WO2019174750A1 - Process for recovering one or more gibberellins from an aqueous medium - Google Patents
Process for recovering one or more gibberellins from an aqueous medium Download PDFInfo
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- WO2019174750A1 WO2019174750A1 PCT/EP2018/056715 EP2018056715W WO2019174750A1 WO 2019174750 A1 WO2019174750 A1 WO 2019174750A1 EP 2018056715 W EP2018056715 W EP 2018056715W WO 2019174750 A1 WO2019174750 A1 WO 2019174750A1
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- fermentation broth
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P27/00—Preparation of compounds containing a gibbane ring system, e.g. gibberellin
Definitions
- the invention relates to a process for recovering one or more gibberellins from an aqueous medium. Furthermore, the invention relates to a process for producing gibberellin 7 (GA7). Also, the invention relates to a process for purifying or enriching gibberellin 7 (GA7) which is contained in a product comprising GA7 and gibberellin 4 (GA4).
- Gibberellins are plant hormones that regulate a wide variety of processes within a plant.
- gibberellins are known.
- Gibberellin 3 (GA3), gibberellin 4 (GA4) and gibberellin 7 (GA7) are examples of well-known gibberellins.
- Gibberellins are commercially produced by fermentation of a natural fungus, Gibberella fugikuroi.
- gibberellins often results in mixtures containing different gibberrellins. For instance, fermentation of Gibberella fugikoroi is known to result in mixtures comprising GA3, GA4 and GA7. As different gibberrellins have different properties and preferred applications, it is often desired to obtain a product containing a particular gibberellin in a high amount relative to other gibberellins. To this end various production and purification processes have been proposed in the art.
- CN107177659 describes a process aiming to obtain a product having high level of GA7 relative to GA4.
- a particular strain, fermentation broth method for analyzing the gibberillins present in the fermentation broth (by extracting the broth with methanol and subsequent analysis by HPLC), it does not teach how to recover gibberellins from the fermentation broth in an efficient manner, let alone how to recover a product from the fermentation broth which is rich a particular gibberellin.
- CN107177659 aims to obtain a relatively high level of GA7 relative to GA4, it is silent regarding the levels of GA3 obtained.
- GA7 gibberellin 7
- the invention provides a process for recovering one or more gibberellins from an aqueous medium, the process comprising:
- the process enables recovery of gibberellin from an aqueous medium in an efficient manner. Furthermore, gibberellins may be separated based on their solubility in the oil. More in particular, the process enables separation of gibberellin 4 (GA4) and/or gibberellin 7 (GA7) from gibberellin 3 (GA3). Such separation has the advantage that any subsequent purification or enrichment steps may be avoided or carried out to a lesser extent.
- the invention provides a process for producing gibberellin 7 (GA7), said process comprising:
- the conditions conducive to produce GA7 from said fermentation broth include the use of 1 wt% oil in the fermentation medium, or more.
- Preferred amounts are 2-10 wt%, preferably 2-5 wt%
- said oil is or comprises cotton seed oil
- the total amount of oil added to the fermentation broth is at least 80 g/l, preferably at least 120 g/l, more preferably at least 150 g/l; and/or (iv) said fermentation broth comprises an aqueous phase and an oil phase, said oil phase comprising said oil; and (b) comprises recovering gibberellin 7 from said oil phase.
- the process according to the second aspect and in particular the use of an oil as defined therein, enables to obtain GA7 in a product having an increased the GA7/GA4 ratio.
- the GA7/GA4 ratio refers to the ratio of the amounts (expressed in weight) of GA7 to GA4 present in the product.
- An increased GA7/GA4 ratio has the advantage that any subsequent purification or enrichment steps may be avoided or carried out to a lesser extent.
- the invention provides a process for purifying or enriching gibberellin 7 (GA7) which is contained in a product comprising GA7 and gibberellin 4 (GA4), said process comprising dissolving said product in a solvent, and inducing crystallization of GA7 from said solvent.
- GA7 gibberellin 7
- GA4 gibberellin 4
- the processes according to the first, second and/or third aspect of the invention are combined.
- the process according to the first aspect of the invention may, for instance, advantageously be used to recover GA7 produced in accordance with the process according to the second aspect.
- the process according to the third aspect of the invention may, for instance, be used to purify or enrich GA7 produced or recovered by the process according to the first and/or second aspect of the invention.
- FIGURE 1 shows a flow chart of an exemplary embodiment of the process according to the first aspect of the invention.
- First aspect recovering one or more gibberellins from an aqueous medium
- a process for recovering one or more gibberellins from an aqueous medium comprising:
- the invention is not limited to the recovery of any particular gibberellin.
- the one or more gibberellins are selected from the group consisting of gibberellin 4 (GA4), gibberellin 7 (GA7) and combinations thereof. More preferably, the one or more gibberellins are selected from the group consisting of GA4, GA7 and combinations thereof, most preferably GA7. It has been found GA4 and GA7 are preferentially extracted into the oil or oil phase. This enables at least partial separation of gibberellins which preferentially remain in the aqueous phase, in particular GA3. Of course, it is possible that the one or more gibberellins are recovered together with further gibberellins.
- the aqueous phase may be any aqueous phase which comprises one or more gibberellins to be recovered.
- the aqueous phase may, for instance, comprise one or more gibberellins selected from the group consisting of gibberellin 4 (GA4), gibberellin 7 (GA7) and combinations thereof, more preferably selected from the group consisting of GA4, GA7 and combinations thereof, most preferably GA7.
- the aqueous medium further comprises GA3.
- the aqueous medium comprises i) GA4 and/or GA7 and (ii) gibberellin 3 (GA3).
- GA3 gibberellin 3
- the aqueous medium is a fermentation broth, has been derived from a fermentation broth, or is comprised in a fermentation broth.
- the aqueous medium and/or fermentation broth may comprise microorganisms which have produced the one or more gibberellins.
- the microorganisms are Gibberella fujikuroi microorganisms.
- the oil may be any suitable oil into which the one or more gibberellins may be extracted.
- the oil is or comprises a natural oil.
- the oil may for instance be a vegetable oil, a marine oil, a microbial oil or a mixture thereof.
- the oil is or comprises a vegetable oil.
