EP3143155A1

EP3143155A1 - Process for preparing carotenoids by submerged fermentation with mixed cultures of (+) and (-) strains of the fungus blakeslea trispora

Info

Publication number: EP3143155A1
Application number: EP15721715.9A
Authority: EP
Inventors: Robert Reinhard Pätz; Thomas Papert; Vivien Peter; Sarah Polage
Original assignee: Hochschule Anhalt
Current assignee: JAECKERING RESEARCH GMBH
Priority date: 2014-05-12
Filing date: 2015-05-12
Publication date: 2017-03-22
Also published as: CN106715708A; DE102014208876B4; US20170081693A1; WO2015173236A1; IL248893A0; DE102014208876A1

Abstract

The invention concerns a simple and effective process for preparing ß-carotene or lycopene by submerged fermentation with mixed cultures of (+) and (-) strains of the fungus Blakeslea trispora, characterized by high productivity of the B. trispora strains. The high productivity is achieved by virtue of the inventive regime, which acknowledges both the morphological state of the hemi-strains with their filamentous growth and the growth state when the strains are mixed (referred to as mating).

Description

Process for the preparation of carotenoids by submerged fermentation with mixed cultures of (+) and (-) strains of the fungus Blakeslea trispora

The invention relates to a simple and effective method for the production of β-carotene or lycopene by submerged fermentation with mixed cultures of (+) and (-) strains of the fungus Blakeslea trispora, which is characterized by a high productivity of B. trispora strains. The high productivity is achieved by the method according to the invention, which takes into account the morphological state of the filamentous half-growths and the growth state in the mixing of the strains (the so-called mating).

Carotenoids are natural dyes that cause a yellow to reddish color and are common in vegetables. They were and are due to their properties as antioxidants and as a precursor for vitamin A subject of many studies. Due to their antioxidant effect they are many diseases such. For example, cancer, arteriosclerosis, rheumatism or Alzheimer's disease. Lycopene (occurring, for example, in tomatoes) has the greatest antioxidant potential and is considered to be the most effective protection against the particularly reactive singlet oxygen. Industrially, the carotenoids are used as food additives and coloring agents e.g. As used in margarines, oils, sauces, soups and fruit juices, but also used as feed additives.

Carotenoids can be produced chemically and biotechnologically. By means of biotechnological production, carotenoids of more complex structure and also the naturally occurring conformational isomers are very easily accessible. The industrial biotechnological process for the production of β-carotene is based on the use of the alga Dunaliella salina or the fungus Blakeslea trispora, wherein using B. trispora, a mixed fermentation of the (+) and (-) strains is carried out to obtain a to obtain maximum yield of beta-carotene.

The preparation of β-carotene by fermentation of B. trispora in numerous patent documents is described in the prior art, for example in EP 1 367 131 A1, WO 93/20183 A1 or WO 02/010429 A1. All described fermentation processes are based on the principle that the (+) and (-) half-stems are inoculated into the bioreactor together. WO 93/20183 A1 describes the production of β-carotene using selected mutant B. trispora strains under specific fermentation conditions. WO 02/010429 A1 discloses a fermentation process with defined pH control during fermentation and the addition of soy lecithin to the culture medium to increase the production of β-carotene with respect to other carotenoids. The fermentation conditions described in EP 1 367 131 A1 provide for both a programmed addition of oxygen and / or β-ionone and a control of the vegetative growth phase of the strains used. US Pat. No. 5,422,247 A and WO 2005/030976 A2 disclose further fermentation processes which comprise a regulation of the pH value and, if appropriate, of the oxygen partial pressure. The literature also describes the use of chemical components to increase the product. However, such ingredients may be toxic and their nutritional or drug regulatory approval may be in question.

It is therefore an object of the present invention to provide an effective process for the preparation of carotenoids, in particular β-carotene and lycopene, which gives good product yields without requiring any chemical additives. In addition, it should be as inexpensive as possible and in the implementation simple and inexpensive and allow a reduction in fermentation duration. The object of the invention is a method for producing a carotenoid selected from ß-carotene or lycopene by submerged fermentation with mixed cultures of (+) and (-) - strains of the fungus Blakeslea trispora and recovery of the carotenoid from the obtained biomass or from the oil phase of Fermentation broth characterized in that first the (-) and (+) strains are pre-cultured separately until full morphological formation, then the (-) strain is inoculated into the bioreactor and during the exponential growth of the (-) -) strain of the (+) strain is added to the (-) strain in a volume ratio of 1: 5 to 1: 100 (also referred to as mating ratio) to induce carotenoid formation and both strains together without pH regulation and without regulation of oxygen partial pressure at 18 to 24 ° C, if necessary, until the end of carotenoid formation (idiophase) fermented.

