Use of a defoamer for maintaining dispersed morphology in submerged fungal fermentation

Abstract

The present invention relates to the use of a defoaming agent for preventing pellet morphology of thermophilic fungi when grown at acidic pH in chemically defined media. The invention pertains to processes for producing a fermentation product, wherein the thermophilic fungus, e.g. a Rhizomucor species, is grown in submerged culture at acidic pH in a chemically defined medium and wherein the strain is cultured in the presence of a defoaming agent. The defoaming agent can be a vegetable oil such as olive or sun flower oil and the fermentation product can be single cell protein in the form of biomass of the thermophilic fungus for use as a dietary source of protein.

Claims

1. A process for producing a fermentation product, wherein the process comprises the steps of: a) growing a strain of a thermophilic fungus in submerged culture in a chemically defined medium, at a pH of less than 5.0, whereby the strain converts nutrients in the medium to the fermentation product and wherein the strain is cultured in the presence of a defoaming agent, preferably a food-grade defoaming agent; and, b) optionally, recovery of the fermentation product produced in step a).

2. The process according to claim 1, wherein the defoaming agent is at least one of an oil-based defoamer, a polyalkylene glycol-based defoamer and a silicon-based defoamer.

3. The process according to claim 1, wherein the defoaming agent comprises a vegetable oil, preferably an edible vegetable oil, wherein more preferably, the vegetable oil is selected from the group consisting of canola (rapeseed) oil, coconut oil, corn oil, cottonseed oil, olive oil, palm oil, palm kernel oil, linseed oil, peanut oil, safflower oil, soya bean oil, sunflower oil and high-oleic sunflower oil, of which olive oil or high-oleic sunflower oil are preferred.

4. The process according to claim 1, wherein the defoaming agent is present and maintained in the chemically defined medium at a concentration of at least 25 ppm (w/v), wherein preferably the defoaming agent is fed continuously or intermittently to the chemically defined medium.

5. The process according to claim 1, wherein the fungal strain is a strain of a fungal genus selected from the group consisting of Rasamsonia, Talaromyces, Penicillium, Acremonium, Humicola, Paecilomyces, Chaetomium, Rhizomucor, Rhizopus, Thermomyces, Myceliophthora, Thermoascus, Thielavia, Mucor, Stibella, Melanocarpus, Malbranchea, Dactylomyces, Canariomyces, Scytalidium, Myriococcum, Corynascus, and Coonemeria, of which the genus Rhizomucor is preferred.

6. The process according to claim 7, wherein the fungal strain is of the species Rhizomucor pusillus, preferably the strain is Rhizomucor pusillus strain CBS 143028, or a strain that is a single colony isolate and/or a derivative of strain CBS 143028.

7. The process according to claim 1, wherein the carbon source in the chemically defined medium consist of at least one of a hydrophilic carbon source and the defoaming agent, wherein preferably the hydrophilic carbon source consists of carbohydrate or an organic acid, and wherein more preferably the defoaming agent is a vegetable oil.

8. The process according to claim 1, wherein the chemically defined medium consists of a carbon source consisting of at least one of carbohydrate and organic acid; a nitrogen source consisting of at least one of urea, ammonia, nitrate, ammonium salts; and minerals, wherein preferably no vitamins are added to the medium.

9. The process according to claim 1, wherein step a) of the process, is carried out as is a fed-batch process, a repeated fed-batch process or a continuous process, preferably a carbon-limited process.

10. The process according to claim 1, wherein the fermentation product is single cell protein in the form of biomass of thermophilic fungus, wherein the process optionally comprises a step b) of recovery of SCP from the medium in the form of biomass of the thermophilic fungus grown in step a).

11. The process according to claim 10, wherein the biomass is recovered from the medium by at least one of sieving, filtration and decantation, whereby preferably the dry matter concentration of the sieved, filtered or decanted biomass (cake) is at least 12% (w/v), and wherein more preferably, the biomass cake is further dried by pressing residual water out.

12. The process according to claim 11, wherein the biomass cake is milled and further dried to a biomass powder by warm air, by freeze drying, preferably under vacuum, or by flash drying, preferably to a water content of no more than 5% (w/w).

