SK16952001A3 - Utilization of pit emulsions in fermentation processes - Google Patents

Abstract

The invention relates to the utilization of O/W emulsions in fermentation processes, said emulsions containing at least water, emulsifying agents and an oil phase that contains one or more compounds selected from the groups consisting of: a) fatty acid alkyl ester and/or b) triglycerides of vegetable origin, wherein the emulsions are produced according to the PIT method and the emulsions have an average drop size ranging from 50 to 400 nm.

Description

The present invention relates to the use of emulsions in a fermentation process which have been produced by the PIT process.

BACKGROUND OF THE INVENTION

Microbiological processes are increasingly used in the synthesis of complex natural substances or other organic compounds, such as antibiotics. This is a matter of metabolism under anaerobic or aerobic conditions, in which microorganisms, in particular bacteria or fungi, are involved. For such processes, various, not always mutually limited terms such as "bioconversion", "biotransformation" or "fermentation" are used in the scientific world. The latter term is also used in the present application for processes in which microorganisms, preferably bacteria, are used for the conversion or synthesis of chemical compounds.

In developing and optimizing fermentation processes, the reaction medium in which the microbiological exchange takes place is particularly important. The reaction medium, almost always an aqueous solution or dispersion, affects in particular the yield and efficiency of the process. To enable successful metabolism to the desired products, microorganisms such as nutrients need carbon, nitrogen and certain trace elements in bound form, for example calcium, iron, phosphorus or zinc. Furthermore, it is necessary to regularly maintain a certain, mostly narrow temperature range and pH. For more details, see the textbook by W. Crueger / A. Crueger, Biotechnology - Lehrbuch der angewandten Microbiology,

2nd edition 1984, R. Oldenbourg Verlag. In particular, Chapter 5 of this work deals with the basics of fermentation techniques. This part of the literature is therefore also expressly part of the publication of the present application. As nutrients for. microorganisms are used in addition to energy-rich sugars or derivatives thereof in many processes in addition to natural fats or oils, as well as derivatives of these classes of substances, such as glycerin, glycerides, fatty acids or fatty acid esters. Of course they must not

- Cultivation media contain no substances that could adversely affect the metabolism of the micro-organism.

For example, DE 37 38 812 A1 discloses a microbial process for the preparation of alpha-omega-dicarboxylic acids, wherein the bacteria of the Candida tropicalis strain convert methyl laurane into the desired dicarboxylic acids. The conversion is carried out in aqueous medium at pH 6.0 and 30 ° C. In addition to microorganisms, the medium contains, as an energy source, glucose, as an emulsifier, ethoxylated sorbitan monooleate, yeast extract, steeping water and sources of inorganic nitrogen and phosphorus. Methyl lauran is then added to the medium. The type of emulsion that is formed in a fermenter or in which methyl laurane is added to the medium is not apparent from the specification. EP 0 535 939 A1 discloses a process for the production of omega-9 multiply unsaturated fatty acids, wherein in an aqueous culture medium the desired multiply unsaturated fatty acids produce suitable microorganisms in the presence of sugar as a source of energy and inorganic or organic nitrogen sources as well as methyl esters. of fatty acids.

However, it is also known to use only fatty substances of the type mentioned above as energy sources. This is of particular interest from an economic point of view, since such fatty substances are generally cheaper than sugars, starches and the like. Park et al. (Park et al., Journal of Fermentation and Bioengineering, Vol. 82, No. 2, 183-186, 1996) disclose a fermentation process for the production of tylosin using microorganisms of the strain Streptomyces fradiae in an aqueous medium containing as the sole carbon source rapeseed oil in the starting amount of about 60g / l.