- the vegetable oil is selected from the group consisting of palm oil, soybean oil, rapeseed oil, sunflower oil, peanut oil, cottonseed oil, palm kernel oil, coconut oil, olive oil, com oil, linseed oil, rice bran oil, safflower oil, sesame oil and mixtures thereof.
- the oil is or comprises cottonseed oil, palm oil, soybean oil, rapeseed oil or
- the oil is or comprises cottonseed oil.
- the oil comprises a certain type of oil, this generally means that more than 50 wt% of said oil is the specified oil, preferably more than 70% and even more preferably more than 90 wt%.
- the oil is an oil comprising esters of fatty acids, for instance glycerides
- the oil comprises triglycerides.
- the oil is or comprises cottonseed oil.
- the oil comprises C8 to C18 unsaturated fatty acids.
- Contacting the aqueous medium with the oil may be effected in any suitable manner.
- said contacting may comprise mixing the aqueous medium with the oil.
- the aqueous medium and the oil are comprised in a fermentation broth.
- the oil has been acted or acts as a carbon source for the microorganisms present in the fermentation broth.
- the aqueous phase and the oil phase can be separated in any suitable manner.
- separating the aqueous phase and the oil phase comprises filtration or gravity separation, more preferably centrifugation. Centrifuges known to the skilled person may be used.
- the one or more gibberellins may recovered from the oil phase in any suitable manner.
- recovering the one or more gibberellins from the oil phase comprises contacting the oil phase with a solvent. This may result in precipitation of the one or more gibberellins.
- the precipitated gibberellins may be recovered.
- Any suitable solid-liquid separation technique may be used, for instance filtration or gravity separation (e.g. centrifugation) said recovering said one or more gibberellins from said oil phase comprises contacting said oil phase with a solvent in which the oil is soluble and/or miscible, resulting in precipitation of said one or more gibberellins.
- the solvent may be any suitable solvent. Suitable solvents include solvents in which the oil is soluble or miscible and which result in precipitation of the one or more gibberellins.
- the solvent is a hydrocarbon solvent, Preferably, said hydrocarbon solvent is or comprises a C5-C12 alkane, more preferably a C5-C8 alkane, most preferably hexane or heptane.
- the process according to the invention comprises recovering GA3 from the aqueous phase.
- GA3 is recovered from the aqueous phase by solvent extraction. Any water immiscible solvent in which GA3 is soluble may be used.
- an extraction solvent is used which is or comprises ethyl acetate.
- GA3 may be recovered from the extraction solvent using known techniques, for instance by evaporation of the solvent, preferably by crystallization. GA3 may also be recovered by concentration of the aqueous medium, resulting in precipitation of GA3, and recovering the precipitated GA3 by filtration or centrifugation. GA3 may also be recovered by emulsion liquid membrane technology (J. Membrane Sci (2010) 348:91-98).
- the aqueous medium is preferably a fermentation broth, has been derived from a fermentation broth, or is comprised in a fermentation broth.
- the invention further provides a process for producing one or more gibberellins, the process comprising culturing microorganisms in a fermentation broth
- the fermentation broth comprises a carbon source which comprises said oil.
- the culturing results in an aqueous phase and an oil phase, and the process further comprises recovering the one or more gibberellins from the oil phase.
- a process for producing gibberellin 7 comprising: (a) culturing a microorganism capable of producing GA7 in a fermentation broth under conditions conducive to produce GA7, said fermentation broth comprising a carbon source which comprises an oil; and
- the fermentation broth comprises a carbon source which comprises an oil.
- the oil is or comprises a natural oil.
- the oil may for instance be a vegetable oil, a marine oil, a microbial oil or a mixture thereof.
- the oil is or comprises a vegetable oil.
- the vegetable oil is selected from the group consisting of palm oil, soybean oil, rapeseed oil, sunflower oil, peanut oil, cottonseed oil, palm kernel oil, coconut oil, olive oil, corn oil, grape seed oil, linseed oil, rice bran oil, safflower oil, sesame oil and mixtures thereof.
- the oil is or comprises cottonseed oil, palm oil, soybean oil, rapeseed oil or combinations thereof. Most preferably, the oil is or comprises cottonseed oil. It was found that these oils are particular preferred to obtain an increased GA7/GA4 ratio.
- the oil is an oil comprising esters of fatty acids, for instance glycerides
- the oil comprises triglycerides.
- the oil comprises C8 to C18 unsaturated fatty acids.
- the oil comprises a certain type of oil
- the conditions conducive to produce GA7 from said fermentation broth include the use of 1 wt% oil in the fermentation medium, or more, preferably 2 wt% of oil or more. Significant improvement has been seen when using a few percent of oil in the medium.
- preferred amounts are 2-10 wt%, preferably 2-5 wt% of oil.
- At least 25 wt.%, more preferably at least 50 wt.%, more preferably at least 75 wt.%, more preferably at least 90 wt.%, more preferably 100 wt.%, of the total amount of carbon source added to the fermentation broth is an oil.
- the total amount of oil added to the fermentation broth is at least 80 g/l, more preferably at least 120 g/l, more preferably at least 150 g/l.
- the fermentation broth comprises an aqueous phase and an oil phase, said oil phase comprising said oil; and (b) comprises recovering GA7 from said oil phase.
- the carbon source may comprise further compounds.
- the carbon source may further comprise starch, for instance com starch and/or potato starch, and/or a sugar, for instance lactose.
- said carbon source comprises corn starch.
- the fermentation broth may comprise any suitable nitrogen source. Suitable nitrogen sources are known to the skilled person.
- the fermentation broth may comprise a nitrogen source comprising flour (or meal), peanut meal, corn steep powder, urea, cottonseed flour (or meal), yeast extract or combinations thereof.
- the fermentation broth comprises a nitrogen source which comprises cotton seed flour (or meal) or soy flour (or meal).
- Suitable levels for the dissolved oxygen content are known to the skilled person.
- culturing is effected at a the dissolved oxygen content in the fermentation broth of at least 20%, such as from 20% to 100%.
- the fermentation has a first stage and a subsequent stage, and during said subsequent stage the dissolved oxygen content (D.O.) is higher than during the first stage. This may for instance be achieved the fermentation has a first stage and a subsequent stage, wherein the agitation during the subsequent stage is greater than in the first stage.