13. An SCP product comprising biomass of a thermophilic fungal strain as defined in claim 6, wherein at least one of: a) the biomass is obtained by a process comprising the steps of: i) growing a strain of a thermophilic fungus in submerged culture in a chemically defined medium, at a pH of less than 5.0, whereby the straining converts nutrients in the medium to the fermentation product and wherein the strain is cultured in the presence of a defoaming agent, preferably a food-grade defoaming agent; and ii) optionally, recovery of the fermentation product produced in step a); and b) the biomass comprises at least 1 ppm of a food-grade defoaming agent, and wherein preferably, the SCP product comprises biomass cake with dry matter concentration of at least 12% (w/v), or a biomass powder with a water content of no more than 5% (w/w/).

14. A food or beverage product, a pet food product or animal feed comprising an SCP product as defined in claim 13.

15. (canceled)

Description

DESCRIPTION OF THE FIGURES

[0079] FIG. 1

[0080] Difference in growth morphology of Rhizomucor pusillus. 25 ml of culture broth in displayed in plates after 19 hours of growth in 200 ml media in shake flasks at 46° C., in an orbital shaker at 200 rpm. From left to right with increasing amount of olive oil as indicated: 0, 25, 50, 100, 143 ppm. Upper and lower rows represent duplicates.

EXAMPLES

Example 1

[0081] Growing Rhizomucor pusillus at Low pH with Varying Carbon Sources

[0082] For preculture of Rhizomucor pusillus strain CBS 143028, the strain is inoculated in 35 ml of a preculture medium pH 5.5 containing the defined mineral medium containing KCl 0.17 gr/L, KH.sub.2PO.sub.4 1.3, Na.sub.2HPO.sub.4 0.4, Citric acid 0.5 gr/L, MgSO.sub.4.7 aq 0.7 gr/L, FeSO.sub.4.7 aq 0.03 gr/L, CaCl.sub.2.2 aq 0,035 gr/L, ZnSO.sub.4.7 aq 0.04 gr/L, MnCl.sub.2.4 aq 0,004, CuSO.sub.4.5 aq 0,0005 gr/L, CoCl.sub.2.6 aq 0,0005 gr/L, Na2B.sub.4O.sub.7.10 aq 0,003 gr/L, KI 0,0003 gr/L, Na.sub.2MoO.sub.4.2 aq 0,0005 gr/L, 11 g Dextrose per l; 4 g (NI-14).sub.2SO.sub.4 per l; and 7.5 g tartaric acid per l. The culture is incubated for 24 hours at 46° C., in a 250 ml Erlenmeyer flask with air permeable stop with baffles, in an orbital shaker at 200 rpm. Under these conditions (pH 5.5, pure dextrose as carbon source) the strain grew well with a dispersed morphology.

[0083] Next 1 ml of the above preculture was used to inoculate 35 ml of a pH 3.5 medium containing a complex carbon source: 400 g potato liquefact per l (peeled potatoes were mashed at 7.5% dry matter and liquefied using Veretase™ of BASF at 95 C, 60 minutes, pH 4.5). The medium further contained 4 g (NH.sub.4).sub.2SO.sub.4 per l; 7.5 g tartaric acid per l, per l, 1.6 g (NH.sub.4).sub.2PO.sub.4 per l, ZnSO.sub.4.7 aq 0.05 gr/L. The incubation is continued for 24 hours at 46° C. in a 250 ml baffled Erlenmeyer flask with air permeable stop. Under these conditions (pH 3.5, complex carbon source) the strain grew well with a dispersed morphology.