In the fermentation process, among other things, the oxygen content of the medium or of the fermentation mixture plays a decisive role. Oxygen plays a role of substrate in aerobic processes. It is critical whether oxygen can be transferred from the gaseous to the liquid phase containing the microorganisms that is sufficient for the process. An important parameter is the specific exchange surface, which is usually determined by the oxygen exchange coefficient to _La (cf. section of the Crueger literature, chapter 5, page 71 et seq.). The adjustment of the optimum oxygen supply is typically carried out by stirring the fermentation mixture, whereby oxygen or air is mixed into the liquid, so that at the boundary surface f

-3the gas exchange. Naturally, significant mechanical energy input by vigorous mixing, as reported by Park et al., Can also damage part of the culture and thus reduce process yield. In addition, dead microorganisms are themselves further degraded and can cause the formation of degradation products by poisoning a culture that prevents economical production. It is known from the work of Goma and Rols (G. Goma, JL Rols, Biotech. Let., Vol. 13, No. 1, pp. 7-12, 1991) that the use of soybean oil in the fermentation process of antibiotic production leads to an improvement in the coefficient. exchange of oxygen to _La , which can lead to an increase in the yield of the whole process with the same energy input (mixing).

It is an object of the present invention to improve the fermentation process so that, on the one hand, the use of cost-effective carbon sources is possible and, on the other hand, a sufficient oxygen supply of the microorganisms is ensured without impermissible mechanical loading of the microorganisms by mixing. A method should be found to minimize mechanical energy input in the fermentation process without reducing the yield. More preferably, it should be possible to increase the yield despite reduced energy input.

The use of special, fine oil-in-water (O / W-emulsions) emulsions has been found to solve the above task.

The essence of the invention

The problem has been solved by using oil-in-water (O / W-emulsions) emulsions in the fermentation process, the emulsions comprising at least water, emulsifiers as well as an oil phase and the oil phase containing one or more compounds from the group consisting of:

(a) alkyl esters of fatty acids; and / or

b) triglycerides of vegetable origin, wherein the emulsions were produced by the PIT method and have a droplet size in the range of 50 to 400 nm.

It is known that oil-in-water (O / W) emulsions which have been produced with non-ionic emulsifiers and are stabilized may, as a rule, reversible phase reversal upon heating, i.e. during a certain temperature interval, the emulsion type changes from o / v r * r

-4 to v / o (water in oil). Since the oil changes to an external, continuous phase, the conductivity of the emulsion drops to zero. The mean value of the temperature between the maximum and the zero conductivity of the emulsion when the temperature is raised is called the phase reversal temperature (PIT) and the emulsions produced in this way are called PIT-emulsions.

It is known that the position of PIT is dependent on many factors, such as the type and volume of the oil component phase, the hydrophilicity and emulsifier structure, and the composition of the emulsifier system.

Essential to the fineness of the PIT-emulsion is the process for its production. As a rule, the aqueous and oil phases are mixed with emulsifiers and then heated to a temperature higher than PIT. The conductivity must drop to zero. Subsequently, the emulsion is recooled to the initial temperature (typically room temperature, approximately 20 ° C). In this case, the emulsion used according to the invention is formed only when it is exceeded and subsequently dropped below the PIT.

It is known that only PIT-emulsions are formed which, when reversed, form a low surface tension microemulsion phase between the oil and water or a lamellar liquid crystal phase. The decisive step here is always the reverse of the cooling process. .

DE 38 19 193 A1 discloses a process for producing low-viscosity O / W emulsions by phase reversal techniques. Here, this technique applies to mixtures that contain an oil component, a nonionic emulsifier and a co-emulsifier in an aqueous medium. The oil component comprises from 50 to 100 wt. % of special mono or diesters, from 0 to 50 wt. % triglycerides of C 6 -C 22 fatty acids and optionally from 0 to 25 wt. hydrocarbon oil. DE 38 19 193 A1 does not disclose any components other than those mentioned and does not disclose the intended use of the emulsions produced.

DE 41 40 562 A1 discloses a process for the preparation of O / W emulsions by the PIT principle, wherein the polar oil components are heated together with an emulsifier system comprising non-ionic emulsifiers having a range of HLB values from 10 to 18 in the presence of co-emulsifiers of C12-C22- of fatty alcohols and / or Guerbet alcohols above the PIT-emulsion temperature and then re-cooled to give a fine emulsion.

DE 1936 35 553 A1 discloses emulsifier systems for the production of fine PITemulsions containing as essential constituents fatty acid ethoxylates and partial glycerides.