- the first and subsequent stage may be a growth and a production stage, the growth stage generally being less than 50% of the time, and the production stage being more than 50% of the total fermentation period.
- the growth time is 20-40% of the total fermentation time, while the production time preferably is 60-80 % of the time.
- the total fermentation time is about 10 days
- the growth time may be 3 days
- the production, subsequent period may be 7 days.
- Suitable temperatures for effecting cultivation are known to the skilled person.
- culturing is effected at a temperature of between 25 °C and 35 °C, preferably wherein during a first stage (preferably having a duration of between 48 to 96 hours) the temperature is maintained between 25 °C and 30 °C, preferably between 27 °C and 29 °C and in a subsequent stage (preferably having a duration of between 120 to 240 hours) the temperature is maintained between 31 °C and 35 °C, preferably between 32 °C and 34 °C.
- Culturing may be effected at values for the pH known to the skilled person.
- culturing is effected at a pH of between 3 and 7.
- Any microorganism capable of producing GA7 may be used.
- the microorganism capable of producing GA7 may be used.
- the microorganism capable of producing GA7 may be used.
- the microorganism capable of producing GA7 may be used.
- a process for purifying or enriching gibberellin 7 (GA7) which is contained in a product comprising GA7 and gibberellin 4 (GA4) said process comprising dissolving said product in a solvent, and inducing crystallization of GA7 from said solvent.
- this process enables to obtain a product having an increased GA7/GA4 ratio.
- the process may comprise dissolving a first product having a first GA7/GA4 ratio, and crystallizing from the solvent a second product having a second GA7/GA4 ratio, wherein the second GA7/GA4 ratio is greater than the first GA7/GA4 ratio, and wherein the GA7/GA4 ratio refers to the ratio of the amounts (expressed in weight) of GA7 to GA4 present in the product.
- any suitable solvent enabling to obtain an GA7/GA4 ratio may be used.
- the solvent is selected from the group consisting of an ether, ketone, ester, and combinations thereof. More preferably, the solvent is selected from the group consisting of methyl t-butyl ether (MTBE), acetone, Methyl isobutyl ketone (MIBK), 3-methyl-2-butanone, cyclohexanone, and ethyl acetate.
- the solvent is or comprises MTBE as this results in a high GA7/GA4 ratio in combination with a high yield.
- Crystallizing may be induced by methods known to the skilled person. Preferably, crystallization is induced by cooling.
- Example 1 involves the use of a production medium comprising 188 g/L of cottonseed oil.
- Comparative experiment A involves the use of a production medium comprising 140 g/L of com starch. Both experiments involve the use of ATCC strain 12616 (a Gibberella fujikuroi strain) as production organism.
- a fermentation was performed using the same medium as described in example 1 (comprising 188 g/L of cotton seed oil). An assessment of the gibberellin content of the various phases of the broth was conducted. The results are presented below.
- GA4 and GA7 can be recovered from the fermentation broth by a process as illustrated in figure 1.
- GA7 is increased relative to GA4 when using 3% of oil.
- the same strain as used in example 3 was used in this experiment.
- carbon source either 20% com starch was used, or 20% corn starch and 3% of proflo oil.
- the N-source was 1.5 wt% yeast powder and l.5wt% cotton seed flour.
- the fermentation time was 10 days.
- the pH was 4.7-4.8. Results are given in table 7.
- MIBK methyl isobutyl ketone
- Crystallization was induced by a controlled cooling ramp from reflux to l0-20°C, over 5-10 hours with slow agitation.
- the table shows that the recrystallized products have a higher GA7/GA4 ratio than the starting product (which had 35% GA7). MTBE not only had the highest GA7/GA4 ratio, but also had the highest percentage of solid recovery.
- the three batches were then recrystallized again from MBTE improving the ratio from GA7/4:
Abstract
The invention relates to a process for recovering one or more gibberellins from an aqueous medium, the process comprising (a) contacting said aqueous medium with an oil to obtain an aqueous phase and an oil phase, resulting in extraction of said one or more gibberellins into the oil phase; and (b) recovering said one or more gibberellins from said oil phase. The invention further relates to a process for producing gibberellin 7 (GA7), said process comprising: (a) culturing a microorganism capable of producing GA7 in a fermentation broth under conditions conducive to produce GA7, said fermentation broth comprising a carbon source which comprises an oil; and (b) recovering GA7 from said fermentation broth.
Description
PROCESS FOR RECOVERING ONE OR MORE GIBBERELLINS FROM AN AQUEOUS MEDIUM
FIELD OF THE INVENTION
[0001] The invention relates to a process for recovering one or more gibberellins from an aqueous medium. Furthermore, the invention relates to a process for producing gibberellin 7 (GA7). Also, the invention relates to a process for purifying or enriching gibberellin 7 (GA7) which is contained in a product comprising GA7 and gibberellin 4 (GA4).
BACKGROUND OF THE INVENTION
[0002] Gibberellins (or gibberellic acids) are plant hormones that regulate a wide variety of processes within a plant. Nowadays, over 120 gibberellins are known. Gibberellin 3 (GA3), gibberellin 4 (GA4) and gibberellin 7 (GA7) are examples of well-known gibberellins.
Gibberellins are commercially produced by fermentation of a natural fungus, Gibberella fugikuroi.
[0003] The production of gibberellins often results in mixtures containing different gibberrellins. For instance, fermentation of Gibberella fugikoroi is known to result in mixtures comprising GA3, GA4 and GA7. As different gibberrellins have different properties and preferred applications, it is often desired to obtain a product containing a particular gibberellin in a high amount relative to other gibberellins. To this end various production and purification processes have been proposed in the art.
[0004] CN107177659 describes a process aiming to obtain a product having high level of GA7 relative to GA4. A particular strain, fermentation broth method for analyzing the gibberillins present in the fermentation broth (by extracting the broth with methanol and subsequent analysis by HPLC), it does not teach how to recover gibberellins from the fermentation broth in an efficient manner, let alone how to recover a product from the fermentation broth which is rich a particular gibberellin. Moreover, whilst CN107177659 aims to obtain a relatively high level of GA7 relative to GA4, it is silent regarding the levels of GA3 obtained.
