w/75 mL of 70 vol % first carbon 70 vol % EtOH 1 kg crude (dry ethanol dose, wash basis) with ethanol 12 Add second Add 0.3 kg C/kg crude (dry 6.2 g Acticarbone ENO-PC dose of basis), mix 2 h at ambient carbon + temperature mix 14 Filter out Vacuum filter over Celite, wash Precoat with 1 g Celite 512 second with 3.6 L of 70 vol % EtOH/kg Filter, wash w/75 mL of 70 vol % carbon crude (dry basis) ethanol dose, wash with ethanol 15 Slowly add Add 45 L water per kg crude (dry 516 mL filtrate water, cool basis) to adjust ethanol content to Add 930 mL of water and hold to 25 vol %, dropwise for ~2-4 h crystallize (overnight). Cool to 5-10° C. for 3 h 16 Filter out Vacuum filter, wash with 5 L water/kg Vacuum filter (12-25μ filter paper) product, crude (dry basis) Wash with 200 mL water water wash 17 Dry Vacuum oven overnight Dry overnight in vacuum oven 12.2 g dried product Exceeded purity + color specifications

EXAMPLE 2

Full Downstream Process Tested at 8 L Broth Scale. Yeast Removed by 100 kDa Ultrafilter

(13) 8 L of broth was taken from the product of a fermentation using resveratrol-producing yeast strain EVST21811 available from Evolva SA. The sample contained 166 g of trans-resveratrol. 0.5 L of 30% NaOH was added to increase the pH to 11 and the material was mixed for 1 hour. The sample was then difiltered on an Alfa Laval M20 tangential flow filtration unit, using an Alfa Laval 0.6 m.sup.2 GR40PP filtration element. The GR40PP filter had a nominal molecular weight cutoff of 100 kDa. The filter was operated at 3 to 4 bar trans-membrane pressure. 46 L of permeate were collected, and the retentate was concentrated to 2 L at the end of the experiment. The permeate was then concentrated to 9 L using the same tangential flow filtration unit, with an Alfa Laval RO98pHt filtration element. The filter was operated at 25 to 30 bar trans-membrane pressure. 60 mL of 96% H.sub.2SO.sub.4 were added to neutralize the pH to 7.0 and precipitate crude resveratrol. The resulting crude resveratrol solids were filtered out of the suspension using a pressure filter, and were washed with 1.4 L of deionized water. This yielded 520 g of wet crude resveratrol. A sample of the material was analyzed and it was found to contain 63 wt % water, and 70 g resveratrol per 100 g of dry matter. So a bulk separation yield of 135 g resveratrol was calculated, corresponding to 86% of the resveratrol contained in the fermentation broth.

(14) A sample of 23.2 g of the crude resveratrol was purified, following the same approach described in Example 1. However in this case, the crude material had a higher purity (70% versus 45% in Example 1) and only a single carbon treatment was necessary to purify the material to target specifications. After the ethanol crystallization, the “semi-crude” resveratrol solids contained 97.6 g of resveratrol per 100 g of dry sample, a much greater purity than that in Example 1 (which was 72%). Without being bound by any particular theory, it is believed to be likely due to the removal of proteins by the ultrafiltration; likely some proteins are soluble at pH of 11 and permeate the micron-rated filter, and then co-precipitate with resveratrol upon acid precipitation. Subsequently those proteins may impair purification process performance. The semi-crude product was analyzed and contained 5.25 g of resveratrol, 87.5% of the resveratrol fed to the crystallization process.

(15) The “semi crude” resveratrol sample was then dissolved in ethanol and contacted with a single carbon dose of 3.0 g (CECA Acticarbone ENO-PC), and stirred for 2 hours before removing the carbon by vacuum filtration. Water was added to crystallize the product. The final product contained 5.12 g of resveratrol, 97.6% of the resveratrol contained in the semi-crude material. The purification yield was therefore 85%, and the overall downstream process yield was 73%. The product contained 98.7 g of resveratrol per 100 g of dry matter. Its colour was analysed and the Yellowness and Whiteness Indices were 15 and 37, respectively.

