EP1670929A2

EP1670929A2 - Method for producing a carotinoid-containing biomass

Info

Publication number: EP1670929A2
Application number: EP04786874A
Authority: EP
Inventors: Arnulf Christner; Klaus-Dieter Menzel; Michael Cyrulies; Claudia Menschel; Andreas Kretschmer; Reinhard PÄTZ
Original assignee: BIOSYNERGY GmbH
Current assignee: Luna Consulting & Management GmbH
Priority date: 2003-09-26
Filing date: 2004-09-26
Publication date: 2006-06-21
Also published as: WO2005030976A2; WO2005030976A3; DE112004002328D2; CN1886519A

Abstract

The invention relates to the production of a carotinoid-containing biomass. The aim of the invention is to provide a method for producing carotinoid-containing biomass that can be realized with a low level of complexity on a large scale and without antibiotics. To this end, the invention provides that non-genetically modified heterothallic fungi with an increased synthesis and storage capacity of ß-carotene are cultured in submerged cultures, whereby the biomass is propagated separately according to (+) and (-) pair type in multiple stages exclusively from spores. Biomass of the (+) pair type or culture liquid of the (+) pair type or homogenized biomass of the (+) pair type or, instead of the (+) pair type, gamone-type stimulators or lactones or trisporic acid or trisporic acid derivatives is/are added to the biomass of the (-) pair type after completion of the last biomass propagation phase before the production fermentation, and this submerged mixture is transferred into the nutrient solution of the product fermentation after a maturing time lasting form hours. The nutrient solution is composed in such a manner that at time tw, the growth phase of the biomass is terminated by phosphorous limitation, the synthesis of ß-carotene is additionally triggered by creating a short-term stress situation, and the solvent-free extraction of the ß-carotene from the resulting dry biomass is made possible by a phosphorous limitation during the growth phase.

Description

Process for the production of biomass containing carotenoids

The present invention relates to a method for producing carotenoid-containing biomass.

Carotenoids are natural coloring and active substances that are formed in the plant organism as protective or colored pigments.

In the animal organism, some of them are converted into vitamin A and as such have numerous special effects in the central building and operating metabolism. For example, ß-carotene is a precursor of vitamin A and is converted into the vitamin in the animal organism. The animal organism is unable to synthesize vitamin A without the added precursors. ß-Carotene is a fat-soluble substance and can therefore only get into the body in combination with fats and oils. The special effects of vitamin A were already known 2000 years ago in China, where e.g. In the case of eye problems or night blindness, the consumption of fresh colored fruit and green vegetables has been recommended.

Humans and animals have a high natural need for ß-carotene, since this substance or substances formed from it in the body stabilize physical performance and ward off harmful environmental effects. The minimum daily requirement is given as 0.8 mg - 1 mg vitamin A = 5 mg - 6 mg ß-carotene (DGE 2000). According to Heseker et al. (VERA series, vol. III, 2nd edition, Wiss. Fachverlag Dr. Fleck, Niederkleen, 1994) the actual daily ß-carotene intake in the Federal Republic of Germany is 1.5 mg - 2.0 mg. According to epidemiological studies, there is a high correlation between low ß-carotene plasma levels and the risk of coronary heart disease and myocardial infarction (Kardinaal et al., Euramic study, Lancet 342, 1379-84, 1993).

But ß-carotene is not only important as an active substance, rather the desire for beauty and ι4 is expressed in cosmetic and pharmaceutical products corresponded to a healthy lifestyle and therefore also used the color properties of the ß-carotene.

However, ß-carotene is not only important in the human field. Ss-Carotene also plays an important role in animal nutrition. Common feed such as clover and alfalfa contain ß-carotene, but the proportion in the plants decreases due to the degradation of the soil due to the industrial production methods.

In nature, ß-carotene is synthesized exclusively by plant organisms and by some microorganisms. Substantial ß-carotenes are contained in carrots (namesake), peppers, cherries, various algae, but also in palm oil and green tea. In this context, essential generally means <0.1% in dry biomass.

Obtaining natural ß-carotene producers on a technical scale is very complex and, above all, requires very large acreage and the processing of large-scale biomass quantities. For this reason, in the middle of the last century, the search for chemical synthesis options and the development of them for application began.