SUMMARY OF THE INVENTION
[0005] It is an object of the invention to provide a process enabling an efficient recovery of one or more gibberellins from an aqueous medium. It is a further object of the invention to provide a process for the production of GA7 enabling to obtain GA7 in a high amount relative to GA4 and/or GA3. It is a further object of the invention to provide a process for purifying or enriching gibberellin 7 (GA7) which is contained in a product comprising GA7 and gibberellin 4 (GA4).
[0006] In a first aspect the invention provides a process for recovering one or more gibberellins from an aqueous medium, the process comprising:
(a) contacting said aqueous medium with an oil to obtain an aqueous phase and an oil phase resulting in extraction of said one or more gibberellins into the oil phase; and
(b) recovering said one or more gibberellins from said oil phase.
[0007] The process enables recovery of gibberellin from an aqueous medium in an efficient manner. Furthermore, gibberellins may be separated based on their solubility in the oil. More in particular, the process enables separation of gibberellin 4 (GA4) and/or gibberellin 7 (GA7) from gibberellin 3 (GA3). Such separation has the advantage that any subsequent purification or enrichment steps may be avoided or carried out to a lesser extent.
[0008] In a second aspect, the invention provides a process for producing gibberellin 7 (GA7), said process comprising:
(a) culturing a microorganism capable of producing GA7 in a fermentation broth under conditions conducive to produce GA7, said fermentation broth comprising a carbon source which comprises an oil; and
(b) recovering GA7 from said fermentation broth
[0009] In a preferred embodiment, the conditions conducive to produce GA7 from said fermentation broth include the use of 1 wt% oil in the fermentation medium, or more.
[0010] Preferred amounts are 2-10 wt%, preferably 2-5 wt%
[0011] In another preferred embodiment, in the process according to the second aspect:
(i) said oil is or comprises cotton seed oil; and/or
(ii) at least 25 wt.%, preferably at least 50 wt.%, more preferably at least 75 wt.%, more preferably at least 90 wt.%, more preferably 100 wt.%, of the total amount of carbon source added to the fermentation broth is an oil; and/or
(iii) the total amount of oil added to the fermentation broth is at least 80 g/l, preferably at least 120 g/l, more preferably at least 150 g/l; and/or
(iv) said fermentation broth comprises an aqueous phase and an oil phase, said oil phase comprising said oil; and (b) comprises recovering gibberellin 7 from said oil phase.
[0012] It was found that the process according to the second aspect, and in particular the use of an oil as defined therein, enables to obtain GA7 in a product having an increased the GA7/GA4 ratio. As used herein, the GA7/GA4 ratio refers to the ratio of the amounts (expressed in weight) of GA7 to GA4 present in the product. An increased GA7/GA4 ratio has the advantage that any subsequent purification or enrichment steps may be avoided or carried out to a lesser extent.
[0013] In a third aspect, the invention provides a process for purifying or enriching gibberellin 7 (GA7) which is contained in a product comprising GA7 and gibberellin 4 (GA4), said process comprising dissolving said product in a solvent, and inducing crystallization of GA7 from said solvent.
[0014] It was found that the process according to the third aspect of the invention enables to obtain a product having an increased GA7/GA4 ratio.
[0015] Advantageously, the processes according to the first, second and/or third aspect of the invention are combined. The process according to the first aspect of the invention may, for instance, advantageously be used to recover GA7 produced in accordance with the process according to the second aspect. The process according to the third aspect of the invention may, for instance, be used to purify or enrich GA7 produced or recovered by the process according to the first and/or second aspect of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGURE 1 shows a flow chart of an exemplary embodiment of the process according to the first aspect of the invention.
DETAILED DESCRIPTION OF THE INVENTION
First aspect: recovering one or more gibberellins from an aqueous medium
[0017] According to the first aspect of the invention, there is provided a process for recovering one or more gibberellins from an aqueous medium, the process comprising:
(a) contacting said aqueous medium with an oil to obtain an aqueous phase and an oil phase resulting in extraction of said one or more gibberellins into the oil phase; and
(b) recovering said one or more gibberellins from said oil phase.
[0018] The invention is not limited to the recovery of any particular gibberellin. Preferably, the one or more gibberellins are selected from the group consisting of gibberellin 4 (GA4), gibberellin 7 (GA7) and combinations thereof. More preferably, the one or more gibberellins are selected from the group consisting of GA4, GA7 and combinations thereof, most preferably GA7. It has been found GA4 and GA7 are preferentially extracted into the oil or oil phase. This enables at least partial separation of gibberellins which preferentially remain in the aqueous phase, in particular GA3. Of course, it is possible that the one or more gibberellins are recovered together with further gibberellins.
[0019] The aqueous phase may be any aqueous phase which comprises one or more gibberellins to be recovered. Thus, the aqueous phase may, for instance, comprise one or more gibberellins selected from the group consisting of gibberellin 4 (GA4), gibberellin 7 (GA7) and combinations thereof, more preferably selected from the group consisting of GA4, GA7 and combinations thereof, most preferably GA7.
[0020] Preferably, the aqueous medium further comprises GA3. Thus, in a particularly preferred embodiment, the aqueous medium comprises i) GA4 and/or GA7 and (ii) gibberellin 3 (GA3). As discussed, the process according to the invention enables the GA4 and/or GA7 to be preferentially extracted into the oil or oil phase, thereby resulting in an at least partial separation of GA4 and/or GA7 from GA3.
[0021] Preferably, the aqueous medium is a fermentation broth, has been derived from a fermentation broth, or is comprised in a fermentation broth. The aqueous medium and/or fermentation broth may comprise microorganisms which have produced the one or more gibberellins. Preferably, the microorganisms are Gibberella fujikuroi microorganisms.
[0022] The oil may be any suitable oil into which the one or more gibberellins may be extracted. Preferably, the oil is or comprises a natural oil. The oil may for instance be a vegetable oil, a marine oil, a microbial oil or a mixture thereof. Preferably, the oil is or comprises a vegetable oil. Preferably, the vegetable oil is selected from the group consisting of palm oil, soybean oil, rapeseed oil, sunflower oil, peanut oil, cottonseed oil, palm kernel oil, coconut oil, olive oil, com oil, linseed oil, rice bran oil, safflower oil, sesame oil and mixtures thereof.