The separate Vorkultivierung of the strains is carried out in a preferred embodiment of the invention in each case at 26 - 30 ° C, particularly preferably at 28 ° C. Separate pre-cultivation of the (-) strain is carried out for about 55 hours. After about 55 hours, the (-) strain is transferred to the bioreactor. The fermentation of the (-) - strain in the bioreactor is also preferably carried out at 26-30 ° C, more preferably at 28 ° C. The Cultivation of the (-) strain in the bioreactor is carried out for about 13 to 14 hours (trophophase).

It is also particularly preferred that the co-fermentation of the (+) and (-) strain (idiophase) be carried out for carotenoid formation at 22 ° C.

According to the present invention, cultivation of the (-) half-stem is carried out on a reactor scale until complete hyphae formation (trophophase) and only then the (+) half-stem is added as an induction measure during the exponential growth of the (-) strain. As a result, the fermentation process can be shortened in time and costs can be saved. In addition, there is a temperature shift of between 26-30 ° C to 18 to 24 ° C between trophy and idiophase. The lower temperature does not inhibit the vitality of the fungal hybrids, but does promote the stability of the product during fermentation. For the successful cultivation with good product yields no regulation of pH or oxygen content is required in the present process. Since there is no need to control the pH, no use of acids or bases is required and costs are saved.

Fumigation rate, rotational speed and temperature are kept constant during the entire product formation (idiophase), which obviously favors adaptation of the fungi to the prevailing conditions. The onset of product formation is characterized by a simultaneous decrease in BTS (biosolute substance) content and pH. The termination criterion of carotenoid formation can be detected by a significant increase in the pH. That is, after a pH rise of 0.5 to 0.8, carotenoid formation is stopped.

The stirring rate during the separate pre-cultivation in the shaking flask of the strains is preferably 130 to 156 rpm. It is preferred according to the invention, the stirring rate during the cultivation of the (-) - strain in the bioreactor (trophophase) to 230 to 350 rpm, more preferably 244 to 344 rpm, adjust.

The gassing volume flow during the trophophase is preferably about 1 vvm (volume of air / volume of fermenter / minute).

In the idiophase, ie in the phase of product formation also referred to as the phase of the common fermentation of the (+) - and (-) - strains after induction, are the Conditions for the stirring rate and the gassing volume flow the same as in the trophophase.

The mating ratio, expressed as the volume ratio (in each case (+) strain to (-) strain), in a particular embodiment of the invention is 1: 5 to 1:20, particularly preferably 1: 5 to 1:10.

As a substrate or fermentation substrate, in the idiophase of the process of the present invention, ie in the product formation phase also referred to as the phase of the common fermentation of the (+) and (-) strains after induction, the substrates well known for B. trispora are used, in particular those containing carbohydrates and organic nitrogen compounds simultaneously. Lipids can also be added to the substrate. Starch or sugar are preferably used in the substrate according to the invention as carbohydrates. It has been shown that, in particular with sugar-containing substrates with a sugar content of 10 to 50 g / L, good yields of carotenoid are obtained. Sugar-containing substrates preferably comprise molasses, beer wort or by-products of the starch industry or combinations thereof. Preferably, the substrate for the phase of the common fermentation of the (+) - and (-) strains after induction contains lipids. The lipids are preferably one or more oils. The oil or oils are preferably one or more native oils. In particular, vegetable oils, preferably native vegetable oils, can be used in the substrate. Examples of suitable vegetable oils are olive oil, rapeseed oil, corn oil and sunflower oil.

If lycopene is to be prepared by the process according to the invention, preference is given to adding a cyclase inhibitor, preferably imidazole and / or an imidazole derivative, to the substrate during the joint fermentation of the (+) and (-) strain (idiophase). The process of the invention allows the production of lycopene without addition of possibly toxic chemicals such. Cyclaseinhibitoren. In this case, lycopene is preferably obtained from the oil phase of the fermentation broth.

In the process of the invention, it is possible to use all known B. trispora half-strains which are publicly available in culture collections, such as e.g. For example, the (-) strain DSM 2388 and the (+) strain DSM 2387 or the (-) strain ATCC 14272 and the (+) strain ATCC 14271. In one embodiment of the invention, the strain BS 01 (FIG. -), in the International Depositary Office of the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) in Braunschweig, Germany under the number DSM 16755, and the strain BS 01 (+), which is deposited there under the number DSM 16754, are used.

In a very particular embodiment of the invention, the method is characterized by the following steps:

(1) The (-) strain is precultured separately for about 55 h and the (+) strain for about 68 h in an Erlenmeyer flask at 28 ° C. and 145 rpm.

(2) After about 55 h, the (-) strain is transferred to a reactor and fermented for about 13-14 h at 28 ° C, a gassing rate of about 1 vvm and a stirrer speed of 244 to 344 rpm.

(3) After addition of the (+) strain, the cultivation temperature is adjusted to 22 ° C. The volume ratio between (+) and (-) strain is 1: 9. The stirrer speed preferably remains at 244 to 344 rpm.