Today, ß-carotene is produced on an industrial scale by 90% by chemical synthesis. However, processes in which ß-carotene is obtained from natural sources are becoming increasingly important because it has been established that the human and animal physiological effects of ß-carotene, despite their low proportion of ß-carotene, in part of <1%, are essentially due to the natural ones Accompanying substances of ß-carotene, which are also carotenoids, is positively influenced.

Since the growing and increasing demand for natural ß-carotene from traditional agricultural production cannot be met, the possibility of industrial biotechnological synthesis of ß-carotene has been working for about 20 years now, with organisms in question come, their performance in terms of ß-carotene synthesis significantly> 1% of the dry biomass can be increased and in particular the fermentation or algae cultivation are accessible for the biotechnical processes.

In general, extensive research has shown that algae (e.g. Dunaliella salina, Chlorella spec, Spirulina spec. Etc.), micro-fungi such as Blakeslea trispora, Choanephora cucurbitarum and Phycomyces blakesleeanus, bacteria such as Flavobacterium, Corynebacterium, Mycobacterium and Brevibacterotor and yeast are not only synthesize β-carotene, but can even produce an excess of carotenoids under certain fermentation and cultivation conditions (Lampila et al., Mycopatholigia 90, 65, 80, pp. 65-80, 1985).

Of the carotenoids, particular attention has been paid to the formation of β-carotene. Up to 20% ß-carotene (De Baets, Vandedrinck, Vandamme, Encyclopedia of Mikrobiology, Vol. 4, 837 - 853, 2000) can be accumulated in the dry biomass in the algae Dunaliella. However, their economic breeding is only possible successfully under tropical conditions in creamy waters. However, the algae biomass concentration in the medium is approximately ten times lower than that of fungal or bacterial cultures and large areas have to be cultivated in order to obtain industrially interesting ß-carotene yields.

For this reason, development is increasingly focusing on microorganisms that simultaneously achieve biomass concentrations> 20 - 30 g / 1 and a high concentration of ß-carotene. Wild strains or selected selectants that have undergone mutagenesis are generally used for this. The derived and selected high-performance mutants are cultivated and used for fermentative biosynthesis when optimal process conditions are set. Known methods of mutagenesis are treatment with nitrosoguanidines of different concentrations alone or, for example, in combination with UV radiation (Cubero et al., 1993, Kuntschikova, 2000).

The highest yield of ß-carotene (3 - 4 g / 1) is obtained according to Weide, Paca, Knorre ("Biotechnology", Gustav-Fischer Verlag, 1987) in the fermentation of a mixed culture of the half-strains of Blakeslea trispora (+) - and (-) - Strains (high-performance mutants) The β-carotene formation of the (-) strain is promoted by trisporic acid (Bu'Lock et al., Arch. Microbiol. 97, 239-244, 1974). The fermentation is running 28 ° C and lasts 6 - 8 days.

The breeding of the different sexual half-strains of Blakeslea trispora was originally developed by Hesseltine and Anderson (US-2 865 814, US-2 890 989) and further developed by various companies.

Additions of thiamine, isoniazid and ß-ionon require the carotene synthesis (Vandamme E. J., Biotechnology of Vitamins, Pigments and Growth Factors, 31 - 33, Elsevier Science Publishers LTD, 1989).

The importance of trisporic acid lies in the fact that, together with the genetic information, this acid requires the formation of an excess of ß-carotene in the (-) - semi-strain (Caglioti L., et al, Tetrahedron Suppl., 7, 175 - 187, 1966). A targeted reduction of trisporic acid by chemicals leads to reduced ß-carotene formation (Lampila). A beneficial effect is obtained by adding abscissic acid (such as ß-carotene to a terpene), ß-ionone (terpene) (see, among others, Feofilova, Arbuzov Mikrobiologija Vol. 44, 3, 1975, alpha-ionone (terpene) and vitamin A (terpene) achieved (US 005328845 of July 12, 1994). The addition of these substances accelerates the RNA translation and leads to an increased synthesis of the enzymes de novo which form carotenoids. Other additives examined are penicillin, retinol, Kerosene, aromatics such as dimethyl phthalate and veratrol, and nitrogen-containing heterocyclic compounds such as iproniazid (in addition to isoniazid see above).