The emulsions according to the invention are characterized in particular by their fineness. The droplet size is from 50 to 400 nm. A droplet size in the range of from 100 to 300 nm is preferred, more preferably in the range of from 180 to 300 nm, and most preferably in the range of from 160 to 250 nm. The drop size is assumed to be Gaussian. The measurement is carried out, for example, by light scattering or absorption.

The fineness of the oil droplets leads to a larger surface area between the oil phase and the aqueous phase, thus allowing faster contact between the microorganisms contained in the aqueous phase and the nutrients contained in the oil phase. Large surfaces will also facilitate the exchange of gases, especially oxygen and ΟΟ ₂ . Additionally, the viscosity of the emulsion and thus of the total fermentation medium is also reduced. As a result, it is possible to greatly reduce the agitation rate of the fermentation medium, thereby allowing the yield of the fermentation process to be increased.

According to the invention, the PIT emulsions are metered into an aqueous fermentation medium containing microorganisms and optionally further excipients and additives. The details of this process, in particular the speed and amount of the emulsion to be metered, result from the type of microorganisms and the fermentation process chosen and can be adapted to those of skill in the art.

The PIT emulsions contain, in addition to water, an oil phase which contains compounds selected from the group of alkyl esters of fatty acids a) or natural vegetable oils and their derivatives b). Groups (a) and (b) are hydrophobic, water-insoluble or sparingly soluble compounds which may also serve as nutrients, ie energy sources, for the bacteria used in the fermentation process, but may also be the starting substances, ie substrates, for the desired bioconversion products.

Suitable esters of group a) are more preferably derived from saturated, unsaturated, linear or branched fatty acids having a total of 7 to 23 carbon atoms. They are therefore compounds of formula I

R ¹ -COO-R ² (I)

P P

Where R ¹ is an alkyl radical of 6 to 22 carbon atoms and R ² is an alkyl radical of 1 to 4 carbon atoms, methyl and ethyl radicals being particularly preferred. Most preferred is the use of methyl esters. The methyl esters of the formula I can be obtained in a conventional manner, for example by the esterification of triglycerides with methanol and subsequent distillation. Suitable fatty acids are caproic acid, heptanoic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, nonadecanoic acid, arachic acid and arachic acid. behenic. Unsaturated representatives are, for example, lauroeleic acid, myristoleic acid, palmitoleic acid, petrozelaidic acid, oleic acid, elaidic acid, ricinoleic acid, linoleic acid, linolaidic acid, linolenic acid, gadoleic acid, arachidonic acid and erucic acid. Mixtures of the methyl esters of these acids are also suitable. Particularly preferred is the use of such PIT-emulsions which contain methyl ester from the group methyloleate, methylpalmitate, methyl stearate and / or methylpelargonate. However, it is also possible to use methyl esters based on natural fatty acid mixtures such as are obtained from linseed oil, coconut oil, palm oil, palm kernel oil, olive oil, castor oil, rapeseed oil, sesame oil, soybean oil or sunflower oil oil and sunflower oil always new and old varieties).

Suitable compounds of group b) are natural oils of vegetable origin. In essence, they are mixtures of triglycerides, wherein the glycerin is always completely esterified with long-chain fatty acids. Particularly suitable are vegetable oils selected from the group of peanut oil, coconut oil and / or sunflower oil.

Peanut oil contains on average (based on fatty acids) 54% by weight. % oleic acid, 24 wt. % linoleic acid, 1 wt. % linolenic acid, 1 wt. % arachic acid, 10 wt. palmitic acid as well as 4% stearic acid. The melting point is from 2 to 3 ° C.

Linseed oil contains mostly 5 wt. % palmitic acid, 4 wt. % stearic acid, 22 wt. % oleic acid, 17 wt. % linoleic acid and 52 wt. Linolenic acid. The iodine number ranges from 155 to 205. The saponification number is from 188 to 196 and the melting point is about -20 ° C.

· R.