[0084] In parallel, 1 ml of the above preculture was used to inoculate 35 ml of pH 3.5 medium containing: KCl 0.5 gr/L; KH.sub.2PO.sub.4 4, Na.sub.2HPO.sub.4 1.1, Citric acid 1.5 gr/L, MgSO.sub.4.7 aq 2 gr/L, FeSO.sub.4.7 aq 0.1 gr/L, CaCl.sub.2.2 aq 0.1 gr/L, ZnSO.sub.4.7 aq 0,125 gr/L, MnCl.sub.2.4 aq 0,012, CuSO.sub.4.5 aq 0,00016 gr/L, CoCl.sub.2.6 aq 0,00015 gr/L, Na2B.sub.4O.sub.7.10 aq 0,0009 gr/L KI 0,00009 gr/L, Na.sub.2MoO.sub.4.2 aq 0,00015 gr/L 22 g Dextrose per l; 4 g (NH.sub.4).sub.2SO.sub.4 per l; 7.5 g tartaric acid per l. The incubation is continued for 24 hours at 46° C. in a 250 ml baffled Erlenmeyer flask with air permeable stop. Under these conditions (pH 3.5, pure dextrose as carbon source) the strain grew with a pellet morphology and growth appeared to have slowed down.

[0085] Thus, we observed that while the Rhizomucor fungus grows well in a dispersed morphology on a complex carbon source at low pH (i.e. pH 3.5) and on a chemically defined medium (with dextrose as C-source) at higher pH (i.e. pH 5.5), the combination of a low pH and a chemically defined medium with dextrose as C-source reduced growth of the fungus, which was observed to no longer grow in a dispersed morphology but to form pellets. We next set out to address this issue.

[0086] Rhizomucor pusillus strain CBS 143028 is inoculated in 200 ml of a preculture medium at pH 5.5 containing a defined mineral composition containing KCl 0.5 gr/L; KH.sub.2PO.sub.4 4, Na.sub.2HPO.sub.4 1.1, Citric acid 1.5 gr/L, MgSO.sub.4.7 aq 2 gr/L, FeSO.sub.4.7 aq 0.1 gr/L, CaCl.sub.2.2 aq 0.1 gr/L, ZnSO.sub.4.7 aq 0.125 gr/L, MnCl.sub.2.4 aq 0.012, CuSO.sub.4.5 aq 0.0016 gr/L, CoCl.sub.2.6 aq 0.0015 gr/L, Na2B.sub.4O.sub.7.10 aq 0.009 gr/L KI 0.0009 gr/L, Na.sub.2MoO.sub.4.2 aq 0.0015 gr/L; 11 g Dextrose per l as C-source; 4 g (NH.sub.4).sub.2SO.sub.4 per l as N-source; and 7.5 g tartaric acid per l. The culture is incubated for 24 hours at 46° C., in a 1 l Erlenmeyer flask with air permeable stop with baffles, in an orbital shaker at 200 rpm.

[0087] 1 ml of the preculture is then used to inoculate 35 ml of pH 3.5 medium containing a defined mineral medium as described above comprising 22 g Dextrose per l as C-source; 4 g (NH.sub.4).sub.2SO.sub.4 per l as N-source; 7.5 g tartaric acid per l; and 1.2 g sunflower oil per l.

[0088] The incubation is continued for 96 hours in a 250 ml baffled Erlenmeyer flask with air permeable stop. At the end of fermentation the packed mycelial volume (PMVV) is determined by centrifugation (15 min×g) and growth morphology is judged by eye. Morphology was ranked 1 on a 1 to 5 scale for pellet formation (1 being dispersed and 5 being only round pellets). When sunflower oil was added to the medium the fungus grew well with a dispersed morphology. This can also explain why the fungus grows well and in a dispersed morphology when using a potato liquefact as carbon source because potato is known to contain a small amount (±100 ppm of lipids).

Example 2

[0089] Growing Rhizomucor pusillus with or without Oil

[0090] Rhizomucor pusillus strain CBS 143028 is inoculated in 200 ml of a preculture medium at pH 5.5 containing a defined mineral medium as described above comprising 22 g Dextrose per l as C-source; 4 g (NH.sub.4).sub.2SO.sub.4 per l as N-source; and 7.5 g tartaric acid per l. The culture is incubated for 24 hours at 46° C., in a 1 l Erlenmeyer flask with air permeable stop with baffles, in an orbital shaker at 200 rpm.