Substances that generate oxygen stress act as accelerators of ß-carotene synthesis. By adding surface-active substances (e.g. Span 20: Seon-Won Kim et.al., Journal of Fermentation and Bioengineering, Vol. 84, No. 4 330 -332, 1997) the oxygen transfer into the aqueous medium can be accelerated and thus the Oxygen availability can be increased. Oxygen stress can also be generated if ozone is added or generated directly in the fermentation. The addition of hydrogen peroxide is also described, which causes a substantial increase in ß-carotene formation (Jae-Cheol Jeong et. Al., Biotechnology letters 21, 683-686, 1999).

The influence of ß-carotene synthesis by varying the ingredients of the nutrient solution is also due to a lack of phosphate (Goncharova, O., Konova, L, Biruzova, V., Biochemical and structural features of Blakeslea trispora dependent on medium composition, Microbiology 65, 47 , 1996), which leads to a change in the cell wall in Blakeslea trispora BS01 (+) and BSOl (-).

The choice of carbon substrates also influences the ß-carotene synthesis. It was found that complex substrates are cheapest if they contain carbohydrates and organic nitrogen compounds at the same time (soybean, soybean meal, soybean meal) (Lampila). The addition of fats and oils also improves the β-carotene synthesis (US Pat. No. 5,422,247, EP 1,367,131 Al).

By adding soybean oil, cotton oil or fatty acids from these oils and soapstock (precipitated fatty acids as a product of oil refining), an increased ß-carotene formation was achieved up to 80 times the comparative sample (Lampila). About the influence of different lipids on the formation of Carotenoids of the mixed couple report Pazola et al., Acta Microbiologica Polonica Vol. 17.1, pp 75 -82, 1968. The composition of the lipids is important for the formation of the storage lipid, the solvent for β-carotene in the cell. It was found that the fatty acid spectrum has a decisive influence on the endogenous lipid pool.

The fundamental studies on the sexuality of fungal ß-carotene producers and on carotenoid biosynthesis were carried out on a shake flask scale. There is hardly any description of the application-related production of ß-carotene using an industrial process, but it is known that various companies produce ß-carotene by fermentation. Mutants from Blakeslea trispora and Phycomyces blakesleeanus are used for this.

The processes used today are based on the fermentation approach developed by Ninet and Renaut in 1979 for small-scale fermentation on a 320 1 scale with a fermentation period of 185 hours, whereby the (+) - and (-) - semi-strains are basically separated from each other and only then in the last stage. For this purpose, both half-strains are dosed into the nutrient solution of the fermentation stage, multiplied there together and, by adding a stimulator, the β-carotene biosynthesis is triggered at a specified point in time. Basically, this technology is known for the production of antibiotics and enzymes. There, a multi-stage, aseptic process is always selected, in which biomass is grown from the shake flask in a first fermentor, which is further enriched in a second fermenter (size levels 1:10). The actual synthesis of the target product takes place in the third stage, in that biomass is first generated there and the target product (primary metabolite or secondary metabolite) is produced by changing the fermentation conditions. Common to all procedures is that they are aseptic. To ensure sterility, choose the discontinuous mode of operation. After reaching the At the end of the stage, the fermentor is emptied and sterilized for repeated use. Individual cultures are used in each method described above. It is based on the fact that the microorganisms used are morphologically homogeneous, since it is easier to achieve the required process conditions with such organisms. If they do not meet these requirements, chemical and physical measures are generally taken to influence the microorganisms accordingly.

The disadvantage of all known carotenoid processes is that they can only be carried out on a large industrial scale to a limited extent.

Only one technical process for the production of β-carotene is currently known, only in a 5-1 fermentor by Mantzouridou et al. In 2001 (Biochemical Engineering Journal 3561, 1-13) the influence of the stirring intensity and the gassing rate being investigated and carried out Modeling the key influencing parameters was determined.

The object of the present invention is to provide a process for the production of carotenoid-containing biomass which can be carried out on a large industrial scale without antibiotics.

The object is achieved by a method according to claim 1. Advantageous embodiments of this solution are disclosed in the subordinate claims.