(16) The test parameters and results are summarized in Table 4 below and FIG. 3.

(17) TABLE-US-00004 TABLE 4 Op# Operation Lab parameters Lab example 1 Add base to Adjust pH to 11.0 8 L broth (166 g resveratrol) dissolve Add ~0.06 L of 30 wt % NaOH per 0.5 L of 30% NaOH resveratrol L of broth Mix 1 h Mix 1 h 2 Separate Alfa Laval M20, 0.6 m.sup.2 of GR40PP 8 L broth yeast solids membranes 46 L permeate from liquid 3-4 bar TMP, 10 L/min cross-flow 2 L retentate product No pre-concentration, just diafiltration 5.8 permeate volumes/broth volume Need to add NaOH to maintain pH in retentate 3 Concentrate Alfa Laval M20, 0.6 m.sup.2 of 46 L permeate liquid RO98pHt membranes 9 L concentrate 25-30 bar TMP, 9 L/min cross- flow 5.1x volumetric concentration factor 1.1 L of concentrate per L of broth 4 Neutralize Adjust pH to 7.0 60 mL of 96% H.sub.2SO.sub.4 added pH with ~0.007 L of 96% H.sub.2SO.sub.4 per L of slowly H.sub.2SO.sub.4 concentrate 5 Filter out Pressure filter, wash solids with Pressure filter 9 L of neutralized crude 0.15 L of water per L of slurry slurry filtered +1.4 L of wash water −>520 g wet crude (63% water, 193 g dry matter, 135 g trans-resv) 6 Dissolve Measure dry matter content, add wet crude 25 L of 70 vol % EtOH/kg crude solid in (dry basis), mix 3 h at 55° C. ethanol 7 Filter out Vacuum filter, wash with 5 L of 70 undissolved vol % EtOH/kg crude (dry basis) solids 8 Slowly add Add 110 L water per kg crude (dry water, cool basis) to adjust ethanol content to and hold to 15 vol %, dropwise for ~2-4 h crystallize (overnight). Cool to 5-10° C. for 3 h 9 Filter out Vacuum filter, wash with 5 L water/ semi-crude, kg crude water wash 10 Dissolve Measure dry matter content, add wet semi- 25 L of 70 vol % EtOH/kg crude crude in (dry basis), mix 2 h at ambient ethanol temperature 11 Add first Add 0.3 kg C/kg crude (dry dose of basis), mix 2 h at ambient carbon + temperature mix 12 Filter out Vacuum filter over Celite, wash first carbon with 3.6 L of 70 vol % EtOH/kg dose, wash crude (dry basis) with ethanol 13 Slowly add Add 45 L water per kg crude (dry water, cool basis) to adjust ethanol content to and hold to 25 vol %, dropwise for ~2-4 h crystallize (overnight). Cool to 5-10° C. for 3 h 14 Filter out Vacuum filter, wash with 5 L water/ product, kg crude water wash 17 Dry Vacuum oven overnight

EXAMPLE 3

Effect of pH on Performance During Bulk Separation Under Alkaline Conditions

(18) The selection of pH for the alkaline dissolution of resveratrol is critical. If the pH is too low, resveratrol will have poor solubility. If the pH is too high, resveratrol may decompose or react with other broth components. A series of tests was performed at pH values of 10, 11, and 12 using 1 L samples of a resveratrol-containing broth. The pH of the broth samples was adjusted with NaOH, and the material was mixed for 1 hour before the yeast was filtered out using a micron-rated pressure filter. The residual solids were washed with water, and the resveratrol in the resulting filtrates were precipitated with 37% HCl. The crude resveratrol was then filtered out of the resulting suspensions and vacuum dried.

(19) The results of the tests are summarized in FIG. 3.

(20) At pH 10, the yield was low (18%), and the material was brown. At pH 11 the yield was high (94%), and the material was a light beige colour. At pH 12 the yield was intermediate (72%), and the material was brown. The resveratrol content of the resulting product increased from 56 wt % at pH 10 to 64 wt % at pH 11, and increased further to 72 wt % at pH 12.

