DE3201428A1 - MULTI-STAGE PROCESS FOR PRODUCING FATS AND OILS - Google Patents

Description

The invention relates to a method for producing fats and oils, particularly rich in triglycerides Fats and oils from microbial sources.

It is well known that fats and oils can be produced by cultivating an oil-synthesizing microorganism, which includes algae, bacteria, mold and yeast. Such microorganisms usually synthesize oils and fats Cell metabolic process .. Extensive research is being done has been carried out in an effort to find microorganisms, media and conditions that are economically practical would allow oil production.

One area of fermentation production of fats and oils that has received particular attention is the area the production of cocoa butter substitutes. Cocoa butter

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is a naturally occurring substance that contains large amounts of 1,3-disaturated-2-unsaturated triglycerides. These Triglycerides include 1-stearoyl - ^ - oleoyl-B-palmitoyl triglycerides and 1,3-dipalmitoyl-2-oleoyl triglycerides. A Process for producing triglycerides rich in the above compounds is disclosed in U.S. Patent 4,032 described.

As described in this patent, a cocoa butter substitute is used by cultivating a microorganism from the genus F.ndomyces, Rhodotorula, Lipomyoes or Rhodospor idium among uiTobon Bed i iKjuiKjcn and ung I I cßcndcs ii.ninnel ii dor / rush and Isolate the fats and oils that are saturated with 1,3-dyesaturated-2-unsaturation Triglycerides are rich, obtained from the cells. That in the fermentation processes of the aforementioned patent The medium employed generally comprises a source of assimilable nitrogen and a source of carbon, preferably in the form of an aldose or a di- or polysaccharide. The resulting cells are collected and a mixture of fats and oils is isolated from them, which are rich in 1,3-disaturated, 2-unsaturated triglycerides.

Improvements method described dos in the aforementioned patent are USSlJ 904 Q ^r j3 (8. 5. 1979) described in the copending application. As described in this copending application, it has been found that the yield of fats and oils can be increased and the distribution of the particular fats and oils controlled when the fermentation medium contains a carbon nutrient source in the form of one or more fatty acids having 10-20 carbon atoms. For example, it has been found that the ratio of saturated to unsaturated acid groups of glyceryl oils can be controlled by using the very acids that make up the fatty acid portion of cocoa butter, namely palmitic, oleic and stearic acids.

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Further improvements to the process described in this application are in copending application, USSN (Case No. 3239), co-filed herewith, reporting that yields of the desired Fats and oils, especially those rich in triglycerides, can be increased considerably if the Fatty acid is present in the fermentation medium in the form of an emulsion. As in the aforementioned copending application , the disclosure of which is incorporated into the present application by this reference, has been found that the use of an emulsion, in particular one, the particles of fatty acid with a particle size below 10 μπι contains, effective from yeast for the production of fats and oils can be used in high yields.

While such procedures represent significant improvements in the field, there is still room for further improvement, especially in the field of produced fats and oils.

It is therefore the object of the invention to provide a method for production of fats and oils through fermentation, in which the yields of such fats and oils with a shortened reaction time can be increased considerably. In particular, the invention is intended to provide a method for producing fats and oils, in particular those that are rich in triglycerides, create from microbial sources, with yields of saturated fats and oils are increased compared to conventional methods.

The inventive concepts lie in a method for Production of fats and oils, especially those rich in triglycerides, in the yeast cells in a first stage grown in a growth medium formulated to contain carbon and nitrogen nutrients whereupon the yeast cells are cultured in an accumulation medium which is composed so that there is at least one Contains fatty acid of 10-20 carbon atoms. It was found

that the use of a two-step process increases the production of fats and oils considerably without simultaneous increases in undesirable by-products.

The method according to the invention is particularly suitable for use in the production of fats and oils of the type predominant in cocoa butter. According to one embodiment of the invention it has also been found that the generation of such oils can be increased considerably if the lipid accumulation medium is formulated in such a way that it contains palmitic acid, Oleic acid and stearic acid, preferably in the form of an emulsion, as in copending application USSN (Case No. 3239) filed at the same time.

According to the present invention, the conditions for rapid growth become the yeast cells are optimized in the first stage and the conditions for the production of saturated fats in the second stage separately optimized. Since the optimal conditions of the second stage for the production of saturated fats the rapid growth of the Yeast cells are detrimental, the two-step process offers a significant improvement over the previously known Techniques.