Coconut oil contains from about 0.2 to 1% by weight of fatty acids. % hexanoic acid, from 5 to 8 wt. % octanoic acid, from 6 to 9 wt. % decanoic acid, from 45 to 51 wt. % lauric acid, from 16 to 19 wt. % myristic acid, from 9 to 11 wt. % palmitic acid, from 2 to 3 wt. % stearic acid, less than 0.5 wt. % of behenic acid, from 8 to 10% oleic acid and up to 1% wt. Linoleic acid. The iodine number ranges from

7.5 to 9.5, saponification number from 0.88 to 0.9. The melting point is in the range of 20 to 23 ° C.

Olive oil predominantly contains oleic acid (cf. Lebensmittelchem. Gerichtl. Chem., 39, 112-114, 1985). Palm oil contains about 2% myristic acid, 42% palmitic acid, 5% wt. % stearic acid, 41 wt. % oleic acid, 10 wt. Linoleic acid. Paio-kernel oil typically has the following composition relative to the fatty acid spectrum: 9 wt. % caproic / caprylic / capric acid, 50 wt. % lauric acid, 15 wt. % myristic acid, 7 wt. % palmitic acid, 2 wt. % stearic acid, 15 wt. % oleic acid and 1 wt. Linoleic acid.

Rape oil typically contains about 48 wt. % erucic acid, 15 wt. % oleic acid, 14 wt. % linoleic acid, 8 wt. % linolenic acid, 5 wt. % of icosenoic acid, 3 wt. % palmitic acid, 2 wt. % decenoic acid and 1 wt. docosadienoic acid. Rapeseed oil from the new variety is enriched with unsaturated proportions. Typical proportions of fatty acids are erucic acid 0.5% by weight, oleic acid 63% by weight, linoleic acid 20% by weight, linolenic acid 9% by weight, icosenic acid 1% by weight, palmitic acid 4% by weight, % hexadecenic acid 2 wt. and docosadienoic acid 1 wt.

Castor oil is comprised of from 80 to 85% of ricinoleic acid glyceride; % of oleic acid glycerides, up to 3% linoleic acid glycerides, and up to about 2% by weight; glycerides of palmitic acid and stearic acid.

Soybean oil contains from 55 to 65 wt. % of the total fatty acid content of polyunsaturated acids, in particular linoleic acid and linolenic acid.

The situation is similar for sunflower oil, whose typical spectrum of fatty acids, based on the total fatty acid content, is as follows: about 1% myristic acid, from 3 to 10% by weight. % palmitic acid, from 14 to 65 wt. % oleic acid and from 20 to 75 wt. Linoleic acid.

All of the above data on the amounts of fatty acids in triglycerides are, as is known, dependent on the quality of the raw materials and can therefore fluctuate quantitatively. Particularly preferred are such PIT emulsions which contain a nutrient of group b) selected from the group of coconut oil, sunflower oil and / or rapeseed oil.

Important components of the PIT-emulsions used according to the invention are emulsifiers or emulsifier systems. It is preferred to use nonionic emulsifiers as emulsifiers, especially ethoxylated fatty alcohols and fatty acids.

To form PIT-emulsions, it is preferred to use a two-component emulsifier system comprising one hydrophilic emulsifier (A) and one hydrophobic co-emulsifier (B). Suitable hydrophilic nonionic emulsifiers (A) are substances having an HLB value of about 8 to 18. The HLB value (Hydrophil-LipophilBalance) is a value which is calculated according to

HLB = (100-L) / 5 where L is the weight fraction of lipophilic groups, i. % of fatty alkyl or fatty acyl groups in percentage of ethylene oxide addition products.

The fatty alcohol ethoxylates according to the invention correspond to the general formula II

R ³ -O- (CH ₂ CH ₂ O) _n -H (II) wherein R ³ represents a linear or branched, saturated or unsaturated alkyl radical having 6 to 24 carbon atoms and n is a number from 1 to 50. Particularly preferred are such compounds Particularly preferred are furthermore those compounds of formula II in which R ^{3 is an} alkyl radical of 16 to 22 carbon atoms.