[0091] 1 ml of the preculture is then used to inoculate 35 ml of pH 3.5 medium containing a defined mineral medium as described above comprising 22 g Dextrose per l as C-source; 4 g (NH.sub.4).sub.2SO.sub.4 per l as N-source; 7.5 g tartaric acid per l; and with or without 0.14 g olive oil per l.

[0092] The incubation is continued for 24 hours in a 250 ml baffled Erlenmeyer flask with air permeable stop. At the end of fermentation the glucose concentration is determined by a biochemistry analyser and growth morphology is judged by eye. The addition of olive oil resulted in a 2 times faster consumption of glucose and the morphology was observed to be dispersed in contrast to the pellets in the negative control.

Example 3

[0093] Growing Rhizomucor pusillus with Varying Antifoaming Agents

[0094] Rhizomucor pusillus strain CBS 143028 is inoculated in 200 ml of a preculture medium at pH 5.5 as described in Example 2. The culture is incubated for 24 hours at 46° C., in a 1 l Erlenmeyer flask with air permeable stop with baffles, in an orbital shaker at 200 rpm.

[0095] 1 ml of the preculture is then used to inoculate 35 ml of pH 3.5 medium as described in Example 2, with or without either 0.14 g olive oil per l, 0.14 g polypropylene glycol (PPG) per l or 0.14 g Antifoam C100K per l (KCC Basildon Chem. Comp. Ltd, Abingdon, Oxford, UK).

[0096] The incubation is continued for 19 hours in a 250 ml baffled Erlenmeyer flask. At the end of fermentation the glucose concentration is determined by a biochemistry analyser, pH was determined and growth morphology is judged by eye. The results are summarized in Table 1.

TABLE-US-00001 TABLE 1 Residual glucose and pH after 19 hours incubation of Rhizomucor pusillus. Incubation conditions; 35 ml, 200 rpm, 46° C., start pH 3.5. Error bars are the result of duplicates. defoamer glucose (g/l) PH None 18.1 ± 2.2  3.4 ± 0.1 Olive oil 5.9 ± 4.2 2.9 ± 0.1 C100K 6.5 ± 2.2 2.9 ± 0.1 PPG 6.9 ± 1.8 2.9 ± 0.1

[0097] The addition of antifoaming agent PPG resulted in a 6.9 times higher consumption of glucose, and C100K resulted in a 6.7 times higher consumption of glucose. With both defoaming agents the morphology was observed to be dispersed contrary to the pellets in the negative control without defoaming agent.

Example 4

[0098] Growing a Thermophilic Fungus at Different Concentrations of Antifoaming Agent

[0099] The above experiments showed that the addition of 143 ppm olive or sunflower oil had a positive effect on the dispersed growth morphology of Rhizomucor pusillus at low pH. We next set out to determine whether this effect is also seen at lower concentrations of the antifoaming agent.

[0100] A concentration range from 143 till 25 pmm olive oil was tested in shake flasks under conditions essentially as described in Examples 2 and 3, except that the fungus was grown in 200 ml medium. As olive oil cannot be diluted in aqueous media the lower limit in this experiment is 25 ppm. The flasks were filled with standard media and brought to pH 3.5, sterilized and inoculated with 3% preculture (same media pH 5.5). After 19 hours the different flasks were judged on morphology, glucose concentration and pH were measured. The glucose concentration (starting at 20 g/l) and pH are indicators for growth, as Rhizomucor pusillus consumes the buffer and thereby lowering the pH. Experimental results presented in Table 1 are the product of duplicates.

TABLE-US-00002 TABLE 2 Residual glucose and pH after 19 hours incubation of Rhizomucor pusillus. Incubation conditions; 200 ml, 200 rpm, 46° C., start pH 3.5. Error bars are the result of duplicates. Olive oil ppm glucose (g/l) PH  0  18 ± 0.6 3.5 ± 0.1  25 11.05 ± 0.2  3.1 ± 0.0  50 10.45 ± 0.4  3.1 ± 0.0 100 12.45 ± 0.5  3.2 ± 0.0 143 13.15 ± 1.2  3.2 ± 0.0

[0101] We conclude from Table 2 that the growth of the negative control was more than 4 times slower than ones with the addition of olive oil, even at the lowest concentration tested in this experiment. As 25 ppm was the lowest concentration of olive oil that could reliably be diluted at this scale, we expect that even lower concentration of olive oil, e.g. 10, 5 or even 1 ppm still produce the advantageous effects. The measurements in Table 2 are consistent with the observations by eye as shown in FIG. 1.