Preferably the oil is or comprises cottonseed oil, palm oil, soybean oil, rapeseed oil or
combinations thereof. Most preferably, the oil is or comprises cottonseed oil.
[0023] With respect to the term that the oil comprises a certain type of oil, this generally means that more than 50 wt% of said oil is the specified oil, preferably more than 70% and even more preferably more than 90 wt%.
[0024] Preferably, the oil is an oil comprising esters of fatty acids, for instance glycerides
(e.g. triglycerides) or alkyl (e.g. methyl or ethyl) esters of fatty acids. Preferably, the oil comprises triglycerides. Most preferably, the oil is or comprises cottonseed oil.
[0025] Preferably, the oil comprises C8 to C18 unsaturated fatty acids.
[0026] Contacting the aqueous medium with the oil may be effected in any suitable manner. For instance, said contacting may comprise mixing the aqueous medium with the oil. In a preferred embodiment, the aqueous medium and the oil are comprised in a fermentation broth. Preferably, the oil has been acted or acts as a carbon source for the microorganisms present in the fermentation broth.
[0027] As a result of contacting the aqueous medium with the oil an aqueous phase and an oil phase are obtained, and the one or more gibberellins to be recovered will be extracted into the oil phase. The skilled person will understand that it is not necessary that the entire amount of the one or more gibberellins originally present in the aqueous medium are extracted into the oil phase.
[0028] Often the aqueous phase and said oil phase form an emulsion.
[0029] The aqueous phase and the oil phase can be separated in any suitable manner.
Preferably, separating the aqueous phase and the oil phase comprises filtration or gravity separation, more preferably centrifugation. Centrifuges known to the skilled person may be used.
[0030] The one or more gibberellins may recovered from the oil phase in any suitable manner. Preferably, recovering the one or more gibberellins from the oil phase comprises contacting the oil phase with a solvent. This may result in precipitation of the one or more gibberellins. The precipitated gibberellins may be recovered. Any suitable solid-liquid separation technique may be used, for instance filtration or gravity separation (e.g. centrifugation) said recovering said one or more gibberellins from said oil phase comprises contacting said oil phase with a solvent in which the oil is soluble and/or miscible, resulting in precipitation of said one or more gibberellins.
[0031] The solvent may be any suitable solvent. Suitable solvents include solvents in which the oil is soluble or miscible and which result in precipitation of the one or more gibberellins. Preferably, the solvent is a hydrocarbon solvent, Preferably, said hydrocarbon
solvent is or comprises a C5-C12 alkane, more preferably a C5-C8 alkane, most preferably hexane or heptane.
[0032] It is found that, if the aqueous medium contains GA3, for instance in addition to GA4 and/or GA7, a relatively a relatively large part of the GA3 will be present in the aqueous phase that is formed when the aqueous medium is contacted with the oil. Preferably, the process according to the invention comprises recovering GA3 from the aqueous phase. Preferably, GA3 is recovered from the aqueous phase by solvent extraction. Any water immiscible solvent in which GA3 is soluble may be used. Preferably, an extraction solvent is used which is or comprises ethyl acetate.
[0033] GA3 may be recovered from the extraction solvent using known techniques, for instance by evaporation of the solvent, preferably by crystallization. GA3 may also be recovered by concentration of the aqueous medium, resulting in precipitation of GA3, and recovering the precipitated GA3 by filtration or centrifugation. GA3 may also be recovered by emulsion liquid membrane technology (J. Membrane Sci (2010) 348:91-98).
[0034] As discussed, the aqueous medium is preferably a fermentation broth, has been derived from a fermentation broth, or is comprised in a fermentation broth.
[0035] Accordingly, the invention further provides a process for producing one or more gibberellins, the process comprising culturing microorganisms in a fermentation broth
comprising the aqueous medium under conditions such that one or more gibberellins are produced, and recovering said one or more gibberellins from said aqueous medium by the process according to the first aspect of the invention. Preferably, the fermentation broth comprises a carbon source which comprises said oil. Preferably, the culturing results in an aqueous phase and an oil phase, and the process further comprises recovering the one or more gibberellins from the oil phase.
[0036] Preferred embodiments and features described herein apply to this process mutatis mutandis.
Second aspect: process for producing gibber ellin 7 (GA7)
[0037] According to a second aspect of the invention there is provided a process for producing gibberellin 7 (GA7), said process comprising:
(a) culturing a microorganism capable of producing GA7 in a fermentation broth under conditions conducive to produce GA7, said fermentation broth comprising a carbon source which comprises an oil; and
(b) recovering GA7 from said fermentation broth,
[0038] The fermentation broth comprises a carbon source which comprises an oil.
Preferably, the oil is or comprises a natural oil. The oil may for instance be a vegetable oil, a marine oil, a microbial oil or a mixture thereof. Preferably, the oil is or comprises a vegetable oil. Preferably, the vegetable oil is selected from the group consisting of palm oil, soybean oil, rapeseed oil, sunflower oil, peanut oil, cottonseed oil, palm kernel oil, coconut oil, olive oil, corn oil, grape seed oil, linseed oil, rice bran oil, safflower oil, sesame oil and mixtures thereof.
Preferably, the oil is or comprises cottonseed oil, palm oil, soybean oil, rapeseed oil or combinations thereof. Most preferably, the oil is or comprises cottonseed oil. It was found that these oils are particular preferred to obtain an increased GA7/GA4 ratio.
[0039] Preferably, the oil is an oil comprising esters of fatty acids, for instance glycerides
(e.g. triglycerides) or alkyl (e.g. methyl or ethyl) esters of fatty acids. Preferably, the oil comprises triglycerides. Preferably, the oil comprises C8 to C18 unsaturated fatty acids.
[0040] With respect to the term that the oil comprises a certain type of oil, this generally means that more than 50 wt% of said oil is the specified oil, preferably more than 70% and even more preferably more than 90 wt%.
[0041] In a preferred embodiment, the conditions conducive to produce GA7 from said fermentation broth include the use of 1 wt% oil in the fermentation medium, or more, preferably 2 wt% of oil or more. Significant improvement has been seen when using a few percent of oil in the medium.
