Process for the fermentation of Ascomycota

Abstract

The present invention refers to a process for the fermentation of Ascomycota, a beta-glucan produced by the process and the use of the beta-glucan as rheology modifier

Claims

1. Process for the fermentation of Ascomycota comprising the following steps: a) Providing a medium with a dry matter content selected from the range of from 0.1 to 0.3 wt.-% and containing less than 0.1 wt.-% of an organic nitrogen source; b) Inoculating the medium with at least one fungus belonging to the division of Ascomycota with an inoculation density selected from the range of from 0.01 g.sub.CDW/L to 50 g.sub.CDW/L; c) Adding at least one carbon source in a concentration of from 1 to 20 wt.-%; d) Mixing the medium and the at least one fungus according to step d) for a time period of from 24 to 400 hours; e) Removing from 30 to 90 wt.-% of the medium and the at least one fungus; f) Adding from 30 to 90 wt.-% of a medium as defined in step a) additionally containing a carbon source; g) Repeating steps c) to f) from 2 to 100 times; wherein at least one of steps c) to g) is carried out with a relative gas content selected from the range of from 0.005 to 0.5.

2. Process according to claim 1, wherein the cell dry weight CDW of the medium in step b) is selected from the range of from 0.01 to 50 g.sub.CDW/L.

3. Process according to any of the foregoing claims, wherein the nitrogen content of the medium according to step a) is selected from the range of from 0.003 to 0.02 wt.-%.

4. Process according to any of the foregoing claims, wherein the pH is kept in the range of from 4.0 to 6.0 during the whole process.

5. Process according to any of the foregoing claims, wherein the temperature T is kept in the range of from 22 to 30 C. during the whole process.

6. Process according to any of the foregoing claims, wherein the dynamic viscosity of the medium during step d) is selected from the range of from 40 to 600 mPas.

7. Process according to any of the foregoing claims, wherein step b) is carried out for a time period of from 1 minute to 8 hours.

8. Beta-glucan produced by a process according to any of claims 1 to 7.

Description

EXAMPLES AND FIGURES

[0074] 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

[0075] FIG. 1 shows the influence of dry matter and nitrogen content on product formation

[0076] FIG. 2 shows the influence of temperature on product formation

[0077] FIG. 3 shows the influence of pH on product formation

[0078] FIG. 4 shows the influence of the C source on product formation

[0079] FIG. 5 shows a schematic overview of the repeated batch process

[0080] FIG. 6 shows product formation per batch within repeated batch production

[0081] FIG. 7 shows the influence of relative gas content on product formation

[0082] FIG. 8 shows the influence of a starvation phase on product formation

EXAMPLE 1

Influence of Dry Matter and Nitrogen Content

[0083] 100 kg of the following media were prepared and autoclaved at 121 C. for 20 minutes in a Techfors 150-reactor (Infors AG, Bottmingen, Switzerland):

[0084] PDB-Medium (Potato Dextrose Broth, dry matter content 0.4 wt.-%, nitrogen content 0.008 wt.-%; state of the art): 3 g/kg potato extract (Sigma Aldrich, Steinheim, Germany), 1 g/kg Antifoam 204 (Sigma Aldrich, Steinheim, Germany) in tap water (Osin'ska-Jaroszuk et al., Extracellular polysaccharides from Ascomycota and Basidiomycota: production conditions, biochemical characteristics, and biological properties; World J Microbiol Biotechnol. 2015 31:1823-1844),

[0085] PM-Medium (Production Medium, dry matter content 0.5 wt.-%, nitrogen content 0.02 wt.-%); state of the art): 2 g/kg (NH4)2O4 (Sigma Aldrich, Steinheim, Germany), 1 g/kg K2HPO2 (Sigma Aldrich, Steinheim, Germany), 0.5 g/kg KCl (Sigma Aldrich, Steinheim, Germany), 0.5 g/kg MgSO4*7H2O (Sigma Aldrich, Steinheim, Germany), 0.01 g/kg FeSO4*7H2O (Sigma Aldrich, Steinheim, Germany), 0.4 G/kg Yeast extract (Lallemand, Montreal, Canada), 1 g/kg Antifoam 204 in tap water (Youssef et al. Pullulan production by a non-pigmented strain of Aureobasidium pullulans using batch and fed-batch culture; Process Biochemistry 34 (1999) 355-366) and

[0086] OM-Medium (invention): 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).