In the first stage of the process according to the invention, the growth-promoting medium formulated so that it contains both carbon and nitrogen nutrients in such proportions contains that the yeast cells are still in the logarithmic phase, i.e. the number of yeast cells increases logarithmically over time to increase the total number of cells present. The logarithmic phase is thus characterized characterized by a high nitrogen / carbon ratio that makes the Promotes growth in cells while keeping fat accumulation to a minimum. Then in the second stage, in the the fat accumulation is desired, the microorganism, the particular yeast, as described in more detail below, in

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a medium with a high carbon / nitrogen nutrient ratio grown to encourage the accumulation of fats and oils.

While the invention is hereinafter referred to with reference to the generation of fats and oils of the predominant type in cocoa butter is described, i.e. of triglycerides with 1,3-distearoyl-2-oleoyl triglycerides, 1-stearoyl - ^ - oleoyl-B-palmitoyl triglycerides and 1,3-dipalmitoyl-2-oleoyl triglycerides, becomes the Those skilled in the art recognize that the inventive concepts also apply to the production of other fats and oils by fermentation Can find application.

The microorganisms useful in practicing the invention can be characterized as oil synthesizing yeasts will; such yeasts are well known and available in the art. For example, there are a number of them in US Pat. No. 4,032,405, the disclosure of which is incorporated into the present application by this reference will. Particularly preferred for use in practicing the invention are species from the genus Rhodosporidium, Lipomyces, Candida, Ehdomyces, Saccharomyces, Rhodotorula, Trichosporon or Torulopsis.

Such oil synthesizing yeasts are well known and can be obtained from original sources such as conventional techniques using conventional techniques Leaves, plant stems and the like., Are isolated. However, it is generally more convenient to select such yeasts from various To obtain culture preservation / depository agencies, including e.g. the American Type Culture Collection. For economic For reasons, it is generally preferred to use an oil-synthesizing yeast which has a tendency to synthesize and Has storage of large quantities of oil. Yeasts that have the ability to accumulate 20% oil and preferably at least 30% oil on standard culture medium (such as glucose, ammonium salts and minerals) are generally preferred.

The growth and fermentation meds that provide nutrients for growing the particular yeast species to be used, depend somewhat on the for use in the method according to the invention specially selected yeast. In general, such media are dilute aqueous basic solutions that Containing carbon and nitrogen nutrient sources, im generally in amounts below 6% by weight, based on the weight of the medium. Preferred media are generally set this way or buffered so that the pH is in the range between about 4.0 and 9.0, preferably between 5 and 8.5, as it is for the optimal growing of yeast is common.

The nitrogen nutrient source can be any of a variety conventional nitrogen-containing compounds, such as those commonly used as nutrients for microbial growth are used. Preferred nitrogen compounds include asparagine, glutamine, peptones and the like. In addition, can other nitrogen-containing compounds such as ammonium salts and urea can also be used.

A nitrogenous nutrient that is especially useful for use Suitable in the practice of the invention is corn steep liquor, the aqueous liquid that is forms in conventional corn wet milling, where dry corn is soaked in warm, dilute sulfuric acid. Corn swelling liquid is made up of around 25% by weight of crude protein (8 wt .- *% nitrogen) and small amounts of ash, sugar and other beneficial culture components together. During corn source fluid can be used on its own as a cheap, yet complete nitrogen nutrient source, it can be used with other conventional nitrogen nutrient sources well known to those skilled in the art be put together.

The media should also include one or more of the known essentials Metabolic mineral salts include, including the

Salts of potassium, sodium, calcium, magnesium, iron or the like. In addition, secondary nutrients such as vitamins and amino acids are also desirable, especially if the cultivation of the yeast takes lancje.

In practicing the invention, the nitrogen / carbon nutrient ratio is 2: 1, preferably 5: 1 based on the molar ratio of nitrogen to carbon present in the nutrient medium. It was found, that the source of carbon in the growth stage is preferably at least one carbohydrate. The carbohydrate is generally in the form of an aldose (e.g. glucose, hexose, pentose, etc.), of disaccharides such as maltose, sucrose etc., and oligosaccharides, the latter preferably derived from the hydrolysis of starch. Polyhydric alcohols will be suitably used as well, with glycerin often being preferred.