The essence of the invention is that carotenoid-containing, in particular ß-carotene-containing biomass in submerged culture by heterothallic fungi from the Zygomycota strain, order Mucorales, for example Blakeslea trispora (high-performance mutants) is produced on an industrial scale with the addition of trisporic acid derivatives and trisporic acid in ferrous solution-containing cultures , by doing: • Pure spore suspensions are obtained from the (+) - and (-) - half-strains of tropical Blakeslea trispora according to the invention after ten to twelve days of emersed growth on a nitrogen-limited complex medium using ion-free water with a 1% surfactant additive, the spore sediments of 10 ml spore suspension of a test tube culture is resuspended with 1 ml ion-free water and then filled up with 9 ml glycerol and these spore suspensions, which contain non-swollen sporangiospores, are filled into tubes and deposited at - 80 ° C to - 85 ° C, the multiply increased ( -) - Half-stem instead of the (+) - half-stem trisporic acid and / or a trisporic acid cosubstance is added at the time at which the biomass of the (+) - half-stem was added in the previous procedure and the mixture was then subjected to a maturation period before a new biomass production is triggered.

• only the (-) - half-stem is subjected to a conventional multi-stage biomass propagation and is used for ß-carotene synthesis and the (+) - half-stem is grown on a laboratory scale in a continuous, cyclical or discontinuous culture, whereby depending on the procedure, biomass, culture fluid, Biomass homogenate or the gamon-like stimulators or trisporic acid the (-) - half-stem after the completion of the exponential growth phase for the biomass, biomass homogenate and culture fluid in a ratio of 1: 100 and for the gamon-like stimulators or for the trisporic acid in a ratio of 1: 1000 to 1500 as a biosynthesis-stimulating Additives are added. Furthermore according to the invention:

• It is advantageous to use stimulators that support the biosynthesis of ß-carotene. Stimulators can be vitamins and other substances that have structures related to ß-carotene from the chemical structure.

• The nutrient solution in the product fermentor is composed in such a way that, at the end of the induction period, the phosphorus concentration is limited by limitation to limit the biomass growth in the entire culture and the synthesis of β-carotene is triggered. This can also be initiated in connection with the time of the phosphorus limitation by an oxygen shock and / or a temperature reduction of at least 4 ° C.

• The nutrient solution is composed in such a way that both the germination of the spores and the increase in biomass restrict the build-up of chitin in the cell walls and thus achieve better solvent-free extractability of the ß-carotene from the fungal cells (as given, for example, in Example 1).

• The viscosity of the nutrient solution is advantageously adjusted by adding enzymes or enzyme mixtures in such a way that optimal mixing and gas exchange conditions are created, thereby enabling economical operation of the fermentation as a technical process.

Activators can also be used to protect the biosynthesis of β-carotene. These can be substances which have structures related to ß-carotene from the chemical structure. • The processes of biomass production and ß-carotene synthesis are separated from each other by the phosphorus limitation, so that they take place one after the other and thereby significantly limit the fermentation time to <90 to 100 hours with a high ß-carotene yield of> 25 g / kg dry biomass is achieved.

The β-carotene is obtained from the fungal biomass by solvent-free extraction with vegetable oil in a manner known per se.

The present invention describes a process which, using heterothallic mushrooms from the Zygomycota strain, order Mucorales, which are capable of producing β-carotene, permits the industrial production of mushroom biomass containing β-carotene under optimal economic and technical conditions.

The biomass is multiplied exclusively from spores according to the (+) - and (-) - pairing type in several stages.

Biomass of the (-) - pairing type only becomes biomass of the (+) - pairing type or culture fluid of the (+) - pairing type or homogenized biomass of the (+) - pairing type or gamon-like stimulators or lactones or trisporic acid or only after completion of the last biomass propagation phase before the product fermentation Trisporic acid derivatives are added, the product fermentation being started with this submerged mixture after a one to several hour ripening period in the inoculum fermenter. The nutrient solution is composed in such a way that the growth phase of the biomass is completed by P-limitation at time t _w .