The compounds of the formula II are obtained in a known manner by reacting fatty alcohols with ethylene oxide under pressure, optionally in the presence of acidic or basic catalysts. Typical examples are capro alcohol, caprylalcohol, 2-ethylhexyl alcohol, caprinal alcohol, lauryl alcohol, isotridecyl alcohol, myristyl alcohol,

-9cetyl alcohol, palmoleyl alcohol, stearyl alcohol, isostearyl alcohol, oleyl alcohol, elaidyl alcohol, petrozelinyl alcohol, linolyl alcohol, linolenyl alcohol, elaeosteryl alcohol, arachyl alcohol, gadoleyl alcohol, behenyl alcohol, erucyl alcohol and brazidyl alcohol, and their technical mixtures, from Roelen oxosynthesis as well as the monomer fraction in the dimerization of unsaturated fatty alcohols. Preferred are industrial fatty alcohols having 12 to 18 carbon atoms, such as coconut alcohol, palm alcohol, palm kernel alcohol or tallow alcohol.

Fatty acid ethoxylates which are also contemplated as an emulsifier component (A) preferably correspond to formula III

R ⁴ CO ₂ (CH ₂ CH ₂ O) _m H (III) wherein R ⁴ represents a linear or branched alkyl radical having 12 to 22 carbon atoms and a number of from 5 to 50 and more preferably from 15 to 35. Typical examples are addition products 20 to 30 moles of ethylene oxide on lauric acid, isotridecanoic acid, myristic acid, palmitic acid, palmoleic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, petrozelic acid, linoleic acid, linolenic acid, glaeoste acid, elaeoste acid , behenic acid and erucic acid as well as their technical mixtures, which are formed, for example, by pressure digestion of natural fats and oils or by reduction of aldehydes from Roelen oxosynthesis. The use of 20 to 30 moles of ethylene oxide on fatty acids of 16 to 18 carbon atoms is preferred.

The partial glycerides which are suitable for use as an emulsifier component (B) preferably correspond to the general formula IV

CH ₂ O (CH ₂ CH ₂ O) _x -COR ⁵ CH-O (CH ₂ CH ₂ O) _y -H (IV)

CH ₂ O (CH ₂ CH ₂ O) _Z -H

B t!

Wherein COR ⁵ is a linear or branched acyl radical having 12 to 22 carbon atoms and ax, y and z are in total 0 or a number from 1 to 50, more preferably from 15 to 35. Typical examples of partial glycerides useful in the invention are lauric acid monoglyceride , coconut monoglyceride, palmitic acid monoglyceride, stearic acid monoglyceride, isostearic acid monoglyceride, oleic acid monoglyceride and tallow monoglyceride, as well as adducts thereof with 5 to 50 and more preferably 20 to 30 moles of ethylene oxide. The use of monoglycerides or technical mixtures of monoglycerides / diglycerides with a predominant proportion of monoglycerides of the formula IV in which the COR ^{5 is a} linear acyl radical having 16 to 18 carbon atoms is preferred.

Other suitable emulsifiers are, for example, nonionic surfactants from one of the following groups:

(I) Addition products of 2 to 30 moles of ethylene oxide and / or 0 to 5 moles of propylene oxide to linear fatty alcohols having 8 to 22 carbon atoms;

(II) Glycerol monoesters and glycerine diesters and sorbitan monoesters and sorbitan diesters of saturated and unsaturated fatty acids having 6 to 22 carbon atoms and their ethylene oxide addition products;

(III) Alkyl monoglycosides and C 8 -C 22 alkyl oligoglycosides and their ethoxylated analogs;

(IV) Addition products of 15 to 60 moles of ethylene oxide on castor oil and / or hardened castor oil;

(V) Polyol esters and especially polyglycerol esters such as polyglycerol polyyricinoleate or polyglycerol poly-12-hydroxystearate. Mixtures of compounds of several of these classes of substances are also suitable;

(VI) Addition products of from 2 to 15 moles of ethylene oxide on castor oil and / or hardened castor oil;

(VII) Partial esters based on linear, branched, unsaturated or saturated C12 / 22-fatty acids, ricinoleic acid and 12-hydroxystearic acid and glycerin, polyglycerol, pentaerythritol, dipentaerythritol, sugar alcohols (for example sorbitol) as well as polyglucosides );

(VIII) Lanolin alcohols;

r r

-11 (IX) Polyalkylene glycols.