In the second stage, in which the fermentation medium is a lipid accumulation medium, contains the fermentation medium a source of carbon, generally a predominant amount of one or more fatty acids having 10-20 carbon atoms. Without wishing to be bound by any theory, it is believed that yeast cells contain such fatty acids in their metabolism, and so a fatty acid content of the fatty acid medium of at least 10 and preferably 40% or more of the total carbon source is preferred. To this Wise the fatty acids, insofar as they are used to modify the metabolism of yeast cells to produce triglyceride oils Serve with a certain fatty acid content, not by the presence of other carbon nutrient sources in the Masked lipid accumulation medium. As already stated, the lipid accumulation level is characterized by a high one Carbon / nitrogen ratio from; in general it is The ratio of carbon to nitrogen in the second stage is greater than or equal to 10: 1 and preferably greater than or equal to about 50: 1.

The fatty acid (s) used as the carbon source in practicing the invention can be obtained from any of a variety of known sources. For example, palmitic acid (C.giO), stearic acid (C ₁ Q: 0) or oleic acid (Cg: 1) can be obtained commercially, either in the form of the free acid or salts such as the sodium salt. These more common fatty acids can be used alone or in admixture with others. Polyunsaturated fatty acids, such as linoleic acid (C, g: 2),

Linolenic acid (C _{1 Q} : 3) and other fatty acids with 16-20 carbons I ο

Substance atoms can also be obtained in pure form, but are more easily obtained in the cheaper form of commercially available mixtures, as obtained from soap stock.

The composition of the fatty acid used is so important that each fatty acid is a unique type of A shift in the oil-synthesizing metabolism of a given yeast species. When a mixture of fatty acids is used, their combined influence is an interaction that leads to metabolic mixtures of triglycerides, which contain the various fatty acids present in the fermentation medium.

An accurate prediction of the exact yield in the one from one special fatty acid carbon source oil composition to be obtained however, is largely based on empirical evidence. Conventional analysis methods allow the determination the yield in composition of oils produced from any particular carbon source, and thus Routine experiments permit the easy identification of fatty acid carbon suitable for the production of any particular oil sources .

However, some generalizations in the form of general rules have been determined. For example, the present leads

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a fatty acid of a given amount of carbon in the Carbon source usually increases the proportion of triglyceride esters with this fatty acid as a component of the triglyceride. Similar is the degree of saturation and / or unsaturation (and in particular the multiple unsaturation) in the oil produced directly associated with the corresponding one Degree of saturation of the fatty acid composition used as the carbon source. The use of palmitic, oleic and stearic acid as a carbon source promotes the formation of Oils that are very similar to those in cocoa butter.

The conditions under which the yeast is grown in order to produce fats and oils according to the method according to the invention, do not differ from those generally used in the field of Fermontationssyr. Learn angcwandlon. Tm general is the in practicing the invention for production The yeast used in such fats and oils is the same as in conventional processes using the same type of yeast.

The temperature at which the fermentation is carried out is generally in the range from about 20 to 40 ^° C., the higher temperatures in this range favoring the production of saturated oils, while lower temperatures in this range favor the production of unsaturated oils.

Similarly, oxygen can have some influence on yeast cell growth. In general it has been found that growing the yeast cells aerobically increases the final yield of oil produced by the microorganisms.

Once the fermentation has been allowed to go on for the desired time, generally 1 to 7 days and preferably less than 5 days, the yeast cells are separated from the fermentation medium using conventional measures and their oil content is removed. For example, the cells can first be frozen, for example

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or hydrolysis is broken and then the oil is removed from the cell debris with a suitable solvent, preferably a volatile solvent to facilitate subsequent removal of the solvent from the oil will.

As noted above, it is an important concept of the invention that the fatty acids present in the lipid accumulation medium are in emulsified form. This is preferably brought about by adding an emulsifier to the fermentation medium, which with is compatible with the fatty acids used and does not adversely affect the metabolism of the yeast cells. in the Generally, emulsifiers employed in practicing the invention are ionic or nonionic emulsifiers with an HLG over 15.