[0042] In this embodiment, preferred amounts are 2-10 wt%, preferably 2-5 wt% of oil.
With relatively low amounts of oil, in particular with about 5 wt% of oil or lower, a separate oily phase is hardly present.
[0043] In a further embodiment, preferably, at least 25 wt.%, more preferably at least 50 wt.%, more preferably at least 75 wt.%, more preferably at least 90 wt.%, more preferably 100 wt.%, of the total amount of carbon source added to the fermentation broth is an oil.
[0044] Preferably, the total amount of oil added to the fermentation broth is at least 80 g/l, more preferably at least 120 g/l, more preferably at least 150 g/l.
[0045] Preferably, the fermentation broth comprises an aqueous phase and an oil phase, said oil phase comprising said oil; and (b) comprises recovering GA7 from said oil phase.
[0046] It was found that the use of an oil, in particular the preferred oils and the preferred amounts as defined, contribute to increasing the GA7/GA4 ratio of the gibberellins produced and in the product obtained. It will be understood by the skilled person that the features and characteristics may be applied alone and may be combined,
[0047] The carbon source may comprise further compounds. For instance, the carbon source may further comprise starch, for instance com starch and/or potato starch, and/or a sugar, for instance lactose. Process, said carbon source comprises corn starch.
[0048] The fermentation broth may comprise any suitable nitrogen source. Suitable nitrogen sources are known to the skilled person. For instance, the fermentation broth may comprise a nitrogen source comprising flour (or meal), peanut meal, corn steep powder, urea, cottonseed flour (or meal), yeast extract or combinations thereof. Preferably, the fermentation broth comprises a nitrogen source which comprises cotton seed flour (or meal) or soy flour (or meal).
[0049] Suitable levels for the dissolved oxygen content (D.O.) are known to the skilled person. Preferably, culturing is effected at a the dissolved oxygen content in the fermentation broth of at least 20%, such as from 20% to 100%. Preferably, the fermentation has a first stage and a subsequent stage, and during said subsequent stage the dissolved oxygen content (D.O.) is higher than during the first stage. This may for instance be achieved the fermentation has a first stage and a subsequent stage, wherein the agitation during the subsequent stage is greater than in the first stage.
[0050] The first and subsequent stage may be a growth and a production stage, the growth stage generally being less than 50% of the time, and the production stage being more than 50% of the total fermentation period. Preferably, the growth time is 20-40% of the total fermentation time, while the production time preferably is 60-80 % of the time. For example, if the total fermentation time is about 10 days, the growth time may be 3 days, and the production, subsequent period may be 7 days.
[0051] Suitable temperatures for effecting cultivation are known to the skilled person.
Preferably, culturing is effected at a temperature of between 25 °C and 35 °C, preferably wherein during a first stage (preferably having a duration of between 48 to 96 hours) the temperature is
maintained between 25 °C and 30 °C, preferably between 27 °C and 29 °C and in a subsequent stage (preferably having a duration of between 120 to 240 hours) the temperature is maintained between 31 °C and 35 °C, preferably between 32 °C and 34 °C.
[0052] Culturing may be effected at values for the pH known to the skilled person.
Preferably, culturing is effected at a pH of between 3 and 7.
[0053] Any microorganism capable of producing GA7 may be used. Preferably, the
microorganisms Gibberella fujikuroi microorganisms.
Third aspect: process for purifying or enriching gibberellin 7 ( GA7)
[0054] According to a third aspect of the invention, there is provided a process for purifying or enriching gibberellin 7 (GA7) which is contained in a product comprising GA7 and gibberellin 4 (GA4), said process comprising dissolving said product in a solvent, and inducing crystallization of GA7 from said solvent. Surprisingly, this process enables to obtain a product having an increased GA7/GA4 ratio.
[0055] The process may comprise dissolving a first product having a first GA7/GA4 ratio, and crystallizing from the solvent a second product having a second GA7/GA4 ratio, wherein the second GA7/GA4 ratio is greater than the first GA7/GA4 ratio, and wherein the GA7/GA4 ratio refers to the ratio of the amounts (expressed in weight) of GA7 to GA4 present in the product.
[0056] Any suitable solvent enabling to obtain an GA7/GA4 ratio may be used. Preferably, the solvent is selected from the group consisting of an ether, ketone, ester, and combinations thereof. More preferably, the solvent is selected from the group consisting of methyl t-butyl ether (MTBE), acetone, Methyl isobutyl ketone (MIBK), 3-methyl-2-butanone, cyclohexanone, and ethyl acetate. Preferably, the solvent is or comprises MTBE as this results in a high GA7/GA4 ratio in combination with a high yield.
[0057] Crystallizing may be induced by methods known to the skilled person. Preferably, crystallization is induced by cooling.
The invention will now further be illustrated using the following examples, without however being limited thereto.
EXAMPLES
Example 1 and comparative experiment A
[0058] The following media were prepared
CM Plate
Difco Potato Dextrose Agar + 0.5% Bacto Agar, pH 5.5
Autoclave l2l°C and 15 psi for 20 minutes, dispense 20ml per standard petri dish Seed medium:
40 g/L Sucrose
12 g/L Toasted Soy flour (ADM, Nutrisoy, 063-160)
2 g/L Urea (Autoclaved separately, add 0.5 ml of 10% stock per flask)
1,5 g/L Potassium Phosphate, monobasic (Sigma)
0,2 g/L Potassium Sulfate (Wako)
0,2 g/L MgS04 heptahydrate
Adjust pH to 6.8
Dispense 25 ml per 250 ml Erlenmeyer flask, close with 3 layers of milk filter Autoclave 121°C and 15 psi for 20 minutes.
Production Medium
[0059] Example 1 (according to the invention) involves the use of a production medium comprising 188 g/L of cottonseed oil. Comparative experiment A involves the use of a production medium comprising 140 g/L of com starch. Both experiments involve the use of ATCC strain 12616 (a Gibberella fujikuroi strain) as production organism.
