AU617103B2 - Process for the preparation of fats usable as cacao butter substitute or as a component of a cacao butter substitute - Google Patents

Description

COMMONWEALTH OF AUSTRA6 7 orm Patents Act 1952-1969 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: Class Int. Class Application Number: Lodged Complete Application No.

Specification Lodged Publ:shed

S

riority: Related art: a o st o Name of Applicant: SAddress of Applicant: Actual Inventor: Address for Service: TO BE COMPLETED BY APPLICANT WESSANEN NEDERLAND B.V.; Prof E.M. Meyerslaan 2, 1183 AV AMSTELVEEN, The Netherlands Elizabeth Catharina VERBREE, Adriaantje YKEMA, Ira Ida Gerritdina Sagitta VERWOERT, and Henk SMIT Care of COLLISON CO., 117 King William Street, ADELAIDE S.A. 5000 Complete Specification for the invention entitled: Process for the preparation of fats usable as cacao butter substitute or as a component of a cacao butter substitute The following statement is a full description of this invention, including the best method of performing it known to fIm us:

T

r Sa Process for the preparation of fats usable as cacao butter substitute or as a component of a cacao butter substitute.

The invention relates to a process for the preparation of fats usable as cacao butter substitute or as a component of a cacao butter substitute comprising cultivating a fat-producing microorganism and subsequently recovering the fats accumulated in the microorganism.

One of the most valuable vegetable fats is cacao butter. This is a naturally-occurring product recovered from seeds of the cacao tree (Theobroma cacao). More in particular cacao butter is a ft product mainly consisting of 1.3-disaturated-2-unsaturated tri- 4t glycerides, containing stearic acid, oleic acic and palmitic acid as fatty acid components. In the following Table A the average fatty acid composition of cacao butter is illustrated (Fincke A., or, (1965), Handbuch der Kakaoerzeugnisse, page 333, Springer Verlag).

0 4 TABLE A Palmitic acid (C 16) 23 30 by weight Stearic acid (C 18) 32 37 by weight Oleic acid (C'18) 30 37 by weight Linoleic acid (C18) 2 4 by weight Linoleic acid 2 0.3 by weight Linolenic acid (C 2018) 0 0.3 by weight Arachic acid (C 20) 0 0.2 by weight Behenic acid (C 22) 0 0.2 by weight Lignoceric acid (C 24) trace In view of the strongly fluctuating offer, ijality and price of cacao butter having generally recognized unique properties the need for this product is still growing and the natural production of this product cannot or hardly meet the demand. Although the increasing need Tor cacao butter can partly be met with analogues having a comparable triglyceride-composition (cacao butter-substitutes) these substitutes do not give a real contribution to the f i 2 solution of this problem because next to the palmitic acid/oleic acid/palmitic acid-rich (POP-rich) palm oil component, these analogues are based for a considerable part on stearic acid/oleic Ecid/stearic acid-rich (SOS-rich) exotic fats like Sheafat and Tengkawan,the properties, price and availability of which imply a clear restriction. For this reason research has been carried out for an alternative preparation method for cacao butter substitutes respectively for the exotic SOS-rich component thereof which have both a good and constant quality and a low cost price.

It is known that fats and oils can be produced by cultivating oil/fat-producing microorganisms like algae, bacteria, moulds and yeasts. Such microorganisms synthesize the lipids in the ordinary 414 course of their cellular metabolism. Extensive research has been carried out in an effort to find the microorganisms, media and t r I S 15 conditions which would permit an economic production of cacao butter substitutes or components thereof.

All kinds of propositions concerning the preparation of cacao Sbutter equivalents have been disclosed in the prior art. For instance, from Dutch Patent Application 78,04549 a process for the 20 microbial production of fats usable as a cacao butter substitute is known according to which the medium for the fat-producing microorganism contains a mixed carbon nutrient at least consisting of a stearoyl derivative and at least a palmitoyl derivative, an oleoyl derivative and a saccharide. Strains of the genera Candida, Torulopsis, Trichosporon, Pichia and Sporobolomyces are mentioned as Smicroorganisms suitable for the production. However, such a process has the disadvantage that a medium should be applied meeting specific high requirements. Such media are expensive so the process according to above Dutch Patent Application 78,04549 cannot or hardly be used on economic scale.