In the context of the invention, the synthesis of β-carotene can additionally be triggered by generating a short-term stress situation. In addition, a phosphor-rate-limited growth and a phosphorus limitation during the growth phase enable the solvent-free extraction of the ß-carotene by means of vegetable oil from the resulting dry biomass of Blakeslea trispora BSOl (-). The (+) mating types used in the process according to the invention are only induction strains and their biomass is grown in continuous or cyclic culture. The (-) - half strains used in the process according to the invention, on the other hand, are only production strains for carotenoids.

The biomass of the (+) - mating types are grown in cyclical culture. The (-) pairing types, on the other hand, are constantly being increased in several stages. The ratio of biomass of the respective (+) - pairing type to that of the respective (-) - pairing type when mating is 1:10 to 1: 200, preferably 1:45 to 1:75. the phase of derepression of the (-) half-strain is determined by the use of culture fluid, homogenates or synthesis products of the (+) - half-strain in a strain-specific manner and is 1 to 180 minutes, preferably 30 to 90 minutes.

The nutrient solution consists of polymeric nutrients, has a phosphorus limitation, whereby the polymeric carbon substrate is subjected to an enzyme-related liquefaction in the course of the medium sterilization after gelatinization, which greatly reduces the viscosity of the solution, but does not result in the formation of monomeric carbon substrates.

The nutrient solution is composed and prepared in such a way that the biomass growth in the culture proceeds evenly and quickly with reduced chitin biosynthesis and after growth is complete, only substrates for product formation are available due to the phosphorus limitation, so that after further enzymatic digestion by the producing fungus, a second secondary biomass formation is not possible.

It is according to the invention that heterothallic fungi with a ß-carotene synthesis potential> 2.0% of the dry weight are used as ß-carotene formers, in particular Blakeslea trispora and strains derived from it by mutagenesis, the different mating types also being able to come from different stem lines. The high-performance mutants BS01 (+) and BSOl (-) from Blakeslea trispora, which are deposited in the collection of microorganisms and cell cultures GmbH (DSZM), are used as the starting material for the process according to the invention.

It is essential to the invention that the production and auxiliary cultures are basically only drawn from spores. Up to a reactor size of 120 l, both half-strains are treated equally in separate cultivation. Thereafter, the BS01 (+) half-stem is continued in the reactor as a continuous or cyclic culture, the BS01 (-) - half-stem is further increased up to a biomass volume of 5 m ³ . At the end of the growth phase of the BS01 (-) semi-strain, when the nutrient substrate concentrations in the reactor have decreased at this point, trisporic acid derivatives and / or trisporic acid are added to the BS01 (-) biomass in a ratio of 1: 1000 - 1500. The trisporic acid derivatives and / or trisporic acid are obtained from the BS01 (+) semi-stem culture. They can be added as BS01 (+) biomass or homogenate (then in a ratio of 1:10 to 1: 100), culture solution or a separate stimulator mixture. After a decisive contact time of 30-180 minutes under fermentative conditions, the nutrient solution for the β-carotene synthesis in the product fermentor is inoculated with this induced mycelium mixture. The nutrient solution is composed in such a way that after 24 hours the phosphorus rate-limited biomass increase is ended by the phosphorus limitation and the ß-carotene synthesis begins. This is additionally induced by at least three hours of oxygen stress and is predominantly ended after 78-94 hours by immediate solid-liquid separation and subsequent drying.

In the context of the present invention, the lineage and the cultivation of the homozygous mating types BS01 (+) and BSOl (-) of the mutagenically treated Blakeslea trispora are important in order to improve the genetic stability guarantee. The strain was determined by mutagenesis with nitrosoguanidine in a concentration of 500 mg / ml, a spore suspension of 0.02-0.1% for BS01 (+) and in combination with UV radiation with a survivability of the spores of 5-7%. and nitrosoguanidine in the same concentration for BSOl (-). BSOl (-) is characterized by a reduced chitin content in the cell wall, both half-strains split oils into glycerol and fatty acids, but cannot utilize the cleavage products. They are also characterized by a high degree of branching of their hyphae. The asexual reproduction of the sporangiospores takes place separately for the half-strains on a nitrogen-limited medium in order to achieve a sufficiently high sporulation. After morphological differentiation of the sporangiospores after twelve days of growth, the homogeneous spore lawn of both half-strains is washed away with ion-free water using a 1% surfactant additive, the spore sediment is washed again with ion-free water and resuspended with glycerol. This suspension is filled into ampoules and deposited at -80 ° C to -85 ° C. Such master preserves with non-swollen spores as a result of water retention due to the absence of ions are one year of starting material for the fermentations regardless of their volume size.

