Process for the purification of complex biocompositions

Abstract

The present invention relates to a process for the purification of complex biocompositions.

Claims

1. Process for the purification of complex biocompositions comprising the steps a) Providing a complex biocomposition; b) Heating of the complex biocomposition to a temperature of from 50 to 140° C.; c) Separating the heated complex biocomposition into a solid and a liquid phase; d) Subjecting the liquid phase to an ultrafiltration and thereby obtaining a retentate and a permeate.

2. Process according to claim 1, wherein step b) is carried out for a time period of from 5 seconds to 2 hours.

3. Process according to any of the foregoing claims, wherein the separation according to step c) is carried out by use of a filter press.

4. Process according to any of the foregoing claims, wherein the molecular weight cut off MWCO of the ultrafiltration membrane is selected from 5 to 500 kDa.

5. Process according to any of the foregoing claims, wherein at least part of the ultrafiltration is carried out by diafiltration.

6. Process according to any of the foregoing claims, further comprising step e) Heating the retentate of step d) to a temperature of from 70 to 140° C.

7. Process according to claim 6, wherein step e) is carried out for a time period of from 10 seconds to 2 hours.

8. Process according to any of the foregoing claims, further comprising step f1) Precipitation of the liquid phase of step c), or retentate of step d).

9. Process according to any of claims 1 to 7, further comprising step f2) drying of the retentate.

10. Process according to any of the foregoing claims, wherein the complex biocomposition contains at least one biopolymer.

11. Process according to any of the foregoing claims, wherein at least one filtration aid is added before or during step b) or c).

12. Process according to any of the foregoing claims, wherein step c) is carried out at a temperature of from 50 to 80° C.

13. Process according to any of claims 1 to 8 or 10 to 12, wherein step f1) is carried out in a bubble column or airlift reactor.

14. Process according to any of the foregoing claims, further comprising steps g) contacting the permeate of step d) with at least one organic solvent for a time period of from 5 seconds to 30 minutes; h) Separating the permeate and at least one organic solvent into two phases of different density; i) Subjecting the phase of lower density to an evaporation; j) Obtaining a surfactant as the residual of step i.

Description

EXAMPLES AND FIGURES

[0083] In the following, the present invention is described by specific examples and figures. The examples and figures are used for illustrating purposes only and do not limit the scope of the present invention.

List of Figures

[0084] FIG. 1 shows the influence of temperature on filtration

[0085] FIG. 2 shows the influence of acid dosing on filtration

[0086] FIG. 3 shows the extracted surfactant in the liquid phase

[0087] FIG. 4 shows the relative dynamic viscosity at different temperatures

[0088] FIG. 5 shows the dynamic viscosity at different UHT treatments

[0089] FIG. 6 shows an exemplary set up including a heat exchange device

[0090] FIG. 7 shows an exemplary set up for high temperature treatment including a heat exchange device without external cooling and a solid-liquid separation

EXAMPLE 1

Influence of Temperature on Filtration

[0091] 150 kg of a complex biocomposition resulting from fermentation of Aureobasidium pullulans (20° C.) comprising a beta glucan biopolymer was mixed with a suspension of 30 kg DI-water and 7.5 kg filter aid Dicalite BF (Dicalite Europe nv, Gent, Belgium).

[0092] The fermentation was carried out as follows:

[0093] 100 kg of the following medium was prepared and autoclaved at 121° C. for 20 minutes in a Techfors 150-reactor (Infors AG, Bottmingen, Switzerland):

[0094] 50 g/kg Sucrose (Südzucker AG), 0.4 g/kg NaNO3 (Sigma Aldrich, Steinheim, Germany), 0.4 g/kg K2HPO4, 0.4 g/kg NaCl (Sigma Aldrich, Steinheim, Germany), 0.4 g/kg MgSO4*7H2O (Sigma Aldrich, Steinheim, Germany), 0.2 g/kg yeast extract (Lallemand, Montreal, Canada), 1 g/kg Antifoam 204 (Sigma Aldrich).