Emulsifiers in the form of fatty acid derivatives are preferred for this purpose of sorbitol and sorbitol anhydrides. Nonionic emulsifiers, such as those listed under under the trade name "Tween" (Atlas Chemical Industries Inc.) are the polyoxyethylene derivatives of fatty acid partial esters of sorbitol anhydrides, and those which are on the market under the trade name "Span", are the fatty acid partial esters of sorbitol anhydrides. Both types of emulsifiers are approved by the National Medicines Agency; it was surprisingly found that they do not adversely affect the metabolism of the yeast cells in the formation of fats and oils.

In general, only as much emulsifier is required as is required to emulsify the fatty acids present in the lipid accumulation medium sufficient to be used. In general, this amount is in the range of 0.0001 to 1%, based on the weight of the Fermentation medium. The emulsion is preferably by adding the emulsifier to the fatty acid or fatty acids and then sufficient agitation to create a practical

homogeneous medium is generated, with the remaining components of the medium either being added at the time of stirring are or not yet.

In the preferred practice of the invention, the emulsion is made to pH by heating the fatty acid with a buffer in the range of 7-9, after which the fatty acid is autoclaved to sterilize it if necessary. then the emulsifier is added and the mixture obtained is homogenized. The emulsion then becomes rapidly at one rate cooled, sufficient to crystallize the stearic acid particles in very small sizes. In accordance with the Practice of the invention has been found that particle sizes below 10 μπι are particularly suitable to ensure that the fatty acid or acids in the lipid accumulation medium are effectively used.

Having described the basic concepts of the invention, reference will now be made to the following examples, which are illustrated in FIG It is intended to illustrate and not limit the practice of the invention. In these examples, all percentages relate based on weight unless otherwise stated.

example 1

This example illustrates the practice of the invention using a two step process and with stearic acid as a carbon nutrient source.

R. toruloides yeast cells were placed in a 500 ml flask grown containing the following growth medium:

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glucose 5% Peptone 5% Yeast extract 1 % water 300 ml

The pH of the growth medium was 5.0 and the growth was continued into the late logarithmic stage (i.e. no fat accumulation).

The cells were then harvested and converted into a series of emulsions at a 1: 1 cell / lipid ratio (dry weight) given. The emulsion was made in a Teckmar homogenizer been. The control fermentation medium (sample A) had the following composition:

Stearic acid 1 % K ₂ HPO ₄ 0.1% Emulsifier (Tween 20) 0.01% antibiotic 10 ng / ml water 100 ml

Samples B to F were put together with the same composition, with the exception that they also contained the following additives:

Sample B
Sample C
Sample D
Sample E.
Sample F.

Control control control control control

0.1% glucose

0.5% glucose

1.0% glucose

0.2% glycerin

0.1% sterculinic acid

The fermentation of samples A-F took place at an initial pH of 8.0 and a temperature of 28 ° C for 3 days in a shake flask at 200 rpm. Then the oil was extracted, as in Example 1 described and submitted for analysis. The following results were obtained:

Results - Experiment 2 Table 2

sample A control 270 mg », R. 0 467 ., C 53% 204 η 180 524 - E. 511 433 F. mg neutral oil 27% 520 13th 47% 528 20% 18% 52% , 4 51% 43% * '
411 % Conversion on a lipid basis 52% 0 37% , 7 41% % Conversion on - 39 43% - 13% 9% , 6 43% 36% Lipid + CH ₂ O-base - 47% 36 , 5 - 0.3 26% , 4 0.5 0.1 - C: 12 0.4 - 0 , 5 , 3 0.5 0.6 , 5 0.6 0.3 1 C: 14 , 5 0.5 0 ,8th 0 , 5 13.4 14.9 0 , 7 12.1 9.7 0.4 C: 16-0 10 , 3 11.5 0 , 3 13th , 2 1.8 2.4 10 , 5 0.5 2.2 11.3 C: 16: 1 0 , 3 1.0 0 , 0 0 , 0 38.6 23.8 1 , 2 36.6 29.4 0.4 C: 18-0 39 , 3 42.2 7th , 5 40 , 0 32 41.9 32 , 4 39.2 44.3 53 C: 18: 1 38 ,1 35.2 54 , 7 36 , 6 0.6 5.2 41 , 4 3.5 7.7 25.4 C: 18: 2 6th , 5 0.9 36 , 6 1 ,1 0.5 0.5 7th , 6 0.6 0.5 3.2 C: 20 0 , 4 0.6 0 ,1 0 , 4 0.1 0.8 0 ,1 0.5 1.7 0.6 C: 18: 3 1 , 3 0.1 33 , 5 0 , 0 0.5 0.6 2 ,8th 0.5 0.6 0.6 C: 22 0 , 7 0.4 , 6 0 11.2 8.0 0 4.8 2.8 0.4 unknown 2 ,1 7.3 , 6 7th , 5 54.5 41.8 2 51.6 41.2 4.5 saturated overall 50 , 6 55.5 , 7 , 2 33.6 44.3 44 39.6 46.5 66.0 monounsaturated total 38 , 5 36.2 , 9 36 0.8 5.9 43 3.9 9.4 25.7 polyunsaturated total 7th 1.0 1 30.4 49 9 41 57 3.7 theoretical Jcd number # 33 34 56 29