All the features shown in the description, the following exemplary embodiments and claims can be essential to the invention both individually and in any combination with one another.

The process according to the invention is to be explained in more detail below on the basis of the two exemplary embodiments for cultivating Blakeslea trispora BS01 (+) and BSOl (-), without restricting the invention to this fungus, since other fungi of the Mucorales order are used in the process according to the invention can. 1st exemplary embodiment

0.3 1 medium with the composition soy peptone 20 g / 1, corn starch 30 g / 1, KH ₂ P0 ₄ 0.5 g / 1, sunflower oil 6.5 ml / 1, vitamin Bl 0.002 g / 1, vitamin B2 0.001 g / 1 after an amylase treatment with 20 mg amylase (BAN 800 MG, Novo Nordisk) per 2 1 medium are filled into 2-1 steep-breast flasks and after autoclaving for 30 min. up to 45 min. at 125 ° C with 1 ml spore suspension with a spore concentration of 2.0 x 10 ⁶ to 4.0 x 10 ⁶ / ml spore suspension. This spore suspension was obtained by washing off a 12-day-old test tube culture inoculated with 0.1 ml of master preserve with 11 ml of ion-free water.

The first stage of propagation takes place in a 2-1 shake flask at 180 rpm. with an amplitude of 10 cm at 27 ° C +/- 1 ° C for 24 h the same for both half-strains. The medium batch for a 30-1 inoculation fermentor consists of soy flour 20 g / 1, corn starch 20 g / 1, KH ₂ P0 ₄ 0.5 g / 1, sunflower oil 1.5 g / 1, vitamin Bl 0.002 g / 1. The soy flour and maize starch in 10 1 tap water is heated in the fermenter to 100 ° C, after cooling to 60 ° C, the potassium dihydrogen phosphate and pre-autoclaved sunflower oil are added and the volume is increased to 22 1. After adjusting the pH to pH = 6.5 to 7.0, sterilization is carried out at 121 ° C for 45 min. The inoculation of the vaccine fermenter is carried out with 0.6 1 shake culture of the BS01 (-) half-strain. Parallel to the start, the BS01 (+) half-stem is cultivated in 2-1 shake flasks in the manner described. After the 24-hour growth, the biosynthesis is induced in the BS01 (-) strain by adding 0.36 l of BS01 (+) culture in the fermenter for 60 to 180 minutes. The cultivation of the BS01 (-) strain biosynthesis-induced by the added BS01 (+) semi-strain proceeds with a constant stirring of 300 rpm. and a ventilation of 0.5 wm at 27.5 ° C. The medium of the 500-1 product fermenter consists of soy flour 25 g / 1, corn flour 10 g / 1, corn starch 40 g / 1, tocopherol-containing oil fraction 2 g / 1. The medium batch of 22.5 kg is stirred with 2.25 g of amylase (BAN 800 MG, Novo Nordisk) in 150 l of tap water; after the pH has been adjusted to pH = 6.8-7.0, it is heated to 73 ° C and after 10 minutes the temperature is raised to 100 ° C. After cooling to 60 ° C., the tocopherol-containing fraction is fed in and the volume is adjusted to 300 l. The medium thus prepared is autoclaved for 45 minutes. at 121 ° C. After setting the starting conditions, 250 rpm, aeration 0.4 wm and 27.5 ° C, the fermented induction culture is transferred to the product fermenter by pressure overlay. With the ventilation rate (up to 1.0 wm) and the stirrer speed, the 300 U / min. does not exceed, the p0 ₂ drop starting from 100% is only allowed to 10% to 20%. From this value, the p0 ₂ value increases to 40% to 60% in 72 hours. The pH value is maintained by two-point control at pH = 7.2 to 7.4 with a 20% lye or 20% acid. The growth with the utilization of proteins up to the phosphorus limitation at the 24th hour takes place with an acidification phase, which is then replaced by an alkalization phase with a moderate release of NH ₄ -N. The primary growth is completed after 24 hours with 30 g biomass / 1 by a phosphorus limitation. At the same time, owing to the induction of ß-carotene biosynthesis in the BS01 (-) half-stem already in the inoculation fermentor, the ß-carotene formation is induced early in the product fermentor from the 20th fermentation hour. The ß-carotene content develops up to 4% of the dry biomass depending on the medium composition and preparation up to the 84th hour. At the time of demolition, the deep red mushroom mycelium is homogeneous and easy to decant. 2. Execution example