(4) A control of the pH value is not carried out. The workup of the biomass is carried out by methods generally known to the person skilled in the art, for example by solid-liquid separation or extraction of the solid, if appropriate after gentle drying of the biomass pellet.

In summary, it should be noted that the present method ensures a high productivity of Blakeslea strains solely by the control of physical parameters, which in the food or drug regulatory approval of carotenoids according to the invention no problems are to be expected since the inventive method no toxic or otherwise dangerous substances are left in the product. With the method according to the invention carotenoid yields are achieved in the dry biomass of at least 2%, preferably from 4 to 6%.

With the method according to the invention, the carotenoid can be obtained not only from the biomass obtained, but can alternatively or additionally be obtained from the oil phase of the fermentation broth.

It is known that in Blakeslea trispora the carotenoid formation increasingly occurs during the idiophase, wherein initially in the (-) - a lipid or oil storage takes place. The carotenoid, in particular the hydrophobic beta-carotene, is incorporated into this intracellular oil phase. After completion of the fermentation, the carotenoid can be removed from the obtained biomass are obtained by the cells are digested and the carotenoid or carotenoid-oil mixture extracted from the intracellular oil phase.

It has surprisingly been found that in carrying out the method according to the invention significant amounts of carotenoid are released from the organisms during the fermentation and discharged into the fermentation broth as a carotenoid-oil mixture. Thus, the inventive method allows the recovery of carotenoid by means of separation of the oil phase of the fermentation broth. There is no cell disruption necessary and the recovered carotenoid can be used without further process steps. The process according to the invention for preparing a carotenoid selected from β-carotene or lycopene by submerged fermentation with mixed cultures of (+) and (-) strains of the fungus Blakeslea trispora and obtaining the carotenoid from the oil phase of the fermentation broth is characterized in that the (-) and (+) strains are pre-cultured separately until full morphological training, then the (-) strain is inoculated into the bioreactor and during the exponential growth of the (-) strain the (+) strain the (-) - strain in the volume ratio 1: 5 to 1: 100 (also referred to as mating ratio) is added to induce carotenoid formation and both strains together without pH regulation and without regulation of the oxygen partial pressure at 18 to 24 ° C, optionally in the phase of carotenoid formation (idiophase), be fermented. Subsequently, the oil phase is separated from the fermentation broth and filtered if necessary, so that macroscopic constituents, ie components with an average diameter of 1 μηι or more, are removed from the oil phase. Thus, a carotenoid-containing oil or oil mixture is obtained, which can be used as such further or which can be subjected to a further purification to obtain even purer carotenoid fractions.

Due to the fact that the organisms release carotenoid formed during the idiophase as a carotenoid-oil mixture from the cell interior, an oil phase is formed in the fermentation broth in the process according to the invention, from which the carotenoid can be obtained, regardless of whether the fermentation substrate itself is a lipid or contains oil or not.

In the process according to the invention, preference is given to using substrates for the production of the carotenoid from the fermentation broth which contain one or more lipids. The lipids are preferably one or more oils. The oil or oils are preferably one or more native oils. In particular, in the Substrate vegetable oils, preferably native vegetable oils used. Examples of suitable vegetable oils are olive oil, rapeseed oil, corn oil and sunflower oil.

In contrast to previous methods, in which the carotenoid is obtained from the biomass obtained and thus are usually designed discontinuous, the inventive method for obtaining carotenoids from the oil phase of the fermentation broth also allows a continuous or at least multi-cyclic process management. In order to ensure a multi-cyclic or continuous process, substrate is again supplied to the fermentation batch after the induction, so that the nutrient supply in the fermentation batch allows further cultivation of the batch. The addition of fermentation substrate can take place, for example, once, repeatedly or regularly.

The addition of fermentation substrate is preferably such that in the fermentation batch, the concentration of the carbon source does not fall below a threshold at which the organisms die off. For example, the first addition of fermentation substrate occurs in a period of 20h to 60h after induction by introduction of the (+) strain.

The addition of fermentation substrate can be repeated or regular. For example, the first addition of fermentation substrate occurs in a period of 20h to 60h after induction by introduction of the (+) strain, while any further addition occurs within a period of 24h to 48h after the previous addition. The time between each additional addition may also be shorter or longer. It is crucial that the substrate content does not decrease permanently or for a longer period of time below a concentration which leads to the inhibition of the fungus.

In a particular embodiment, the method according to the invention for producing a carotenoid selected from β-carotene or lycopene by submerged fermentation with mixed cultures of (+) and (-) strains of the fungus Blakeslea trispora and recovery of the carotenoid from the oil phase of the fermentation broth is characterized by the following steps : (1) The (-) strain is precultured separately for about 55 h and the (+) strain in the conical flask at 28 ° C. and 145 rpm for about 68 h.

(2) After about 55 h, the (-) strain is transferred to a reactor and fermented for about 13-14 h at 28 ° C, a gassing of about 1 vvm and a stirrer speed of 244 to 344 rpm. (3) After induction of carotenoid production by the addition of the (+) strain, the cultivation temperature is adjusted to 22 ° C. The volume ratio between (+) and (-) strain is 1: 9.