Ethylene oxide and / or propylene oxide addition products on glycerol monoesters and glycerine diesters as well as sorbitan monoesters and sorbitan fatty acid esters or on castor oil are known, commercially available products. These are mixtures of homologues whose mean degree of alkoxylation corresponds to the ratio of the amounts of ethylene oxide and / or propylene oxide to the substrate to which the addition reaction has been carried out.

When selecting an emulsifier system, it may be useful to perform a mathematical determination of the PIT of the system. This is particularly true for the potential optimization in the selection of the emulsifier or emulsifier system and its adaptation, on the one hand, to the selection and mixing from the aqueous phase due to other technical considerations and, on the other hand, to the type of oil phase. Appropriate expertise is developed from completely different sectors, in particular in the cosmetics sector. In particular, reference is made to T. H. Förster, W. von Rybinski, H. Tesmann and A. Wadle, "Calculation of optimum emulsifier mixture for phase inversion emulsification" in the International Journal of Cosmetic Science 16, 84-92 (1994). It shows in detail how it is possible to mathematically calculate the phase inversion in concentrates (CAPICO) method for a given three-component system consisting of an oil phase, an aqueous phase and an emulsifier based on an EACN value (equivalent alkane carbon number) ) characteristic of the oil phase. In addition to this publication by Förster et al., There is again an essential literature for the complex of topics opened here, which must be seen in connection with the publication of the publication by Fórster et al. In detail, the selection and optimization of the emulsifiers / emulsifier system are then shown in detail on the basis of a number of examples using the CAPICO method within the EACN concept for optimum setting of the determined phase reversal temperature range values.

Preferred PIT emulsions used according to the invention contain from 20 to 90 wt. % water, more preferably from 30 to 80 wt. % and most preferably from 30 to 60 wt. The residue up to 100 wt. it comprises the oil phase as well as emulsifiers and optionally other auxiliaries and additives. The oil phase itself is preferably present in amounts of from 10 to 80 wt%, most preferably from 40 to 70 wt%. The oil phase preferably comprises exclusively component a) or component b) or a mixture r e

- 12 of these ingredients. Particularly preferred is the use of such emulsions which contain an oil phase and an aqueous phase in a ratio of 1: 1. The emulsifiers or emulsifier systems are preferably present in amounts of from 1 to 25% by weight, more preferably in amounts of from 5 to 20% by weight. % and most preferably in amounts of from 5 to 15 wt. The emulsions used according to the invention preferably have a phase reversal temperature in the range of from 20 to 95 ° C, and most preferably from 40 to 95 ° C.

The described PIT-emulsions can be used according to the invention in fermentation processes of all types. It is possible to use all process arrangements known to the person skilled in the art, for example in batches, semi-continuous as well as continuous fermentation. All fermentation systems known to those skilled in the art are also applicable. For details, see Crueger, pages 50 to 70. The use of microemulsions is not even limited to certain microorganisms, but emulsions can be used to produce or convert all compounds by fermentation known to those skilled in the art. In addition to conventional fermentation processes which are mainly used for the synthesis of antibiotics (cf. Crueger, pages 197 to 242), the emulsions described are also suitable for use in microbial transformation ("bioconversion"), for example for transformation of stereides and sterins, antibiotics and pesticides or vitamins (cf. Crueger, pages 254-273). However, it is preferred to use in the fermentation process for the production of antibiotics, for example chefalosporine, tylosin or erythromycin.

As a rule, the emulsions are metered in an appropriate manner into an aqueous fermentation mixture which contains microorganisms as well as a nitrogen source and trace elements and optionally other auxiliaries, in particular antifoams. Possible sources of nitrogen are, for example: peptone, yeast and malt extract, steep water, urea or lecithins. The trace elements may be present in the form of inorganic salts, for example sodium or potassium nitrate, ammonium nitrate, ammonium sulfate, iron sulfate, etc. It may also be advantageous if the PIT emulsions themselves contain other additives, such as antifoams or nitrogen sources.

DETAILED DESCRIPTION OF THE INVENTION

Various emulsions were produced by mixing the starting materials and heating the mixture above the PIT-temperature as well as cooling to room temperature (20 ° C).