From European Patent Specification 0,005,277 a microbiological process for the preparation of oils and fats is known which is based on: a) preparing a growth medium in which the carbon nutrient comprises fatty acids having from 10 to 20 carbon atoms, b) inoculating said growth medium with oil synthesizing yeast cells like strains of the genera Rhodosporidium, Lipomyces, Candida, Saccharomyces, Endomyces and Rhodotorula, .1 i.

3 c) cultivating said yeast cells under aerobic conditions, a pH between 4.0 and 6.0 and a temperature between 20 40 0

C,

d) separating the cultivated cells from said growth medium and e) recovering the oil from the cultivated cells.

Examples of such fatty acids to be used as carbon nutrient are palmitic acid, oleic acid and stearic acid as well as mixtures thereof. Therefore this last process has also the disadvantage that the production of a cacao butter substitute cannot be carried out o 10 in an economic way in view of the required expensive substrate. In o 10 this respect it is pointed at the phrase mentioned in the main claim indicating that the composition of the oil synthesized in dependent on the ratio of saturated fatty acid to unsaturated fatty Sacid present in the growth medium.

Further processes for the production of fats and oils with the 0 0 help of fat-producing microorganisms requiring at least one fatty acid having 10-20 carbon atoms as carbon nutrient in the growth o medium are known from U.S. Patent Specifications 4,485,172 and o'o 4,485,173. Preferably palmitic acid, oleic acid and stearic acid as .0 well as mixtures thereof are used as carbon nutrient, whereas the 0 20 relative concentration of these acids is adjusted in such a way that cacao butter substitutes can be produced. Strains of the genera Rhodosporidium, Lipomyces, Candida, Endomyces, Saccharo- S4° myces, Rhodotorula, Trichosporon and Torulopsis are mentioned as suitable yeasts. On account of the fact that for the production of cacao butter substitutes growth media should be applied comprising a carbon nutrient with the above specified composition it is stated that the processes known from said U.S. Patent Specifications are not considered practical in an economic way.

In the above mentioned U.S. Patent Specification 4,485,173 also the application of a desaturase-enzym-inhibitor like sterculic acid being a cyclopropene fatty acid is mentioned resulting in triglycerides having a higher stearic acid/oleic acid-ratio. It is stated in this U.S. Patent Specification that such an inhibitor strongly reduces the desaturation of stearic acid into oleic acid.

In this respect it is also pointed at European Patent Application 0,159,137, in which many cyclopropene fatty acids are disclosed as desaturase-enzyme-inhibitors for the production of cacao butter 4 substitutes. ith respect to such desaturase-enzyme-inhibitors which are all fatty acids it is brought to the fore that these acids are inserted into the triglycerides to be formed by the microorganisms and for that reason do form a part of the produced cacao butter substitute. However, this phenomenon is unwanted from a toxicological point of view because from many references like Chemical Abstracts, Vol. 70 (1969) 967,c, Vol. 92 (1980) 20804t, Vol. 65 (1966) 4520e and Vol. 62 (1965) 6866a it appears that after consumption these desaturase-enzyme-inhibitors may cause very detrimental effects in test animals.

Finally from U.S. Patent Specification 4,235,933 a process for the production of oils with the help of an oil-producing strain of Candida curvata is known, in which process a whey permeate obtained by ultrafiltration is used as a cheap substrate. Examples of appropriate microorganisms are the Candida curvata strain D, ATCC No.

20509 and the Candida curvata strain R, ATCC No. 20508. However, on account of a too high content of unsaturated fatty acids the quality of the product obtained according to this process is insufficient with respect to the quality of cacao butter. For that reason this product is merely used in animal feed materials.

The invention relates to a process for an economic production of cacao butter substitutes or components thereof by using as a microorganism a mutant of a yeast from the genera Apiotrichum, Candida, Cryptococcus, Endomyces, Hansenula, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Sporobolomyces, Torulopsis, Trichosporon and Yarrowia or a fusant derived from in total two, three of four, genetically different mutants and/or yeast cells, all belonging to the above mentioned genera, wherein the enzyme system responsable for the conversion of stearic acid into oleic acid is totally or partially genetically blocked.