[0095] After autoclaving, for the rest of the experiment the temperature was controlled at 26° C., the pH was adjusted to 4.5+/−0.25 using 5 M H2SO4 and 5 M NaOH and controlled to 4.5+/−0.25 for the remaining fermentation with 5 M NaOH, the medium was stirred at 52 rpm and aerated with 150 L/min at a headspace pressure of 0.2 bar.

[0096] When constant conditions were reached, each medium was inoculated to a concentration of 0.019 g/kg CDW of Aureobasidium pullulans. The organism was cultivated in the respective medium at the conditions mentioned above for 144 h.

[0097] The mixture was heated in a stirred vessel. Temperature (20° C., 50° C., 75° C.) and heating time of the complex biocomposition were varied according to table 1. Afterwards the mixture was filtered using a filter press (Netzsch Filtrationstechnik GmbH, Selb, Germany) with pressurized air at 2 bar. For the filtration a filter cloth (MarsSyntex PP2442) was used. The produced filtrate mass was recorded versus filtration time. The figure shows the filtration performance which is defined as the produced filtrate mass after 30 min filtration time at the respective temperatures divided by the filtrate mass of the reference No. 1, which was carried out at 20° C. without heating.

TABLE-US-00001 TABLE 1 Preparation of filtration samples Holding time Heating at heating Temperature of No. temperature temperature filtration 1 — — 20° C. 2 50° C. 30 min 50° C. 3 75° C. 30 min 75° C. 4 75° C. 90 min 75° C. 5 75° C. 30 min 20° C.

[0098] FIG. 1 clearly proves that filtration at elevated temperatures improves the filtration performance, which means that the filtration is faster. Comparing experiments No. 3 with No. 5 clearly shows that cooling down to 20° C. after the heating step does not improve the filtration.

EXAMPLE 2

Influence of Acid Dosing on Filtration Performance

[0099] 150 kg of biocomposition (20° C.) with the same composition as used in example 1 was mixed with a suspension of 30 kg DI-water and 7.5 kg filter aid Dicalite BF (Dicalite Europe nv, Gent, Belgium). The mixture was heated in a stirred vessel. Temperature (20° C., 75° C.) and addition of HNO.sub.3 were varied according to table 2. Afterwards the mixture was filtered in a filter press (Netzsch Filtrationstechnik GmbH, Selb, Germany) with a pressurized air at 2 bar. For the filtration a filter cloth (MarsSyntex PP2442) was used. The produced filtrate mass was recorded versus filtration time. FIG. 2 shows the filtration performance which is defined as the produced filtrate mass after 30 min filtration time at the respective temperatures divided by the filtrate mass of the reference No. 1, which was carried out at 20° C. without heating.

TABLE-US-00002 TABLE 2 Preparation of filtration samples with dosing of acid Amount of aqueous Holding time at HNO.sub.3 Heating heating Temperature of No. (w70% HNO.sub.3) pH-value temperature temperature filtration 1 — 4.6 — — 20° C. 2 0.6 L 2.6 — — 20° C. 3 — 4.6 75° C. 30 min 75° C. 4 0.6 L 2.6 75° C. 30 min 75° C.

[0100] FIG. 2 clearly demonstrates that the best filtration performance is achieved by heating up the complex biocomposition, whereas addition of acid, like proposed in the prior art, even decreases the filtration performance.

EXAMPLE 3: OBTAINING A SURFACTANT-RICH LIQUID PRODUCT

[0101] For production of the biopolymer the liquid fraction of experiment No. 3 of the previous example (75° C., no acid) was concentrated by an ultrafiltration unit with a cut-off of 300 kDa (Synder, LX-3A-2540M) with a concentration factor of three.

[0102] Afterwards, 200 mg of solid campher (Alfa Aeser, (1R)-(+)-Campher, 98%) was strewed on the surface of the liquid sample (100 mL) of the obtained permeate. On an aqueous surface without a surfactant the campher moves by transformation of the surface tension. With a surfactant the campher does not move due to the lowered surface tension by the surfactant. For control tap water was used as negative control and a tap water+0.1 wt % cleaning agent containing a surfactant (Ecolab, P3-ultrasil 112) as positive control.