(4) A control of the pH value is not carried out.

(5) After 20h-60h after addition of the (+) strain, new fermentation substrate, optionally with oil, is added to the fermentation broth.

(6) Optional additions of fermentation substrate to the fermentation broth are made, with any further addition occurring within a period of 24 hours to 48 hours after the previous addition of fermentation addition. In the meantime, the fermentation is preferably continued under unchanged conditions.

(7) Separation of the (carotenoid-containing) oil phase from the remaining fermentation broth and optionally filtration of the recovered carotenoid-containing oil or oil mixture.

In the method according to the invention, e.g. by changing environmental conditions, such as refeeding additional nutrients through the addition of fermentation substrate, the secondary metabolites, such as carotenoids, are excreted from the fungi in microbial oil. This process is similar to detoxification and ensures the survival of the fungus. By discharging the intracellular carotenoid oil mixture into the fermentation broth, the carotenoid can be obtained directly as the oil phase of the fermentation broth and a costly and time-consuming separation of the carotenoids from the biomass is no longer necessary. Consequently, no mechanical, thermal, enzymatic or chemical treatment of the biomass must be carried out, the carotenoids can be removed as native carotenoids directly as the oil phase of the fermentation broth. By the process procedure proposed here, the production of carotenoids can be operated as a continuous process in which set higher yields than in the conventional process, especially if the carotenoids are obtained not only from the oil phase of the fermentation broth but also additionally from the biomass.

By repeated feeding with fermentation substrate, the content of carotenoids and xanthophylls in the oil product can be increased in the process according to the invention. In order to allow a continuous or cyclic process management with as many Nachfütterungszyklen, in the inventive method and the one or more additional additions of (+) or (-) half-strain to the fermentation broth are carried out and, accordingly, the oil is further enriched with carotenoids.

It has been found that by means of the process according to the invention, in particular in the production of carotenoids from the oil phase of the fermentation broth, it is possible to prepare oils or oil mixtures which have a content of carotenoid, in particular of native carotenoid, of 400 mg / L or more, preferably 1, 500 mg / l or more, especially from 400 to 1, 500 mg / l and more.

It has been shown that, by means of the process according to the invention, in particular in the recovery of the carotenoids from the oil phase of the fermentation broth, it is possible to prepare oils or oil mixtures which have a content of lycopene, in particular of native lycopene, of 2 mg / L or more, preferably 2 to 20 mg / L and more.

Thus, the method according to the invention allows the production of native carotenoid, which has not been affected by processes during cell disruption. Thus, the present invention also relates to native carotenoid or to an oil or oil mixture containing a native carotenoid, wherein the native carotenoid can be prepared or prepared by a method according to the invention. The carotenoid-containing oil or oil mixture according to the invention preferably has a native carotenoid concentration of 400 mg / l or more. The carotenoid-containing oil or oil mixture according to the invention is preferably characterized in that the concentration of beta-carotene is 20 to 1500 mg / L, more preferably 30 to 1300 mg / L, most preferably from 300 to 1300 mg / L. The carotenoid-containing oil or oil mixture prepared by the process according to the invention preferably has a concentration of lycopene of more than 2 mg / L, more preferably from 2 to 20 mg / L, most preferably from 2 to 15 mg / L.

In a particular embodiment, the carotenoid-containing oil or oil mixture according to the invention has at least 30 mg / L beta-carotene and at least 2 mg / L lycopene, preferably at least 400 mg / L beta-carotene and at least 5 mg / L lycopene.

The carotenoid-containing oil or oil mixture according to the invention can be, for example:

> 0.25 mg / l lutein,

> 2 mg / L beta-cryptoxanthin,

> 2 mg / L lycopene, > 3 mg / L gamma-carotene,

> 0.25 mg / L alpha-carotene, and

> 30 mg / L beta-carotene

contain.

For example, the carotenoid-containing oil or oil mixture according to the invention:

0.25 to 1 mg / L lutein,

2 to 25 mg / L beta-cryptoxanthin,

2 to 15 mg / L lycopene,

3 to 35 mg / L gamma-carotene,

0.2 to 25 mg / L alpha-carotene, and

30 to 1300 mg / L beta-carotene

contain. In particular, the carotenoid-containing oil or oil mixture according to the invention can be, for example:

0.28 to 0.71 mg / L lutein,

2.05 to 24.9 mg / L beta-cryptoxanthin,

2.09 to 14.35 mg / L lycopene,

3.01 to 31, 8 mg / L gamma-carotene,

0.29 to 24.9 mg / L alpha-carotene, and

30.9 to 1295 mg / L beta-carotene

contain. The invention will be explained in more detail by means of examples, without limiting it thereto.

Characters:

FIGS. 1 to 7 show:

Fig. 1: an HPLC chromatogram of the native sunflower oil.