Within the scope of the invention the term "cacao butter substitute" stands forafatty mixture comprising for more than perferably more than 50%, of 1.3-disaturated-2-unsaturated triglycerides containing saturated or unsaturated C 1 6 and

C

18 -fatty acids as fatty acid components. Further the term i _i 1 "component of a cacao butter substitute" stands forafatty mixture comprising for more than 25%, preferably more than 50%, of 1.3-disaturated-2-unsaturated triglycerides, containing saturated and unsaturated forms of C 18 -fatty acids as fatty acid components.

The mutants of fat-producing yeast genera used in the process according to the invention like preferably Apiotrichum curvatum, also called desaturase-mutants, are obtained by means of mutagenation. This mutagenation can be carried out with for instance chemical mutagentia like N-methyl-N'-nitroso-guanidine, ethylmethane sulphonate or by means of radiation with UV-light. The screening of the desaturase-mutant can be performed by the 1 application of nutrient substrates to which oleic acid has been added or not.

Within the scope of the invention at first those mutants are isolated wherein the enzyme "desaturase" catalysing the conversion of stearic acid into oleic acid is inactive. The desaturase-mutants isolated in this way are dependent on the addition of oleic acid to the growth medium. In this respect it is stated that starting from the isolated "absolute" desaturase-mutants wherein the enzyme desaturase is completely inhibited, "partial" desaturase-mutants are isolated (if necessary after induction with a mutagens), wherein the desaturase is only partially inhibited; said partial 'desaturase-mutants do not need oleic acid for their growth.

As seen under a phase-contrast-microscope the morphology of the isolated absolute desaturase-mutants is clearly different from that of the wild type Apiotrichum curvatum (ATCC 20509). Some mutant cells are larger and have a clear tendency to pseudomycelia formation. Under fat producing conditions in the presence of limited amounts of oleic acid one may observe a granular structure in said desaturase-mutants which indicates solid fat at the used cultivation temperature of 30'C; this structure is in contrast to that of the wild type showing intracellular oil drops.

The results with shake cultures and fermentor batches indicate that the absolute desaturase-mutants in the presence of a small amount of oleic acid and partial desaturase-mutants also in the absence of oleic acid do have a normal growth phase and fat production phase.

I_

6 One of the absolute desaturase-mutants of Apiotrichum curvatum ATCC 20509 has been deposited in accordance with the Budapest treaty under the number CBS 199.87 with the Centraal Bureau voor Schimmelcultures, the Netherlands.

On account of the fact that absolute desaturase-mutants of Apiotrichum curvatum need oleic acid for the synthesis of membrane lipids the "partial" revertants of desaturase-mutants are preferred because these revertants are only partially inhibited in their desaturase activity and for that reason capable to grow in the absence of oleic acid. This advantage manifests itself in a favourable influence on the cost price of the growth medium.

The growth media necessary for the cultivation of the specific ri: yeast cells according to the invention include in general dilute aqueous solutions containing carbon and nitrogen nutrients, generally less than 6% by weight, based on the weight of the medium. Usually, sugars like glucose or lactose are used as carbon nutrient and energy source. Ammonium salts are a convenient nitro- 4a gen nutrient, however, use can also be made of other conventional nitrogen-containing compounds like urea, asparagine, glutamine, peptones and the like.

In general, high carbon-to-nitrogen-ratios are favourable in respect of the fat production. The nitrogen promotes the growth and the fission of the yeast cells during the growth phase and the remaining carbon is converted into accumulated lipids. Normally the carbon-to-nitrogen-ratio in the growth medium is between 10 and 100 (g carbon per g nitrogen) and preferably between 20 and Next to carbon and nitrogen the medium should also include the elements essential for the growth like potassium, sodium, magnesium, iron, sulfur, calcium and zinc, if necessary. These elements are added in the salt form.

The growth medium is buffered in such a way, that the pH, dependent on the yeast species, has a value in the range of from to 9.