[0060] Experimental method: Colonies were grown on a CM plate, and each grown colony was macerated in a test tube (130x13 mm) containing 2 ml H20 and 20 glass beads (diameter = 3.0 mm). 0.5 ml of each macerated colony mixture were inoculated into the seed medium (25 ml/250 ml flask) and incubated at 28 °C at 180 rpm for 46 hours. 1 ml of the resulting medium were added into the production medium (25 ml/250 ml flask), and incubated at 28 °C, 180 rpm for 72 hrs followed by incubation at 33 °C, 220 rpm for 168 hrs. Then, 0.25 ml of the resulting whole broth was strongly mixed for 10 minutes with 4.75 ml of ethanol, followed by centrifugation at 1560 g (2600 rpm) for 10 minutes. The supernatant was analyzed by HPLC.
[0061] The results are shown in the table below
[0062] This shows that the process of example 1 (using the oil as carbon source) results in a higher titer of GA7 as well as in a higher GA7/GA4 ratio.
Example 2
[0063] A fermentation was performed using the same medium as described in example 1 (comprising 188 g/L of cotton seed oil). An assessment of the gibberellin content of the various phases of the broth was conducted. The results are presented below.
[0064] This shows that the major part (90.3 resp. 82.8%) of the GA4 and GA7 is found in the emulsified layer (oil phase). In contrast, the oil phase contains only 8.2% of the GA3. Example 3.
[0065] The effect of temperature was investigated using a proprietary strain in two media (see example 1 for media composition, for comparative experiments, 12.5% corn starch was used). The results are presented below:
Table 5 Effect of temperature
FM flasks: n=2 n=2 n=2 n=2 n=l n=2
[0066] An increase of temperature in a second stage of fermentation is found to result in a strong increase in productivity of gibberellins.
Example 4
[0067] The effect of aeration was investigated in the medium with oil (see example 1 for medium composition) using a proprietary strain.
Table 6 Effect of aeriation (dissolved oxygen)
[0068] An increase of aeration in a second stage of fermentation is found to result in a strong increase in productivity of gibberellins.
Example 6
[0069] GA4 and GA7 can be recovered from the fermentation broth by a process as illustrated in figure 1.
Example 7 and comparative experiment B
[0070] GA7 is increased relative to GA4 when using 3% of oil.
[0071] The same strain as used in example 3 was used in this experiment. As carbon source, either 20% com starch was used, or 20% corn starch and 3% of proflo oil. The N-source was 1.5 wt% yeast powder and l.5wt% cotton seed flour. The fermentation time was 10 days. The pH was 4.7-4.8. Results are given in table 7.
Table 7
Example 8
[0072] A mixture of GA4 and GA7 (65% GA4 and 35% GA7) was dissolved and recrystallized from the following solvents (2.5 ml. solvent used for each experiment)
methyl t-butyl ether (MTBE)
acetone
methyl isobutyl ketone (MIBK)
3-methyl-2-butanone
- cyclohexanone
ethyl acetate
Crystallization was induced by a controlled cooling ramp from reflux to l0-20°C, over 5-10 hours with slow agitation.
[0073] The relative amounts of GA4 and GA7 in the solid product obtained were analyzed by HPLC. The results are indicated in the table below:
The table shows that the recrystallized products have a higher GA7/GA4 ratio than the starting product (which had 35% GA7). MTBE not only had the highest GA7/GA4 ratio, but also had the highest percentage of solid recovery.
Example 9
3.0Kg of GA7/GA4 31/69 was recrystallized from MTBE in three 1 kg batches as per the table below:
The combined GA7 content of the recovered material is (512x0.5 + 537x0.49 + 600x0.49) = 830.77g which represents 89.3% of the 930g GA7 contained in the input.
The three batches were then recrystallized again from MBTE improving the ratio from GA7/4:
50/50 to 68/32.
The recovery of available GA7 was 64.3%.
The three batches were then combined and recrystallized from MBTE improving the ration to
84/19.
The recovery of GA7 in the case is 69.6%.
The recovery of GA7 in the case is 69%.
Solids from the liquors of the later experiments were combined and recrystallized three times to give an extra l03g of GA7 94% pure by HPLC.
[0074] This example shows that, starting from a GA7/GA4 31/69 mixture, by applying a few crystallizations, a 94% pure GA7 product can be obtained.
Claims
1. Process for recovering one or more gibberellins from an aqueous medium, the process comprising:
(a) contacting said aqueous medium with an oil to obtain an aqueous phase and an oil phase, resulting in extraction of said one or more gibberellins into the oil phase; and
(b) recovering said one or more gibberellins from said oil phase.
2. Process according to claim 1, wherein said one or more gibberellins are selected from the group consisting of gibberellin 4 (GA4), gibberellin 7 (GA7) and combinations thereof, preferably GA7.
3. Process according to claim 1 or claim 2, wherein said aqueous medium comprises (i) GA4 and/or GA7 and (ii) gibberellin 3 (GA3).
4. Process according to any preceding claim, wherein said oil is or comprises a natural oil, preferably a vegetable oil.
5. Process according to any preceding claim, wherein the oil comprises triglycerides.
6. Process according to any preceding claim wherein the oil comprises C8 to C18 unsaturated fatty acids.
7. Process according to any preceding claim, wherein said oil is or comprises cottonseed oil, palm oil, soybean oil, rapeseed oil or combinations thereof, preferably wherein said oil is or comprises cottonseed oil.
8. Process according to any preceding claim, wherein said aqueous phase and said oil phase form an emulsion.
9. Process according to any preceding claim, wherein said process further comprises
separating the aqueous phase and the oil phase, preferably wherein said separating comprises gravity separation, preferably centrifugation.
10. Process according to any preceding claim, wherein said recovering said one or more
gibberellins from said oil phase comprises contacting said oil phase with a solvent in which the oil is soluble and/or miscible, resulting in precipitation of said one or more gibberellins.
11. Process according to any preceding claim, wherein said recovering said one or more
gibberellins from said oil phase comprises contacting said oil phase with a hydrocarbon
solvent, preferably wherein said hydrocarbon solvent is or comprises a C5-C12 alkane, more preferably a C5-C8 alkane, most preferably hexane or heptane.
12. Process according to claim 10 or 11, wherein said contacting said oil phase with said
hydrocarbon solvent results in precipitation of said one or more gibberellins, and wherein the process further comprises recovering said precipitated one or more gibberellins.