Fig. 2: an HPLC chromatogram of the carotenoid-containing oil after 3 Nachfütterungen with starchy substrate (sunflower oil).

Fig. 3: an HPLC chromatogram of the carotenoid-containing oil after 5 Nachfütterungen with starchy substrate (sunflower oil). Fig. 4: a HPLC chromatogram of the carotenoid-containing oil after 5 Nachfütterungen with glucose-containing substrate (sunflower oil). Fig. 5: an HPLC chromatogram of corn oil.

6 shows an HPLC chromatogram of the carotenoid-containing oil after a post-feeding with starch-containing substrate (maize germ oil). Fig. 7: a HPLC chromatogram of the carotenoid-containing oil after 5 Nachfütterungen with starchy substrate (corn oil).

Examples Example 1:

The half-stems DSM 2387 and DSM 2388 are separated on YPsS solid medium (starch: 15 g / L, yeast extract: 4 g / L, magnesium sulfate: 0.45 g / L, dipotassium hydrogen phosphate: 1.0 g / L, agar: 20 g / L). Spore formation starts within 3 days. From the 3rd day of cultivation, the spores can be rinsed off with a sterile 0.2% Span20 solution.

A 300 mL flask is charged with 200 mL YPsS medium (starch: 15 g / L, yeast extract: 4 g / L, magnesium sulfate: 0.45 g / L, dipotassium hydrogen phosphate: 1.0 g / L, agar: 2 g / L , Thiamine: 0.002 g / L, sunflower oil: 30 g / L). The steam sterilized at 121 ° C for 20 minutes, medium is inoculated to room temperature with 2 ^* 10 ⁶ spores after cooling. The (-) strain is precultured separately for 55 hours and the (+) strain for 68 hours at 28 ° C. The shaking frequency is 145 rpm.

A reactor with a nominal volume of 5 L is filled with 3200 ml of the liquid medium (YPsS without yeast extract) and sterilized for 30 minutes at 121 ° C. After cooling the medium, the sterile yeast extract (360 mL) and thiamine (12 mL) are added. The weight of the individual components of the liquid medium was 3572 mL. After adjusting the cultivation temperature of 28 ° C, the reactor is seeded with 200 mL of DSM 2388 culture. Cultivation takes place for 13 hours so that the fungus is within the exponential growth phase. Subsequently, 400 ml of the DSM 2387 culture are added to induce carotenoid formation. For the idiophase, the temperature is lowered to 22 ° C. Within 36 to 48 hours, an intense orange coloration of the biomass begins. For the cultivation neither a control of the pH value nor the pO2 value is necessary. Fumigation is continuous with 1 vvm at a stirrer speed of 300 rpm.

After completion of the cultivation, the processing of the biomass (solid-liquid separation, extraction of the solid optionally after gentle drying of the biomass pellet) takes place. The yield of β-carotene in the biomass is 3.2 to 6.18%.

Example 2:

A 300 mL flask is charged with 200 mL YPsS medium (wort: 10 g / L, yeast extract: 4 g / L, magnesium sulfate: 0.45 g / L, dipotassium hydrogen phosphate: 1.0 g / L, agar: 2 g / L , Thiamine: 0.002 g / L, sunflower oil: 30 g / L). The steam sterilized at 121 ° C for 20 minutes, medium is inoculated to room temperature with 2 ^* 10 ⁶ spores after cooling. The (-) strain is precultured separately for 55 hours and the (+) strain for 68 hours at 28 ° C. The shaking frequency is 145 rpm.

A reactor with a nominal volume of 5 L is mixed with 3200 mL of the liquid medium (wort: 10 g / L, magnesium sulfate: 0.45 g / L, dipotassium hydrogen phosphate: 1.0 g / L, agar: 2 g / L, sunflower oil: 30 g / L) and sterilized for 30 minutes at 121 ° C. After cooling the medium, the sterile yeast extract (14.292 g / 360 mL) and thiamine (0.0072 g / 12 mL) are added. The weight of the individual components of the liquid medium was 3572 mL. After adjusting the cultivation temperature of 28 ° C, the reactor is seeded with 200 mL of DSM 2388 culture. Cultivation takes place for 13 hours so that the fungus is within the exponential growth phase. Subsequently, 400 ml of the DSM 2387 culture are added to induce carotenoid formation. For the idiophase, the temperature is lowered to 22 ° C. Within 40 to 56 hours, an intense orange coloration of the biomass begins.

For the cultivation neither a control of the pH value nor the pO2 value is necessary. Fumigation is continuous with 1 vvm at a stirrer speed of 300 rpm.

After completion of the cultivation, the processing of the biomass (solid-liquid separation, extraction of the solid optionally after gentle drying of the biomass pellet) takes place. The yield of ß-carotene in the biomass is 1, 8 to 2.3%. Example 3:

The half strains ATCC 14271 and ATCC 14272 are separated on YPsS solid medium (starch: 15 g / L, yeast extract: 4 g / L, magnesium sulfate: 0.45 g / L, dipotassium hydrogen phosphate: 1.0 g / L, agar: 20 g / L). Spore formation starts within 3 days. From the 3rd day of cultivation, the spores can be rinsed off with a sterile 0.2% Span20 solution.