Example 5

[0102] Growing a Thermophilic Fungus in a Fermenter with Olive Oil as Anti-Foaming Agent

[0103] For preculture Rhizomucor pusillus strain CBS 143028 was inoculated in 200 ml of a defined mineral medium at pH 5.5 containing KCl 0.17 g/L, KH.sub.2PO.sub.4 1.3 g/L, Na.sub.2HPO.sub.4 0.4 g/L, citric acid 0.5 gr/L, MgSO.sub.4.7 aq 0.7 gr/L, FeSO.sub.4.7 aq 0.03 gr/L, CaCl.sub.2.2 aq 0.035 gr/L, ZnSO.sub.4.7 aq 0.04 gr/L, MnCl.sub.2.4 aq 0.004, CuSO.sub.4.5 aq 0.0005 gr/L, KI 0.0003 gr/L, 22 g Dextrose per L; 4 g (NH.sub.4).sub.2SO.sub.4 per L; and 7.5 g tartaric acid per L. The preculture was incubated for 24 hours at 46° C., in a 1 L Erlenmeyer flask with air permeable stop with baffles, in an orbital shaker at 200 rpm. The preculture was then used to inoculate a fermenter with 11 L working volume containing the defined mineral medium as described above at a pH of 3.5 and comprising 77 g Dextrose per L as C-source; 1.4 g (NH.sub.4).sub.2SO.sub.4 per L as N-source and supplemented with NH.sub.3 as titrant. The fungus was grown in the fermenter in batch mode for 24 hours. Olive oil was continuously being fed to maintain a concentration of 50 ppm. At the end of the batch, the growth of the fungus was still dispersed and no pellets were formed.

Example 6

[0104] Growing a Thermophilic Fungus in a Fermenter with Anti-Foaming Agent Struktol SB 420

[0105] For preculture Rhizomucor pusillus strain CBS 143028 was inoculated in 200 ml of a defined mineral medium at pH 5.5 containing KCl 0.17 gr/L, KH.sub.2PO.sub.4 1.3 gr/L, Na.sub.2HPO.sub.4 0.4 gr/L, citric acid 0.5 gr/L, MgSO.sub.4.7 aq 0.7 gr/L, FeSO.sub.4.7 aq 0.03 gr/L, CaCl.sub.2.2 aq 0.035 gr/L, ZnSO.sub.4.7 aq 0.04 gr/L, MnCl.sub.2.4 aq 0.004, CuSO.sub.4.5 aq 0.0005 gr/L, KI 0.0003 gr/L, 22 g Dextrose per L; 4 g (NH.sub.4).sub.2SO.sub.4 per L; and 7.5 g tartaric acid per L. The preculture was incubated for 24 hours at 46° C., in a 1 L Erlenmeyer flask with air permeable stop with baffles, in an orbital shaker at 200 rpm. The preculture was then used to inoculate a fermenter with 8 L working volume containing mineral medium at a pH of 3.5 containing KH.sub.2PO.sub.4 2.7 gr/L, MgSO.sub.4.7 aq 1.3 gr/L, CaCl.sub.2.2 aq 0.067 gr/L, KCl 0.33 gr/L, Na.sub.2HPO.sub.4 0.73 gr/L, FeSO.sub.4.7 aq 0.07 gr/L, ZnSO.sub.4.7 aq 0.08 gr/L, MnCl.sub.2.4 aq 0.008, KI 0.0006 gr/L and comprising 5.5 g Dextrose per L as C-source; 0.8 g Urea per L as N-source. The fungus was grown in the fermenter in batch mode for 18 hours. Struktol SB 420 (www.struktol.de) was added to a final concentration of 0.2 gr/L. At the end of the batch, the growth of the fungus was still dispersed and no pellets were formed.