* Values for comparison purposes

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As the above data show, the addition leads to smaller results Amounts of carbohydrates and / or glycerine lead to increased conversion efficiency. The addition of 0.1% glucose or glycerol resulted in maximum conversions and further increases in the glucose content the conversions no longer increased, but actually began to decrease when conversions went up Lipid and sugar as substrate were calculated. Sterculinic acid seemed to increase the conversions too.

A small addition of glucose (0.1-0.5%) led to very small increases in the palmitin and store levels at the expense of Oleic and linoleic acid, while 1% addition of glucose led to slightly increased desaturation at the expense of stearic acid. It appears as if glycerin slightly stimulates desaturation. The addition of small amounts of sterculinic acid resulted in greatly reduced desaturase activity and increased stearic acid values.

Example 2

This example illustrates a two step process of the invention in which fermentation time has been varied.

Cells grown as described in Example 1 were harvested by centrifugation and placed in a series of Brought emulsions at a cell / lipid ratio of 1: 1. Each emulsion had the following composition:

Lipids 1 % Stearic acid 0 .7% Palmitic acid 0 , 3% K ₂ HPO ₄ 0 /1 % Emulsifier 3 drops

Fermentation flasks held 200 ml and were running at 200 rpm and incubated at 32 ° C; the pH was kept at 8.0.

Cells were harvested after 20 hours and 2, 3, 5, 7, 9 and 11 days. The oils were extracted and analyzed. The following results were obtained:

Results - Example 2

Fermentation time (h) 20th 48 37
92.2 72 120 29
89.8 168 j 8th 216 264 i ■ % Conversion to neutral
oil
% Triglyceride - - 1 ! unknown 25 25
97.1 ' 37 41 - 37
89.8 28
85.1 - 27 30
* 89 9 25 21
87 * 16 13
75 * C: 10 - - ' - - - - 0.1 - 5 - - C: 12 [ - . ° ' ⁴ - - 0.5 - 7th - - C: 14 - - J - 0.1 0.1 0.1 - 7th - - unknown 0.6 0.5 - 0.5 0.4 0.1 0.5 7th 0.3 0.4 unknown 0.2 0.2 19.8 0.1 - 20.2 - 4th - 0.1 C: 16 - - 1.1 - - 1.1 - - - C16: 1 27 21.7 1.9 20th 20.5 1.9 20, 18.3 18.7 unknown 1.9 1.2 0.6 1 1.2 0.7 1, 7th 1.1 1.8 unknown 2.0 1.9 29.8 2 1.8 28.2 1, 1.8 1.1 C: 18th 1 0.7 40 0.6 0.6 41.4 0, 0.6 0.6 C18: 1 16.3 30.7 3.2 28.6 30.5 3.9 23, 28.1 22.9 C18: 2 46.2 38.5 0.5 42.2 40.9 0.9 47, 42.6 46.6 C20 3.3 3.2 0.6 3.5 2.7 - 2, 5.2 6.2 C18: 3 0.4 0.6 1.8 0.6 0.5 0.24 0, 0.7 0.6 unknown - 0.2 0.4 0.3 - 0.7 0.5 0.2 C: 22 0.4 0.2 - - - - 0.5 0.6 0.7 0, 0.7 0.7

* Triglyceride is% neutral oil

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In this example, 20 hours after the initial second stage incubation, the palmitin level was high during the Stearing content was relatively low. After 20 hours, the conversion was only partially complete. After 48 hours they were Palmitin and olein levels decreased, while the store level was increased. After 48 hours the composition was similar to cocoa butter and the degree of conversion was also at its maximum. Of this At this point, the conversion efficiency gradually decreased while the composition remained remarkably the same. The percentage triglycerides in the neutral oil was also highest after 48 hours and remained fairly steady around 90%. On the last day the conversion rate, percentage of triglycerides, palmitin and store levels all fell slightly, while the The olein and linoleic levels were slightly increased. The results suggest that once triglycerides are synthesized and are stored, they are little changed, but slowly processed in the metabolism to maintain energy will.