A 300 mL flask is charged with 200 mL YPsS medium (starch: 15 g / L, yeast extract: 4 g / L, magnesium sulfate: 0.45 g / L, dipotassium hydrogen phosphate: 1.0 g / L, agar: 2 g / L , Thiamine: 0.002 g / L, sunflower oil: 30 g / L). The steam sterilized at 121 ° C for 20 minutes, medium is inoculated to room temperature with 2 ^* 10 ⁶ spores after cooling. The (-) strain is precultured separately for 55 hours and the (+) strain for 68 hours at 28 ° C. The shaking frequency is 145 rpm. Two reactors with a nominal volume of 5 L are each filled with 3200 ml of the liquid medium (YPsS without yeast extract) and sterilized for 30 minutes at 121 ° C. After cooling the medium, the sterile yeast extract (360 mL) and thiamine (12 mL) are added. The weight of the individual components of the liquid medium was 3572 mL. After adjusting the culture temperature of 28 ° C, the reactors are each inoculated with 200 mL of the ATCC 14271 culture and the ATCC 14272 culture. There is cultivation for 14 hours so that the fungus is within the exponential growth phase.

In a reactor with a nominal volume of 15 L, 9 L YPsS medium are introduced, sterilized and inoculated with 500 mL ATCC 14272. The fungus is in exponential growth after 14 hours. 1 L of the ATCC 14271 culture is added to induce product formation. For the idiophase, the temperature is lowered to 22 ° C. Within the next 50 to 64 hours, an intense orange coloration of the biomass begins.

After completion of the cultivation, the processing of the biomass (solid-liquid separation, extraction of the solid optionally after gentle drying of the biomass pellet) takes place. The yield of ß-carotene in the biomass is 3.8 to 4.5%. Example 4:

A 300 mL flask is charged with 200 mL YPsS medium (starch: 15 g / L, yeast extract: 4 g / L, magnesium sulfate: 0.45 g / L, dipotassium hydrogen phosphate: 1.0 g / L, agar: 2 g / L , Thiamine: 0.002 g / L, sunflower oil: 30 g / L). The steam sterilized at 121 ° C for 20 minutes, medium is, after cooling to room temperature ^* inoculated each with 2106 spores. The (-) strain is precultured separately for 55 hours and the (+) strain for 68 hours at 28 ° C. The shaking frequency is 145 rpm.

A reactor with a nominal volume of 5 L is filled with 3200 ml of the liquid medium (YPsS without yeast extract) and sterilized for 30 minutes at 121 ° C. After cooling the medium, the sterile yeast extract (360 mL) and thiamine (12 mL) are added. The weight of the individual components of the liquid medium was 3572 mL. After adjusting the cultivation temperature of 28 ° C, the reactor is seeded with 200 mL of DSM 2388 culture. Cultivation takes place for 13 hours so that the fungus is within the exponential growth phase. Subsequently, 400 ml of the DSM 2387 culture are added to induce carotenoid formation. For the idiophase, the temperature is lowered to 22 ° C. Within 36 to 48 hours, an intense orange coloration of the biomass begins. For the cultivation neither a control of the pH value nor the p02 value is necessary. Fumigation is continuous with 1 vvm at a stirrer speed of 300 rpm.

After 24 h after addition of the (+) strain, new fermentation substrate is added to the fermentation broth. The addition of cultivation medium is repeated several times at intervals of 12 to 48 hours, thus enhancing the effect. Within 10 to 48 hours, an intense orange to red color of the sunflower oil sets in. After completion of the cultivation, the workup of the biomass and the oil phase takes place (solid-liquid separation, extraction of the solid optionally after gentle drying of the biomass pellet). The yield of β-carotene in the biomass is 3 to 6%, the content of β-carotene in the oil phase is 500 to 1500 mg L-1 with 7 times adding new fermentation substrate. Example 5:

A 300 mL flask is charged with 200 mL YPsS medium (starch: 15 g / L, yeast extract: 4 g / L, magnesium sulfate: 0.45 g / L, dipotassium hydrogen phosphate: 1.0 g / L, agar: 2 g / L , Thiamine: 0.002 g / L, corn oil: 30 g / L). The steam sterilized at 121 ° C for 20 minutes, medium is, after cooling to room temperature ^* inoculated each with 2106 spores. The (-) strain is precultured separately for 55 hours and the (+) strain for 68 hours at 28 ° C. The shaking frequency is 145 rpm. A reactor with a nominal volume of 5 l is filled with 3200 ml of the liquid medium (YPsS without yeast extract) and sterilized for 30 minutes at 121 ° C. After cooling the medium, the sterile yeast extract (360 mL) and thiamine (12 mL) are added. The weight of the individual components of the liquid medium was 3572 mL. After adjusting the cultivation temperature of 28 ° C, the reactor is seeded with 200 mL of DSM 2388 culture. Cultivation takes place for 13 hours so that the fungus is within the exponential growth phase. Subsequently, 400 ml of the DSM 2387 culture are added to induce carotenoid formation. For the idiophase, the temperature is lowered to 22 ° C. Within 36 to 48 hours, an intense orange coloration of the biomass begins.