(21) Based on the high yield observed at pH 11, this test was repeated several times, using broth samples from several batches. The results are summarized in FIG. 4. The process yield was very reproducible, with results from 90 to 94% shown in FIG. 4. The purity of the resulting crude resveratrol was more variable, and ranged from 46% to 68%.

EXAMPLE 4

Effect of Ultrafilter Molecular Weight Cutoff Rating on Performance During Yeast Filtration Under Alkaline Conditions

(22) When using ultrafiltration for yeast filtration at elevated pH, it has been found that the use of an ultrafilter with a lower molecular weight cutoff significantly increases the purity of the crude resveratrol after acid precipitation, without significant impairment of filtration rate. The stability of the filtrate at pH 11 is also improved with use of an ultrafilter with lower molecular weight cutoff. Both of these attributes are advantageous for processing.

(23) A 129 L broth sample and a 96 L broth sample were processed using an Alfa Laval M38 ultrafilter at pH11. Three ultrafiltration membranes were tested, with molecular weight cutoffs of 5, 25, and 100 kDa. The filtration performance results are summarized in Table 5 below. After adjusting pH to 11, the yeast suspensions were concentrated by a factor of 2.5-3, and then diafiltered while maintaining pH at 11 in the retentate. The filtrates were subsequently concentrated by reverse osmosis and precipitated with sulfuric acid. The resulting crude resveratrol materials were filtered out of suspension and analyzed. Yields were comparable for all three materials, approximately 78%. The purity of the materials was analyzed and the results are summarized in Table 6. The crude resveratrol produced with a 100 kDa ultrafilter contained 71.4% resveratrol. The crude resveratrol produced with the 25 kDa and 5 kDa ultrafilters was comparable in purity (79.8% and 79.2% respectively).

(24) It is advantageous for process intermediate materials to have a high degree of stability, so that they can be processed over time without the need for immediate processing on equipment with high capacity. The various filtrates from the ultrafiltration tests were concentrated by reverse osmosis and held at pH 11 for 3 to 7 days at 5° C. to 8° C. to evaluate their stability. The results are summarized in Table 7 below. The product decomposition is a comparison between the composition at the time of production and that measured 7 days after production. The stability improved monotonically as the molecular weight cutoff of the ultrafilter was decreased from 100 kDa to 25 kDa to 5 kDa. 11%, 7%, and 5% of the resveratrol was decomposed in the respective tests.

(25) TABLE-US-00005 TABLE 5 EVST 21811 w/SAG790LV EFSC 4687 w/J647 2016-125 (129 L) 2016-167 (96 L) Membrane material GR40PP GR60PP GR90PP GR40PP Nominal molecular 100 kDa 25 kDa 5 kDa 100 kDa weight cutoff Surface Area 0.75 m.sup.2 0.75 m.sup.2 0.75 m.sup.2 2.25 m.sup.2 Cross Flow ~25 L/min ~25 L/min Flux 46-29 46-32 42-28 29-36 L/m.sup.2/h L/m.sup.2/h L/m.sup.2/h L/m.sup.2/h Trans-membrane pressure 3-3.2 bar 3-3.2 bar Retentate VCF (volumetric 3x 2.5x concentration factor) Temperature ~30° C. ~30° C. Permeate volumes 44.6 L 44.0 L 46.4 L 101 L Diafiltration Volume 52.7 L 38.8 L Retentate Volume 44.9 L 42.5 L NaOH added 8 L 4.1 L Water flux recovery 97% 47% 52% 82% after cleaning

(26) TABLE-US-00006 TABLE 6 Assay of Crude Precipitate (g trans-resveratrol per g Membrane (MWCO) dry matter) GR40PP (100 kDa) 71.4% GR60PP (25 kDa) 79.8% GR90PP (5 kDa) 79.2%