[0087] Before autoclaving 50 g/kg sucrose (Sdzucker AG) were added to each medium. 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 and aerated at a relative gas content of 0.016 and a headspace pressure of 0.2 bar.

[0088] 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.

[0089] The viscosity as a measure for the product formation 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. The improvement by the self-developed medium is shown in FIG. 1 by the medium cost (sum of the costs for all medium components) and the dynamic viscosity of the fermentation broth after 144 h of cultivation as a measure of product formation. It is shown that the OM medium with a dry matter content of 0.16 wt.-% and a nitrogen content of 0.0066 wt.-% shows highest viscosity (and therefore beta glucan yield) while keeping costs at a minimum level.

EXAMPLE 2

Influence of Temperature on Beta Glucan Yield

[0090] 2 kg of OM medium and 50 g/kg sucrose (Sdzucker AG) were autoclaved at 121 C. for 20 minutes in a Labfors 5 BioEtOH-reactor (Infors AG, Bottmingen, Switzerland) equipped with one Rushton turbine and one pitched blade impeller. After autoclaving, for the rest of the experiment the temperature was controlled to the temperatures ranging from 24 to 32 C. (see FIG. 2, 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 and aerated at a relative gas content of 0.03.

[0091] When constant conditions were reached, the 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. The viscosity as a measure for the product formation 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.

[0092] It is shown in FIG. 2 that best results were achieved at temperatures from 24 to 30 C. with an optimum of 26 C. (dynamic viscosity of the fermentation broth after 144 h of cultivation as a measure of beta glucan yield).

EXAMPLE 3

Influence of pH on Beta Glucan Yield

[0093] 2 kg of OM medium and 50 g/kg sucrose (Sdzucker AG) were autoclaved at 121 C. for 20 minutes in a Labfors 5 BioEtOH-reactor (Infors AG, Bottmingen, Switzerland) equipped with one Rushton turbine and one pitched blade impeller. After autoclaving, for the rest of the experiment the temperature was controlled to 26 C., the pH was adjusted and controlled to the values given in FIG. 3 using 5 M H2SO4 and 5 M NaOH, the medium was stirred and aerated at a relative gas content of 0.03.

[0094] When constant conditions were reached, the 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. The viscosity as a measure for the product formation 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.

[0095] It is shown in FIG. 3 that best results were achieved at a pH from 4 to 6 with an optimum of 5 (dynamic viscosity of the fermentation broth after 144 h of cultivation as a measure of beta glucan yield).

[0096] The improvement of the pH set to 5.0 is shown in FIG. 3 by the dynamic viscosity of the fermentation broth after 144 h of cultivation as a measure of product formation.

EXAMPLE 4

Influence of Different C-Sources on Beta Glucan Yield

[0097] 2 kg of OM medium and different C-sources as given in FIG. 4 to equal 50 g total sugar /kg were autoclaved at 121 C. for 20 minutes in a Labfors 5 BioEtOH-reactor (Infors AG, Bottmingen, Switzerland) equipped with one Rushton turbine and one pitched blade impeller. After autoclaving, for the rest of the experiment the temperature was controlled to 26 C., the pH was adjusted to 4.750.25 using NaOH and H2SO4 and controlled to 4.750.25 using 5 M H2SO4 and 5 M NaOH, the medium was stirred and aerated at a relative gas content of 0.03.

[0098] When constant conditions were reached, the 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. The viscosity as a measure for the product formation 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.

[0099] It is shown in FIG. 4 that best results were achieved using sucrose as C source (dynamic viscosity of the fermentation broth after 144 h of cultivation as a measure of beta glucan yield).