The temperature at which the fermentation is carried out is generally within the range of about 20 to 40°C. Further, oxygen may have a favourable effect on the growth and fat production of the yeast cells.

7 Following up the above cheap whey or whey permeates or molasses can be used as substrate in the process according to the invention. For obtaining an optimum C/N-ratio one may add NH 4 to the growth medium. By using fed batch cultivations and in particular partial recycle cultivations of Apiotrichum curvatum in wheypermeate very high fat production ratios could be achieved so that an economical production of yeast lipids has become possible.

Once the carbon nutrient has been consumed completely or for the major part, the yeast cells are separated from the growth medium by conventional methods and their fat content removed. For example, their cell-structure can be destroyed by freezing, hydrolysis or elevated pressure and then the fat extracted with a suitable solvent.

The analysis data reveal that with the help of the mutants according to the invention one may produce triglycerids having a percentage of saturated fatty acids comparable to that of cacao butter.

Further to the above mentioned desaturase-mutants of Apiotrichum curvatum absolute desaturase-mutants of Saccharomyces cerevisiae ATCC 25657 have been prepared and isolated. These absolute mutants are also dependent on oleic acid for their growth.

Test results obtained with an absolute desaturase-mutant of said Saccharomyces strain, cultivated in the presence of different amounts of oleic acid indicate that the percentage of saturated fatty acids in the yeast lipids can be varied to a saturation ratio comparable to that of cacao butter. With respect to the results obtained with the desaturase mutants of Apiotrichum and Saccharomyces it may be concluded that beyond doubt the process according to the invention for the production of cacao butter substitutes is applicable to any yeast species belonging to the genera listed above.

Next to the above described "absolute" and "partial" desaturase-mutants obtained by means of mutagenisation the invention also relates to the preparation and application of fusants derived from two, three or four, genetically different mutants and/or yeast cells, all belonging to one or more of the genera Apiotrichum, Candida, Cryptococcus, Endomyces, Hansenula, Lipomyces, Pichia, -L 1 1.1 Rhodosporidium, Rhodotorula, Saccharomyces, Sporobolomyces, Torulopsis, Trichosporon and Yarrowia. More in particular these fusants are obtained by fusing protoplasts of the two fusion partners. So a fusant according to the invention is a new yeast strain having the genetic information of both parents and capable of passing it on to its descendants.

Within the scope of the invention fusants are isolated which are derived from an amino acid auxotrophic mutant of Apiotrichum curvatum and a desaturase-mutant of Apiotrichum curvatum (CBS 199.87) as fusion partners. A part of the new strains resulting from this fusion showed a good growth in whey permeate and had a satisfying fat production, the quality of which approached that of cacao butter. Such a fusant has been deposited in accordance with the Budapest treaty under the number CBS 159.88 at the Centraal Bureau voor Schimmelcultures, The Netherlands.

Further fusions have been carried out between strains of different genera, such as Apiotrichum, Saccharomyces and Yarrowia (intergeneric fusions). From literature it is known that fusants often contain the complete genome of one of the parent strains but only a part of the genome (some chromosomes or even some genes) of the other parent strain (Peberdy, J.F. (1987), Microbiol. Sciences 4 108). For that reason a fusant may contain specific genes in duplicate an other genes not. Hybrides according to the invention, produced with the help of intergeneric fusions will show a great diversity, however, the most favourable fusants are easily detectable on the basis of their properties.

The invention is elucidated in the examples mentioned below; these examples should not be interpreted in a restrictive way.

Example 1 A) Isolation procedure used for obtaining "absolute" desaturasemutants derived from the fat-producing yeast strain Apiotrichum curvatum ATCC 20509.

The following Table B lists the parameters applied for the production of desaturase-mutants of Apiotrichum curvatum. The i i a i 9 starting strain was Apiotrichum curvatum ATCC 20509 corresponding to the above mentioned strain Candida curvata ATCC 20509 mentioned in U.S. Patent Specification 4,235,933.