For the cultivation neither a control of the pH value nor the p02 value is necessary. Fumigation is continuous with 1 vvm at a stirrer speed of 300 rpm.

After 30 h after addition of the (+) strain, new fermentation substrate is added to the fermentation broth. The addition of cultivation medium is repeated several times at intervals of 12 to 48 hours, thus enhancing the effect. Within 10 to 48 hours, an intense orange to red color of the sunflower oil sets in. After completion of the cultivation, the workup of the biomass and the oil phase takes place (solid-liquid separation, extraction of the solid optionally after gentle drying of the biomass pellet). The yield of β-carotene in the biomass is 3 to 6%, the content of β-carotene in the oil phase is 400 to 600 mg L-1 with 4 additions of new fermentation substrate. Example 6:

To determine the oil composition with respect to the carotenoids and xanthophylls, an HPLC analysis (Table 1) of the carotenoid-containing oils from Examples 4 and 5 was carried out.

Table 1: HPLC method

t [min] A (acetone) [%] B (water) [%]

0.0 80 20

7.0 100 0

7,5 80 20

12 80 20

T = 30 ° C; V | " _j . = 10 μί; Column: C18 (Agilent XDB); Detection: λ = 450 nm; Flow: 1 ^mL /,

HPLC:

Pump: Merck Hitachi L-6200A

Autosampier: Merck Hitachi AS-4000A

UV-VIS detector: Merck Hitachi L-4250

Interface: Agilent Interface 35900E

Software: OpenLAB

To determine the concentration of the carotenoids and xanthophylls in the oil of the invention, stock solutions of the existing standards (Table 2) were prepared. For this purpose, a defined amount was dissolved in oil or acetone (Carl Roth GmbH + Co. KG) and stored in a brown bottle. Before the HPLC analysis, the stock solutions were filtered to ensure a particle-free solution. On the basis of the determined calibration line (concentration to area) it was possible to deduce the concentration from the peak area in the carotenoid-containing oil. Table 2: Standards

To analyze the carotenoid-containing oils, the oil, but also parts of the fermentation broth and biomass, would be separated by decanting (pouring off). By centrifuging the sample at 3044 g for 5 min, a clear separation between the biomass, fermentation broth and oil phase could be obtained. The oil samples were withdrawn and filtered for HPLC determination (pore size 0.45 μηη, PTFE syringe filter, Carl Roth GmbH + Co. KG) to ensure a particle-free sample. This approach is particularly suitable for the laboratory scale, in a conversion in pilot plant scale separators and decanters, for liquid-liquid separation, should be used.

Exemplary spectra of the oils studied are shown in Figs. 1-7. 1 shows a HPLC chromatogram of the native sunflower oil; 2 is a HPLC chromatogram of a carotenoid-containing oil according to Example 4 after 3 Nachfütterungen with starchy substrate (sunflower oil). 3 is a HPLC chromatogram of a carotenoid-containing oil according to Example 4 after 5 Nachfütterungen with starchy substrate (sunflower oil). 4 is a HPLC chromatogram of a carotenoid-containing oil according to Example 4 after 5 Nachfütterungen with glucose-containing substrate (sunflower oil); 5 shows a HPLC chromatogram of the maize germ oil; 6 is a HPLC chromatogram of a carotenoid-containing oil according to Example 5 after a post-feeding with starch-containing substrate (corn oil); and FIG. 7 shows an HPLC chromatogram of a carotenoid-containing oil according to Example 5 after 5 restorations with starch-containing substrate (maize germ oil).

In order to be able to carry out an exact quantification of the carotenoids and xanthophylls, the composition of the oils used was determined in the first step. The peak areas determined were from the peak areas of the carotenoid-containing oil samples subtracted. The significant peaks were detected as lutein, β-cryptoxanthin, lycopene and α-, β- and Y-carotene (Table 3).

Table 3: Minima and maxima of the content determined by HPLC in mg L ^-1 at different cultivation conditions.

The minima and maxima values represent in each case the lowest and highest value of all carotenoid-containing oil samples. The β-carotene content of 1295 mg L ^-1 was reached on the reactor scale after repeated feeding 8. In the shake flask experiments the maximum was about 4 to 5 post-feedings at around 700 mg L ^"1 .