(27) TABLE-US-00007 TABLE 7 Nominal Molecular Original After pH Product Weight Concentration Neutralization Decomposi- Cutoff (g/L) (g/L) tion EFSC 21811 100 kDa 15.9 14.2 10.7% GR40PP (100 kDa) GR60PP  25 kDa 26.7 24.89   7% (25 kDa) GR90PP  5 kDa 24.7 23.4   5% (5 kDa) EFSC4687 100 kDa 16.43 14.54 11.5% GR40PP (100 kDa)

EXAMPLE 5

Semi Crude Crystallisation

(28) 20 g of crude resveratrol was dissolved in 500 ml of 70 vol % of ethanol. This was stirred for one hour at 55° C. and filtered through filter paper for the retention of particles having a size greater than 0.2 microns to remove any undissolved solids. These solids were washed with 60 ml 70 vol % ethanol. After filtration 560 ml of filtrate was collected. This was diluted with 560 ml of water to have a 35 vol % ethanol concentration. The temperature was maintained at 55° C. during the water addition. The diluted filtrate was then cooled to 5° C. to 10° C. over a period of 3 to 4 hours. This was done stepwise as follows: 55° C. to 40° C. for one hour, 40° C. to 20° C. for one hour and 20° C. to 5° C. for one hour. 448 ml water was added to adjust the concentration to 25 vol % and the temperature was maintained at 5° C. and stirred for one hour before the crystals were separated by filtration. The crystals were filtered through filter paper rated for retention of particles having a size greater than 0.2 microns and washed with 50 ml of deionised water.

Final Product Crystallisation

(29) 13.68 g of semi crude resveratrol was dissolved in 320 ml of 70 vol % ethanol. 6.5 g of CECA acticarbone ENO-PC was added and the resulting mixture stirred for 2 hours. The carbon was then removed by filtration using filter paper for retention of particles having a size greater than 0.2 microns. The resultant cake was washed with 20 ml of 70 vol % ethanol. 340 ml of filtrate was collected. 136 ml of water was added to form an ethanol concentration of 50 vol % ethanol. The solution was heated to 55° C. for one hour. The solution was then cooled to 5 to 10° C. over a period of 3 to 4 hours. This was done stepwise as follows: 55° C. to 40° C. for one hour, 40° C. to 20° C. for one hour and 20° C. to 5° C. for one hour. 476 ml water was added to adjust the concentration to 25 vol % and the temperature was maintained at 5° C. and stirred for one hour before the crystals were separated by filtration. The crystals were filtered through filter paper rated for retention of particles having a size greater than 0.2 microns and washed with 50 ml of deionised water.

(30) Further details of the filtrations are set out in Table 8

(31) TABLE-US-00008 TABLE 8 Cooling Anti-Solvent Crystallisation Crystallisation Resveratrol 40 g/L 40 g/L Concentration Assay 96.35 95.93 Related Bisnoryangonin 0.39% a/a Bisnoryangonin 0.31% a/a Impurities Dihydro 0.06% w/w Pinosylvin 0.11% w/w Pinosylvin 0.11% w/w Yield 77% 75% Losses in  3%  2% Filtrate Filtration  2.24 35   time (min) Cake ~3 mm ~3 mm Thickness Appearance Crystalline Less Crystalline

EXAMPLE 6

(32) 9 L of microbial fermentation broth comprising resveratrol at pH 6.3 was processed using Alfa Laval M20. Four ultrafilters were tested, GR40PP (100 kDa), GR60PP (25 kDa), GR80PP (10 kDa) and GR90PP (5 kDa). The filtration performance is set out in Table 9.