The two mutant half-strains of Blakeslea trispora are grown in the manner described in Example 1 for the first stage of propagation. The medium and the inoculation of the 30-1 inoculatory fermenter with the BS01 (-) half-strain are also identical to the information given in Example 1. The cultivation of the BS01 (+) semi-strain takes place under continuous and / or cyclic conditions in a 5-1 bioreactor with a net volume of 4 1 culture. The particle-free medium contains vegetable gelatin 5 g / 1, corn steep liquor (100%) 10 g / 1, glucose 5 g / 1 and Rhodigel (Rhodia Food) 0.5 g / 1. The Rhodigel feed to the medium for the purpose of adjusting a viscosity in order to maintain homogeneous fungal growth takes place with a stock solution. To prepare this stock solution, 5 g Rhodigel / 1 are slowly stirred into a water reservoir with an Ultraturrax. The medium is 35 min. autoclaved at 125 ° C after the pH was adjusted to pH = 7.0. The medium is inoculated with 0.3 l shake culture of the BS01 (+) half-strain. After a discontinuous growth phase of 5 hours, the transition to continuous culture control with D = 0.1 h ^{"1 takes place} . The productivity of the culture is 0.5 g / 1 x h. For biosynthesis induction (mating) in the 30-1 seed reactor With a biomass concentration of 8-10 g / 1, the 22 1 (-) culture is fed 0.36 1 BS01 (+) culture, thus interrupting the continuous cultivation of the BS01 (+) semi-strain by a cycle. The induction conditions correspond to those in Example 1. The production conditions are also identical, regardless of the periodic course of the product fermentation with the induction culture, biomass from the BS01 (+) half-strain is available for the production of active biomass, homogenized biomass and the gamon-like and derepressing substances. INTERNATIONAL FORM

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The microorganism referred to in 1 has been received by this International Depository on (date of ice deposit) and an application for the conversion of this initial deposit into a deposit under the Budapest Treaty has been received on (date of receipt of the application for conversion).

V. INTERNATIONAL DEPOSIT

Name: DSMZ-GERMAN COLLECTION OF signature (s) of the person (s) authorized to represent the international depositary MIKROORGANISMEN UND ZELLKULTUREN GmbH or the authorized officer (s): Address: Mascheroder Weg I b D-38124 Braunschweig

Date: 2004-10-06 If Rule 6.4 (d) applies, this is the time when the status of an international depositary was acquired. Form DSMZ-BP / 4 (single page) 12/2001 INTERNATIONAL FORM

BIOSYNERGY GmbH Margarethenhain 7 04579 Espenhain RESPONSIBILITY FOR LIFE issued in accordance with Rule 102 by the INTERNATIONAL DEPOSIT BODY specified below

I DEPOSIT π LABELING OF THE MICROORGANISM

Name BIOSYNERGY GmbH INPUT NUMBER 04579 Espenham, assigned by the INTERNATIONAL DEPOSIT AGENT Margarethenhain 7

Address DSM 16755 Date of filing or forwarding ¹ 2004-09-23 m VITALITY CERTIFICATE

The viability of the microorganism mentioned under II is on 2004-09-27 ^! At that point the microorganism was checked

() ³ viable () ³ no longer viable

TV CONDITIONS UNDER WHICH THE VITALITY EXAMINATION HAS BEEN CARRIED OUT ⁴

V INTERNATIONAL DEPOSIT

Name DSMZ-GERMAN COLLECTION OF the person (s) authorized to represent the international depository MIKROORGANISMEN UND ZELLKULTUREN GmbH or the employee (s) authorized by her

Address Mascheroder Weg lb D-38124 Braunschweig (, Cist? Date 2004-10-06 Indication of the date of first filing If a new filing or a redirection has been made, specification of the date of the last new filing or redirection In the rule 10 2 Letter a number u and, in the cases provided, indicate the most recent life test Check if applicable Fill in if the information has been requested and if the results of the test were negative