The content of carotenoids in the oil phase was dependent on the process strategy. After one or two additional replenishment, the content of, for example, β-carotene was about 50 to 100 mg L ^-1 . The content of β-carotene in the oil could be increased by repeated replenishment with starch, molasses or glucose-containing medium with four to five replenishment to about 500 to 800 mg L ^"1 and with a 7 to 10maligen Nachfütterung to over 1000 mg L ^{" 1.} The distance of the feeding was in the current investigations between 20 and 50 hours, however The content of carotenoids and xanthophylls is mainly dependent on the number of post-feeds, the oil used and the substrate.

The main proportion of carotenoids was about 80 to 97% ß-carotene, the detection of other carotenoids was dependent on the substrate used, oil used and number of Nachfütterungen. Above all, the number of Nachfütterung lowers the proportion of other carotenoids and thus increases the percentage of ß-carotene, which can be done by synthesis processes, a change in the content of carotenoid-containing oil.

Claims

claims

1 . Process for the preparation of a carotenoid selected from β-carotene or lycopene by submerged fermentation with mixed cultures of (+) and (-) strains of the fungus

Blakeslea trispora and recovery of the carotenoid from the biomass obtained and / or from the oil phase of the fermentation broth,

characterized in that

first the (-) - and the (+) - strain are pre-cultured separately until full morphological formation, then the (-) strain is inoculated into the bioreactor and during the exponential growth of the (-) - strain of (+) - Strain is added to the (-) strain in a volume ratio of 1: 5 to 1: 100 to induce carotenoid formation and both strains are fermented together without pH regulation and without regulation of the oxygen partial pressure at 18 to 24 ° C.

2. The method according to claim 1, characterized in that both strains are fermented after induction together without pH regulation and without regulation of the oxygen partial pressure at 18 to 24 ° C until the end of carotenoid formation.

3. The method according to claim 1 or 2, characterized in that

the separate precultivation of the strains and the cultivation of the (-) strain in the bioreactor are carried out at 26-30 ° C.

4. The method according to any one of claims 1 to 3, characterized in that the (+) - the strain (-) - in the volume ratio 1: 5 to 1: 20, preferably 1: 5 to 1: 10, is added.

5. The method according to any one of claims 1 to 4, characterized in that the common fermentation of the (+) - and (-) - strain is carried out for carotenoid formation at 22 ° C.

6. The method according to any one of claims 1 to 5, characterized in that the substrate for the common fermentation of the (+) - and (-) - contains tribe carbohydrates and organic nitrogen compounds.

7. The method according to claim 6, characterized in that serving as carbohydrate-containing fraction of the substrate starch, molasses or beer wort.

8. The method according to claim 1 to 7, characterized in that the substrate for the common fermentation of the (+) - and (-) - contains strain of lipids, preferably one or more oils, more preferably one or more native oils, most preferably Native vegetable oils, in particular, for example, olive oil, rapeseed oil, corn oil and / or sunflower oil.

9. The method according to any one of claims 1 to 8, characterized in that in the case of the production of lycopene, a cyclase inhibitor is added to the substrate for the common fermentation of the (+) - and (-) - strain, preferably imidazole and / or an imidazole derivative ,

10. The method according to any one of claims 1 to 9, characterized in that in the common fermentation of the (-) - and (+) - strain gassing rate, stirrer speed and temperature are kept constant.

1 1. Process according to one of Claims 1 to 10, characterized in that the (-) strain DSM 2388 and the (+) strain DSM 2387 or the (-) strain ATCC 14272 and the (+) strain ATCC 14271 are used ,

12. The method according to any one of claims 1 to 1 1, characterized in that the carotenoid-containing oil phase is separated from the remaining fermentation broth.

13. The method according to claim 12, characterized in that the obtained carotenoid-containing oil phase is filtered.

14. The method according to any one of claims 1 to 13, characterized in that the method is operated continuously.

15. The method according to any one of claims 1 to 14, characterized in that the fermentation batch after induction substrate is supplied again.

16. The method according to claim 15, characterized in that the renewed addition of substrate takes place in a period of 20h to 60h after induction of the fermentation batch by introducing the (+) - strain.

17. The method according to claim 16, characterized in that further additions of substrate are carried out, whereby preferably each further addition takes place within a period of 24h to 48h after the previous addition of substrate.

18. Oil or oil mixture containing native carotenoid, characterized in that the oil or oil mixture is prepared by a process of claims 1 to 17.

19. oil or Olgemisch according to claim 18, characterized in that the oil or oil mixture has a concentration of native carotenoid of 400mg / l or more.

20. Oil or Olgemisch according to any one of claims 18 or 19, characterized in that the oil or oil mixture has a concentration of at least 400 mg / L beta-carotene and at least 5 mg / L lycopene.

21. Oil or oil mixture according to claim 18, characterized in that the oil or oil mixture contains:

> 0.25 mg / l lutein,

> 2 mg / L beta-cryptoxanthin,

> 2 mg / L lycopene,

> 3 mg / L gamma-carotene,

> 0.25 mg / L alpha-carotene, and

> 30 mg / L beta-carotene.