TABLE B Isolation method of absolute desaturase-mutants 4 4I 14 4 4 0 4 4 4 44 4 4 4 4 4 4404 Starting strain: 10 Mutagens: dose: incubation medium: growth phase: density of suspension: 15 incubation time: incubation temperature: number of treated cells: survival: number of desaturase mutants: mutant frequence: Apiotrichum curvatum ATCC 20509 N-methyl-N'-nitroso-guanidine (NG) 500-1000 microgram NG/ml 0.1 M of phosphate buffer, pH logarithmic E660 30-180 min.

30 0

C

2.10 9 0.0014 0.025% 6 1 per 16.000 A 1 per 80.000 B) Isolation procedure used for obtaining "partial" desaturasemutants starting from an absolute desaturase-mutant of the fatproducing yeast strain Apiotrichum curvatum ATCC 20509.

The following Table C lists the parameters applied for obtaining the partial desaturase-mutants. The absolute desaturasemutant of Apiotrichum curvatum ATCC 20509 deposited under number CBS 199.87 has been used as starting strain.

I I 1 sl TABLE C Isolation method of partial desaturase-mutants Starting strain: Mutagens: dose: incubation medium: growth phase: density of suspension: incubation time: incuabtion temperature: survival: (double) mutant frequence: Absolute desaturase-mutant CBS 199.87 N-methyl-N'-nitroso-guanidine (NG) 200-500/ug NG/ml 0.1 M phosphate buffer pH logarithmic

E

6 6 0 30-120 min.

0.00008 1 per 140,000 tot 1 per 1,500,000 Example 2 Application of the isolated absolute desaturase-mutant and partial desaturase-mutants isolated as well as an analysis of the produced products.

In a continuously stirred fermentor provided with an aeration pipe and a pH controlling device a pure culture of an absolute or of a partial desaturase mutant of Apiotrichum curvatum ATCC 20509 was inoculated on a medium having the following composition: 4 a Glucose Oleic acid Tween 80

NH

4 Cl KH2PO4 Na 2 HP04 MgSO 4 Yeast extract CaC12 FeC13 ZnSO 4 Water 10 g 0-0.12 g 0-0.3 g 0.25 g 3.5 g 1,0 g 0.75 g 0.75 g 50 mg 5 mg 0.5 mg 1 liter 11 The cells were cultivated at a pH of 5.0 and a temperature of and were harvested after the glucose present in the growth medium was consumed. The lipids were extracted with organic solvents and analysed for their triglyceride-composition and fatty acid distribution (according to methods generally known in the art).

The following Table D shows the test results of an absolute desaturase-mutant according to the invention, cultivated in the S 10 presence of different amounts of oleic acid and of four partial desaturase-mutants according to the invention in respect of the results obtained with the wild type (Apiotrichum curvatum ATCC r. I 20509).

I 4 tt4 I 4 to .4.4 4 4 f0 '1 4° '144 f 4 TABLE D Fatty acid compositions of mutants of Apiotrichum curvatum in which the activity of the enzyme desaturase is inhibited totally or partially with the fatty acid composition of the wild type ATCC 20509.

Strain ATCC CBS CBS CBS R22.7 R22.14 R22.50 R22.102 Cacao 20509 199.87 199.87 199.87 butter g oleic acid/g glucose 0 0.042 0.080 0.120 0 0 0 0 g Tween 80/g glucose 0 0.105 0.20 0.30 0 0 0 0 g fat/g glucose 0.12 0.16 0.19 0.23 0.12 0.12 0.12 0.12 fatty content 30 40 53 58 30 30 30 fatty acid composition: C14 0.53 0.78 0.70 0.84 0.57 1.48 1.08 1.17 C16 28.10 21.95 21.20 19.20 12.83 30.10 22.60 25.80 23-30 C16.1 0.39 0.63 1.16 1.91 C18 13.90 52.13 45.40 35.70 35.51 29.60 32.60 41.30 32-37 C18.1 43.70 9.73 18.20 28.70 39.47 28.00 36.70 16.40 30-37 C18.2 9.57 7.17 7.74 8.58 5.45 5.70 1.13 7.77 2-4 C18.3 1.74 1.97 1.19 1.01 1.07 1.18 0.27 3.62 0-0,3 0.31 0.93 0.80 0.62 2.05 0.37 1.16 1.09 0-1 C22 0.30 0.77 0.70 0.56 0.68 0.57 0.74 0.75 0-0,2 C24 1.05 2.84 2.24 1.94 0.52 1,08 0.93 1.36 trace SFA* 44.25 79.48 71.23 59.11 53.01 64.39 59.67 70.74 60 3 TGL** 95 87 94.3 96.3 95 95 95 95 96 *%SFA saturated fatty acids **%TGL triglycerides Example 3 In this example further details are given concerning the distribution of the saturated an unsaturated fatty acids on the 1and 3-positions and the 2-position respectively of the glycerol molecule for the fat products obtained by means of the mutants according to the invention and the wild type Apiotrichum curvatum ATCC 20509 respectively. Such a distribution is considered important because for vegetable lipids like cacao butter it is characteristic that unsaturated fatty acids (oleic acid) are preferred localized on the 2-position of the glycerol molecule. The absolute desaturase-mutant CBS 199.87 and two partial desaturasemutants i.e. the revertants R33.192 and R38.11 are used as the mutants according to the invention.