EXAMPLE 5

Repeated Batch Production

[0100] 2 kg of OM medium and 50 g/kg sucrose (Sdzucker AG) were autoclaved at 121 C. for 20 minutes in a Labfors 5 BioEtOH-reactor (Infors AG, Bottmingen, Switzerland) equipped with one Rushton turbine and one pitched blade impeller. After autoclaving, for the rest of the experiment the temperature was controlled to 26 C., the pH was adjusted to 4.750.25 using NaOH and H2SO4 and controlled to 4.750.25 using 5 M H2SO4 and 5 M NaOH, the medium was stirred and aerated at a relative gas content of 0.03.

[0101] When constant conditions were reached, the medium was inoculated to a concentration of 0.019 g/kg CDW of Aureobasidium pullulans. The organism was cultivated at the conditions mentioned above. When the pH increases above 5, 75% of the medium were harvested and the fermenter was refilled with fresh OM medium (composition as defined above). In the first nine cycles an additional growth phase was included by adding 5% of the working volume of a growth medium (30 g/kg sucrose (Sdzucker AG) and 20 g/kg yeast extract (Lallemand, Montreal, Canada) for 2 days. In batches 10 to 12 no growth phase was included. This cycle of harvesting and refilling was repeated for several times, as shown in FIG. 5. The viscosity of samples as a measure for the product formation 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.and a temperature of 20 C.

EXAMPLE 6

Influence of Relative Gas Content

[0102] 0.8 L of OM medium and 50 g/kg sucrose (Sdzucker AG) were autoclaved at 121 C. for 20 minutes in a DasGip-reactor (DasGip Bioblock stirrer vessel, Eppendorf AG, Hamburg, Germany) equipped with two Rushton turbines, or 2 L of OM medium and 50 g/kg sucrose were autoclaved at 121 C. for 20 minutes in a Labfors 5 BioEtOH-reactor (Infors AG, Bottmingen, Switzerland) equipped with one Rushton turbine and one pitched blade impeller and one pitched blade impeller, or 100 kg of OM medium and 50 g/kg sucrose (Sdzucker AG) were autoclaved at 121 C. for 20 minutes in a Techfors 150-reactor (Infors AG, Bottmingen, Switzerland), or 25 m.sup.3 of OM medium and 50 g/kg sucrose (Sdzucker AG) were sterilized by ultra-high temperature treatment (150 C., 10 minutes) and transferred into a 30 m.sup.3 stirred tank reactor. For the rest of the experiment the temperature was controlled to 26 C., the pH was adjusted to 4.750.25 using NaOH and H2SO4 and controlled to 4.750.25 using 5 M H2SO4 and 5 M NaOH, the medium was stirred at specific power inputs ranging from 100 to 12.000 W/m.sup.3 and aerated with 0.5 to 1.5 volumes of air per volume of medium and per minute.

[0103] When constant conditions were reached, the medium was inoculated to a concentration of 0.019 g/kg CDW of Aureobasidium pullulans. The organism was cultivated at the conditions mentioned above.

EXAMPLE 7

Influence of Starvation Phase

[0104] 2 kg of OM medium (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)) were autoclaved at 121 C. for 20 minutes in a Labfors 5 BioEtOH-reactor (Infors AG, Bottmingen, Switzerland) equipped with one Rushton turbine and one pitched blade impeller. After autoclaving, for the rest of the experiment the temperature was controlled to 26 C., the pH was adjusted and controlled to 4.75 using 5 M H2SO4 and 5 M NaOH, the medium was stirred and aerated at a relative gas content of 0.017.

[0105] When constant conditions were reached, the 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. After 0 h, 2 h and 4 h, respectively, a sterile sucrose solution was added to the medium in a single shot to reach a sucrose concentration of 50 g/kg. The viscosity as a measure for the product formation 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.

[0106] It is shown in FIG. 8 that best results were achieved implementing a starvation phase of 4 hours (dynamic viscosity of the fermentation broth after 144 h of cultivation as a measure of beta glucan yield).