Example 3

This example illustrates a two step process in accordance with the invention for producing oils similar to those of FIG Cocoa butter.

Using the procedure of Example 2, yeast cells were made grown, harvested and at a cell / lipid ratio 1: 1 added to a range of emulsions.

A number of emulsions of the following composition were produced:

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Lipids 1%

Emulsifier (Tween 20) 0.01%

Glycerine 0.2%

K ₂ HPO ₄ 0.1%

Palmitin / 0.1% stearic acid (45/55)
pH 6.5

Sample A) incubated in flasks at 200 rpm at 32 ° C. for 72 hours a volume of 200 ml

B) like A, only incubated with Lipomyces Starkeyii

C) like A, only stirring increased to 300 rpm and the volume reduced to 100 ml and finished in 48 h, no glycerin

D) like C, only lipids increased to 3% and Tween 20 increased 0.03% increased

E) like D, only 0.6% carbohydrate added.

A. Results C. D. E. B. % Conversion to 60% 47% 36% 45 Triglyceride 37% FSZ - - 0.1 0.1 C: 12 0.5 - 0.3 0.4 0.4 C: 14 22.0 0.3 25.0 35.3 31.2 C: 16 1.3 42.0 2, '> 2.1 2.2 C: 16: 1 1.9 5.0 1.4 1.7 1.8 unknown 30th 2.4 14th 9.8 13.1 C: 18th 37 10.5 47.4 45.0 45.8 C: 18: 1 5.1 38.4 6.4 3.2 2.9 C: 18: 2 0.5 1.0 0.4 0.3 0.4 C: 20 0.6 0.1 0.6 0.3 0.3 C: 18: 3 0.2

It was observed that Sample A had a conversion rate of

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60% and the fatty acid content was quite similar to cocoa butter. Sample B using another organism (L. Starkeyii), did not have a similar composition, which shows certain differences between different yeasts.

Samples C, D, and E were at higher aeration / agitation rates (lower volume, higher number of rpm) enriched with fat and show a significant decrease in the stearate content, which shows that these factors can also control the fatty acid composition of obtained butter.

It will be understood that various changes and modifications may be made in the details of the composition, operation and processing can be made without deviating from the inventive concept, in particular as defined in the claims

Claims (11)

Claims 1. A method for producing fats and oils, characterized by growing yeast cells in a growth medium which a source of absorbable nitrogen and a source for assimilable carbon, the ratio of nitrogen to carbon being such that the yeast cells in grow their logarithmic phase by harvesting the yeast cells so grown and bringing them into contact with at least a fatty acid in a lipid accumulation medium for promotion the production of these fats and oils. 2. The method according to claim 1, characterized in that a growth medium containing at least one carbohydrate is used. 3. The method according to claim 2, characterized in that as Carbohydrate a saccharide is used. 4. The method according to claim 1, characterized in that the yeast cells are grown aerobically. 5. The method according to claim 1, characterized in that the fatty acid in the lipid accumulation medium in the form an emulsion is used. 6. The method according to claim 5, characterized in that the fatty acid used with a particle size below 10 μπι will. 7. The method according to claim 1, characterized in that a fatty acid having 10 to 20 carbon atoms is used. 8. The method according to claim 1, characterized in that a stearic acid / palmitic acid mixture is used. 9. The method according to claim 1, characterized in that a stearic acid / palmitic acid / oleic acid mixture is used. 10. The method according to claim 5, characterized in that the fatty acid has been over-emulsified with an emulsifier with an HLG. 11. Process for the production of fats and oils, as they " characteristically found in cocoa butter, characterized by growing yeast cells in a growth medium, which contains a source of assimilable nitrogen and a source of assimilable carbon, where the ratio of nitrogen to carbon is such that the yeast cells grow in their logarithmic phase, by harvesting the yeast cells thus grown and bringing them into contact with an emulsion of at least one fatty acid 10 to 20 carbon atoms to produce the fats and oils.