13. Process according to any preceding claim, wherein the process further comprises
recovering GA3 from said aqueous phase, preferably by solvent extraction, preferably using an extraction solvent selected from ethyl acetate, and recovering gibberellin GA3 from the extraction solvent, preferably by crystallization.
14. Process according to any preceding claim, wherein said aqueous medium is a fermentation broth, has been derived from a fermentation broth, or is comprised in a fermentation broth.
15. Process according to any preceding claim, wherein said aqueous medium comprises
microorganisms which have produced said one or more gibberellins, preferably wherein said microorganisms are Gibberella fujikuroi microorganisms.
16. Process for producing one or more gibberellins, the process comprising culturing
microorganisms in a fermentation broth comprising an aqueous medium under conditions such that one or more gibberellins are produced, and recovering said one or more gibberellins by the process according any preceding claim.
17. Process according to claim 16, wherein said fermentation broth comprises a carbon source which comprises said oil.
18. Process according to claim 16 or 17, wherein an aqueous phase and an oil phase are obtained, and wherein the process further comprises recovering the one or more gibberellins from the oil phase.
19. Process according to any preceding claim, wherein a product comprising G4 and G7 is recovered, and wherein the process further comprises purifying or enriching G7 by the process according to any one of claims 42 to 47.
20. Process for producing gibberellin 7 (GA7), said process comprising:
(a) culturing a microorganism capable of producing GA7 in a fermentation broth under conditions conducive to produce GA7, said fermentation broth comprising a carbon source which comprises an oil; and
(b) recovering GA7 from said fermentation broth.
21. Process according to claim 20, wherein said oil in said fermentation broth is 2 wt% or more, and preferably 10 wt% or less.
22. Process according to any one of claims 20 to 21, wherein at least 25 wt.%, preferably at least 50 wt.%, more preferably at least 75 wt.%, more preferably at least 90 wt.%, more preferably 100 wt.%, of the total amount of carbon source added to the fermentation broth is an oil.
23. Process according to any one of claims 20 to 22, wherein the total amount of oil added to the fermentation broth is at least 80 g/l, preferably at least 120 g/l, more preferably at least 150 g/l.
24. Process according to any one of claims 20 to 23, wherein said fermentation broth comprises an aqueous phase and an oil phase, said oil phase comprising said oil; and (b) comprises recovering gibberellin 7 from said oil phase.
25. Process according to any one of claims 20 to 24, wherein said oil is or comprises a natural oil, preferably a vegetable oil.
26. Process according to any one of claims 20 to 25, wherein the oil comprises triglycerides.
27. Process according to any one of claims 20 to 26, wherein the oil comprises C8 to C18
unsaturated fatty acids.
28. Process according to any one of claims 20 to 27, wherein said oil is or comprises
cottonseed oil, rapeseed oil, soybean oil, rapeseed oil or combinations thereof.
29. Process according to any one of claims 20 to 28, wherein said oil is or comprises
cottonseed oil.
30. Process according to any one of claims 20 to 29, wherein said carbon source further
comprises starch, for instance com starch and/or potato starch, and/or a sugar, for instance lactose.
31. Process according to any one of claims 20 to 30, wherein said carbon source comprises com starch.
32. Process according to any one of claims 20 to 31, wherein said fermentation broth comprises a nitrogen source comprises flour (or meal), peanut meal, com steep powder, urea, cottonseed flour (or meal), yeast extract or combinations thereof.
33. Process according to any one of claims 20 to 31, wherein said fermentation broth comprises a nitrogen source which comprises cotton seed flour (or meal).
34. Process according to any one of claims 20 to 31, wherein said fermentation broth comprises a nitrogen source which comprises soy flour.
35. Process according to any one of claims 20 to 34, wherein the fermentation has a first stage and a subsequent stage, and wherein during said subsequent stage the dissolved oxygen content (D.O.) is higher than during the first stage.
36. Process according to any one of claims 20 to 35, wherein culturing is effected at a
temperature of between 25 °C and 35 °C, preferably wherein during a first stage (preferably having a duration of between 48 to 96 hours) the temperature is maintained between 25 °C and 30 °C, preferably between 27 °C and 29 °C and in a subsequent stage (preferably having a duration of between 120 to 240 hours) the temperature is maintained between 31 °C and 35 °C, preferably between 32 °C and 34 °C.
37. Process according to any one of claims 20 to 36, wherein said culturing is effected at a pH of between 4 and 7.
38. Process according to any one of claims 20 to 37, wherein said microorganisms are
Gibber ella fujikuroi microorganisms .
39. Process according to any one of claims 20 to 38, wherein said recovering is by a process according to any one of claims 1 to 19.
40. Process according to claim 21, or 25-39, wherein said oil is present in an amount of 2-5 wt%.
41. Process according to any one of claims 20 to 40, wherein a product comprising G4 and G7 is recovered, and wherein the process further comprises purifying G7 by the process according to any one of claims 42 to 47.
42. Process for purifying or enriching gibberellin 7 (GA7) which is contained in a product comprising GA7 and gibberellin 4 (GA4), said process comprising dissolving said product in a solvent, and inducing crystallization of GA7 from said solvent.
43. Process according to claim 42, wherein said solvent is selected from the group consisting of an ether, ketone, ester, and combinations thereof.
44. Process according to claim 42 or 43, wherein said solvent is selected from the group
consisting of methyl t-butyl ether (MTBE), acetone, Methyl isobutyl ketone (MIBK), 3- methyl-2-butanone, cyclohexanone, ethyl acetate and combinations thereof.
45. Process according to claim 44, wherein said solvent is or comprises MTBE.
46. Process according to any one of claims 42 or 45, wherein the process comprises dissolving a first product having a first GA7/GA4 ratio, and crystallizing from the solvent a second product having a second GA7/GA4 ratio, wherein the second GA7/GA4 ratio is greater than the first GA7/GA4 ratio, and wherein the GA7/GA4 ratio refers to the ratio of the amounts (expressed in weight) of GA7 to GA4 present in the product.
47. Process according to any one of claims 42 to 46, wherein said crystallizing is induced by cooling.
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