Form DSMZ-BP / 9 (single page) 12/2001

Claims

claims

1. Process for the production of ß-carotene-containing biomass based on the microbiological synthesis of heterothallic fungi modified by mutagenesis with increased synthesis and storage capacity of ß-carotene in submerged cultures, characterized in that • the biomass is separated exclusively from spores according to (+) - and ( -) - Mating type is multiplied in several stages, • the spores of the (+) - and (-) - mating type are stored in sealed tubes after washing with ion-free water with glycerol protection at - 80 ° C to - 85 ° C, • to the biomass of ( -) - Pairing type after completion of the last biomass propagation phase before product fermentation Biomass of the (+) - pairing type or culture fluid of the (+) - pairing type or homogenized biomass of the (+) - pairing type or gamon-like stimulators or lactones or trisporic acid or trisporic acid derivatives are added and • this submerged mixture after a time-dependent one to five hour mating or he induction time is transferred to the nutrient solution of the product fermentation, the nutrient solution being composed in such a way that at the time t _w the phosphorus-rate-limited growth phase of the biomass is completed solely by phosphorus limitation, • the synthesis of ß-carotene in the product culture additionally by generating a three- up to seven-hour temperature and oxygen stress situation is triggered, • With a phosphorus limitation only after the phosphorus rate-limited growth phase without further addition of inorganic phosphorus compounds to the medium, the solvent-free extraction of the ß-carotene from the resulting dry biomass is made possible depending on the strain.

2. The method according to claim 1, characterized in that the (+) - mating types are only induction strains and their biomass is grown in continuous and cyclical culture.

3. The method according to claim 1, characterized in that the (-) - half-strains are only production strains for carotenoids.

4. The method according to claim 1, characterized in that the biomasses of the (+) - mating types are grown in cyclical culture and only the (-) - mating types are continuously multiplied in new stages.

5. The method according to claim 1, characterized in that the ratio of biomass of the respective (+) - pairing type to that of the respective (-) - pairing type when mating is 1:10 to 1:200, preferably 1:45 to 1:75 ,

6. The method according to claim 1, characterized in that the mating time or the maturing time when using culture fluid, homogenates or synthesis products is determined depending on the microorganisms used and is 1 to 180 minutes, preferably 30 to 180 minutes.

, A method according to claim 1, characterized in that the nutrient solution consists of polymeric substrates, has a phosphorus limitation, during the preparation after gelatinization of the polymeric carbon substrate is subjected to an enzyme-related liquefaction, which greatly reduces the viscosity of the solution, but does not cause monomeric carbon substrates.

8. The method according to claim 1, characterized in that the nutrient solution is composed and prepared in such a way that the biomass growth in the culture proceeds uniformly and rapidly with reduced chitin biosynthesis and only growth substrates are available for the product formation after growth has ended due to the phosphorus limitation, so that after further enzymatic digestion by the producing fungus, secondary biomass development is not possible.

9. The method according to claim 1, characterized in that stressors are used during the fermentation.

10. The method according to claim 9, characterized in that the stressors are excess oxygen and / or antioxidants.

11. The method according to claim 1, characterized in that stimulators are used.

12. The method according spoke 11, characterized in that the stimulators are vitamins and / or precursors of vitamins and / or structures related to ß-carotene from the chemical structure.

13. The method according to claim 12, characterized in that the structures related to the ß-carotene are ß-ionone, trisporic acid, carotenoids, fragments of carotenoids and / or lactones.

14. The method according spoke 1, characterized in that heterothallic mushrooms with a ß-carotene synthesis potential> 2.0% of the dry weight are used as the ß-carotene.

15. The method according to claim 1 to 14, characterized in that the heterothallic fungi Blakeslea trispora and strains derived therefrom by mutagenesis.

16. The method according spoke 15, characterized in that the heterothallic mushrooms are Blakeslea trispora BS01 (+) and BSOl (-).

17. The method according spoke 14, characterized in that the different types of Paarang heterothallic mushrooms come from a stem line.

18. The method according to claim 14, characterized in that the different mating types of the heterothallic mushrooms come from different stem lines.