(33) TABLE-US-00009 TABLE 9 Filtration summary GR40PP GR60PP GR80PP GR90PP Nominal 100 kDa 25 kDa 10 kDa 5 kDa molecular weight cutoff Surface Area 0.108 m.sup.2 0.072 m.sup.2 0.072 m.sup.2 0.072 m.sup.2 Cross Flow   ~9 L/min TMP 4-5 bar Flux rates 50 66 42 42 VCF1 (L/m.sup.2/h) VCF2 44 75 42 58 (L/m.sup.2/h) Diafiltration 72 75 108  117  Temperature ~25° C.   Diafiltration 9 L Volume Retentate 1 L Volume Assay 70.72% 86.57% 87.94% 85.68%

(34) The filtration was carried out after adjusting the pH to 11 and then diafiltrated while maintaining the pH of the retentate at 11. The filtrates from each membrane was collected separately and precipitated with sulfuric acid. The yields for all four membranes were comparable at 98.61%. The purity of the material was calculated. The results are set out in for the bulk separation in Table 10 and after ultrafiltration in Table 11.

(35) TABLE-US-00010 TABLE 10 Resveratrol Concentration/ Volume/ in Crude Yield/ Assay Weight Resveratrol Filtrate Losses Broth 19.92 g/L 9 L 179.3 g

(36) TABLE-US-00011 TABLE 11 Resver- atrol in Concentration/ Volume/ Resver- Crude Yield/ Assay Weight atrol Filtrate Losses UF 1.77 g/L   1 L  1.77 g  0.9% Retentate GR40PP 70.72% 71.1 g 50.28 g 0.5 g 98.64% GR60PP 86.57% 79.6 g 68.91 g 0.2 g GR80PP 87.94% 31.9 g 28.05 g 0.02 g  0.7% (in GR90PP 85.68% 34.5 g 29.56 g 0.6 g filtrates)

(37) The crude material from GR80PP (10 kDa) and GR90PP (5 kDa) was processed into decolouring without going into pre-crystallisation. The carbon treatment was effective in removing colour inducing impurities along with other impurities including bisnoryangonin. The crude material contained about 1% of bisnoryangonin and was removed with the carbon treatment. The results are set out in Table 12. The crude intermediate was dissolved in 70 vol % ethanol at 40 g/L and heated to 55° C. for one hour to dissolve everything into solution. The solution was treated with CECA ActiCarbone ENO-PC in three doses (1.5 kg of carbon for 1 kg of resveratrol). The suspension was stirred for 2 hours at 40° C. and the carbon was removed by vacuum filtration using black ribbon filter paper. The carbon cake was washed with 5× volumes to 1 g of resveratrol 70 vol % ethanol. The filtration and washing was repeated for each of the doses with carbon. The results are set out in Table 13.

(38) TABLE-US-00012 TABLE 12 Bulk Separation 98.6%   98.6%   98.6%   98.6%   Yield Crude Assay 87.94%   87.94%   85.68%   85.68%   Carbon Dose 1.5 kg/1 kg 1.5 kg/1 kg 1.5 kg/1 kg 1.5 kg/1 kg In Three Steps In Three Steps In Three Steps In Three Steps Carbon and 70% 67% 71% 68% Crystallization Yield Final Assay 98.45% 98.81% 98.35% 99.41% Colour YL24 WI 14 YL 12 WI 51 YL 15 WI 43 YL 14 WI 43 Specification L*91 a*-2 b* 14 L*93 a*-1 b* 7 L*94 a*-3 b* 9 L*93 a*-2 b* 8 Impurity Profile Coumaric acid Coumaric acid Coumaric acid Coumaric acid 0.09% w/w 0.14% w/w 0.09% w/w 0.15% w/w Dihydroresveratrol Dihydroresveratrol Dihydroresveratrol Pinosylvin 0.33% w/w 0.3% w/w 0.3% w/w 0.07% w/w Bisnoryangonin Bisnoryangonin Bisnoryangonin Impurity Impurity Impurity 0.05% a/a 0.07% a/a 0.07% a/a Pinosylvin Pinosylvin Pinosylvin 0.07% w/w 0.06% w/w 0.07% w/w Overall Yield 69% 66% 70% 67%

(39) TABLE-US-00013 TABLE 13 Before Carbon Treatment (% a/a) Crude resveratrol GR80PP 1.25 Crude resveratrol GR90PP 1.25 After Carbon Treatment (% a/a) Final crystals GR80PP Not detected Final crystals GR90PP Not detected