TABLE-US-00003 TABLE 3 Test setup: Evidence of surfactant No. Sample Campher test 1 Water Negative control Negative 2 Water + surfactant Positive control Positive 3 Permeate Sample Positive

[0103] Table 3 above clearly proves that by carrying out the ultrafiltration step, a permeate is obtained which contains a surfactant. Since the surfactant was produced by fermentation, the surfactant can be considered as bio-surfactant. Therefore, it can be demonstrated that the inventive process is able to obtain a surfactant-rich liquid product.

EXAMPLE 4 EXTRACTION OF THE SURFACTANT

[0104] The permeate of the previous example was mixed with an equal volume ethyl acetate (Sigma Aldrich, Steinheim, Germany) and then the two liquid phases were separated by centrifugation (15 min at 14.000 g). The phase with the lower density was then evaporated in a Heidolph rotary evaporator at 40° C. and 150-250 mbar until a solid residual remained in the evaporator. The residual was dissolved in DI-water at pH 5 resulting in a dry matter concentration of 0.1 wt.-%. After shaking a formation of foam was observed.

[0105] FIG. 3 clearly shows that the invented process is able to produce a surfactant which leads to foam formation in water.

EXAMPLE 5 STABLE VISCOSITY AT DIFFERENT TEMPERATURE

[0106] Viscosity of the retentate after ultrafiltration (Synder, LX-3A-2540M, cut-off 300 kDa) of the liquid phase of experiment No. 3 of the example 2 (75° C., no acid) was measured with a rotational rheometer and coaxial cylinder according to DIN 53019 using a Malvern Kinexus Lab+-rheometer (Malvern Panalytical Ltd., Almelo, Netherlands) at a shear rate of 20 s.sup.−1 and a temperature of 20° C., 50° C. and 80° C. The figure shows the relative dynamic viscosity which is defined as the viscosity at the measurement temperature divided by the dynamic viscosity at 20° C.

[0107] FIG. 4 clearly shows that there is no decrease of the viscosity by increased temperatures. This is in contrast to most biopolymers which typically show a decrease of viscosity by increasing the temperature.

EXAMPLE 6 VISCOSITY AFTER UHT TREATMENT

[0108] The retentate after ultrafiltration (Synder, LX-3A-2540M, cut-off 300 kDa) from the previous example was treated with an UHT (Armfield FT74XTS) at a temperature between 110 and 135° C. with a holding time of 15 s and cooled afterwards to 20° C. within 50 s. The samples were measured with a rotational rheometer and coaxial cylinder according to DIN 53019 using a Malvern Kinexus Lab+-rheometer (Malvern Panalytical Ltd., Almelo, Netherlands) at a shear rate of 20 s.sup.−1 and a temperature of 20° C. The figure shows the relative dynamic viscosity which is defined as the viscosity with UHT treatment divided by the dynamic viscosity without UHT treatment.

[0109] FIG. 5 clearly proves that the UHT treatment does not reduce the viscosity after cooling. Therefore, it can be concluded that the UHT has no negative influence on the biopolymer.

EXAMPLE 7 VISCOSITY AT HIGH TEMPERATURE UNDER STIRRING

[0110] The mixture was heated in a stirred vessel as described in example 2 but the holding time at heated temperature was extended to 14 hours at 70° C.

[0111] Viscosity of the mixture before and after the heating process was measured with a rotational rheometer and coaxial cylinder according to DIN 53019 using a Malvern Kinexus Lab+-rheometer (Malvern Panalytical Ltd., Almelo, Netherlands) at a shear rate of 20 s.sup.−1 at a temperature of 20° C. FIG. 8 shows the relative dynamic viscosity which is defined as the viscosity before heating compared to the viscosity after the heating process.

[0112] FIG. 8 clearly shows that there is no decrease of the viscosity with increased temperature and at stirred conditions for 14 hours. This is in contrast to most biopolymers, which typically show a decrease of viscosity with increasing temperature.