-13 (procedure according to DE 38 19193 A1). The PIT temperature was determined by conductivity measurement. Drop size was measured using a Coulter N4 Plus Submicron Particle Sizer. The measuring angle was 90 °. The results are shown in Tables 1a and 1b. The emulsions are suitable, for example, as separate sources of nutrients in the fermentation process and can be added directly to the aqueous fermentation mixture.

Table 1a

% weight. % weight. % weight. % weight. % weight. % weight. % weight. % weight. rapeseed oil 34 45 45 45 45 40 40 45 Water 55 37 37 37 37 44 44 37 castor oil, ethoxylated with 40 moles EO per mol castor oil 5 3 behenyl alcohol + 10 EO 2.3 3 5 4 3 3 3. glycerínolejan 3.7 2 1 1 hydrogenated castor oil, ethoxylated with 7 mol EO per mole of castor oil 15 15 11 13 13 12 13 PIT 84 ° C 82 ° C 68 ° C 61 ° C 68 ° C 72 ° C High: 57 ° C 75 ^e C drop size 318 nm 293 nm 230 nm 187 nm 195 nm 229 nm 175 nm 296 nm

Table 1b

% weight. % weight. % weight. % weight. % weight. metylolejan 45 45 sunflower oil 45 40 coconut oil 34 Water 45 45 55 37 44 C16 / 18-fatty alcohol + 12 EO 5 7 castor oil, ethoxylated with 40 moles EO per mol castor oil 4 behenyl alcohol +10 EO 2.8 3 3

-14e e · e r *> · · p e r y r y r

glycerol monostearate 3 glycerínolejan 4.2 hydrogenated castor oil, ethoxylated with 7 mol EO per mole of castor oil 15 12 C16-fatty alcohol + 6 EO 5 PIT 73 ’C 74 ’C 62 ’C 68 ’C 57 ’C drop size 201 nm 216 nm 156 nm 219 nm 180 nm

r r r r r r r r r C C C C C

Claims (10)

Use of oil-in-water emulsions in a fermentation process comprising at least water, emulsifiers as well as an oil phase comprising one or more compounds selected from the group consisting of: (a) alkyl esters of fatty acids; and / or b) triglycerides of vegetable origin, wherein the emulsions were produced by the PIT method and have a droplet size in the range of 50 to 400 nm. The use according to claim 1, wherein the oil phase comprises as component a) fatty acid methyl esters. Use according to claim 1 or 2, wherein the emulsions have a mean droplet size in the range from 100 to 300 nm, more preferably from 180 to 300 nm and most preferably from 160 to 250 nm. Use according to claims 1 to 3, wherein the emulsions contain water in amounts of from 20 to 90% by weight, more preferably from 30 to 80% by weight. % and most preferably from 30 to 60 wt. Use according to claims 1 to 4, wherein the emulsions comprise the oil phase in amounts of from 10 to 80% by weight, more preferably from 40 to 70% by weight. Use according to claims 1 to 5, wherein the emulsions comprise a fatty acid methyl ester of the general formula I R ¹ -COO-R ² O) wherein R ¹ is an alkyl radical of 6 to 22 carbon atoms and R ² is a methyl radical. Γ r Γ -167. Use according to claims 1 to 6, wherein the emulsions comprise in the oil phase methyl oleate, methyl palmitate, methyl stearate and / or methylpelargonate. Use according to claims 1 to 7, wherein the emulsions comprise in the oil phase coconut oil, sunflower oil and / or rapeseed oil. Use according to claims 1 to 8, wherein the emulsions comprise an emulsifier system composed of a hydrophilic emulsifier having an HLB value of 8 to 18 in combination with a hydrophobic co-emulsifier. Use according to claim 9, wherein emulsions are used whose emulsifier system has a quantitative ratio between 10:90 and 90:10 between the hydrophilic emulsifier and the co-emulsifier. Use according to claims 1 to 10, wherein emulsions are used which contain emulsifiers in amounts of from 1 to 25% by weight, more preferably in amounts of from 5 to 20% by weight. % and most preferably in amounts of from 5 to 15 wt.