For the determination of the position of the fatty acids in the obtained fat products use has been made of a IUPAC-method i.e.

IUPAC 2.210: "Determination of fatty acids in the 2-position in the triglycerides of oils and fats".

It appears from Table E illustrated below that for Apiotrichum curvatum ATCC 20509, the absolute desaturase-mutant CBS 199.87 and the partial desaturase-mutants R33.192 and R38.11 the unsaturated fatty acids do preferably have the 2-position of the glycerol molecule which is also the case in cacao butter.

I TABLE E Table E-l. fatty acid position determination A.curvatum ATCC 20509 Table E-2. fatty acid position determination CBS 199.87 fatty by weight in acid triglycerides by weight on the 2-position fatty acid by weight in triglycerides by weight on the 2-position C16 C16.1 C18 C18.1 C18.2 C18.3 28.1 13.9 43.7 9.6 1.7 C16 C16.1 C18 C18.1 C18.2 C18.3 19.2 1.9 35.7 28.7 8.6 1.0 83.7 12.5 0.4 56.3 21.9 1.1 Table E-3. fatty acid position determination partial desaturase-mutant R33.192 Table E-4. fatty acid position determination partial desaturase-mutant R38.11 fatty by weight in acid triglycerides by weight on the 2-position fatty acid by weight in triglycerides by weight on the 2-position C16 C16.1 C18 C18.1 C18.2 C18.3 C24 16.9 3.3 27.9 30.3 13.3 2 2.6 3.0 2.0 60.7 28.6 2.4 0.3 C16.1 C18 C18.1 C18.2 C18.3 C24 33.6 0.2 17.4 34.4 9.5 1.5 3.2 0.3 1.8 74.2 16.1 1.1 I ~1~II~C~C~ Example 4 A) Isolation procedure used for obtaining desaturase-mutants of the yeast strain Saccharomyces cerevisiae ATCC 25657.

The following Table F lists the parameters applied for the production of desaturase-mutants of Saccharomyces cerevisiae ATCC 25657.

TABLE F Isolation method of absolute desaturase-mutants o0 0 9o o 0 0 I o n e 0 0000 00 000oo 0 00 O Oee 0a.

0000 0 0 00 r0 OQ e starting strain: mutagens: dose: incubation medium: growth phase: density of suspension: incubation time: incubation temperature: inactivation of EMS: inactivation time: inactivation temperature: number of treated cells: survival: number of desaturase-mutants: mutant frequence: B) Fatty acid composition of the Saccharomyces cerevisiae ATCC Saccharomyces cerevisiae ATCC 25657 ethylmethane sulphonate (EMS) 0.1 M of phosphate buffer pH logarithmic E660 120 min.

5% of sodium thiosulphate 30 min.

room temperature 250,000 15 3 1 per 6250 fats produced by desaturase-mutants of 25657.

The following Table G shows the test results obtained with an absolute desaturase-mutant of Saccharomyces cerevisiae (UFA3), cultivated in the presence of different amounts of oleic acid and with the wild type Saccharomyces cerevisiae ATCC 25657.

Th- 16 TABLE G Strain 4*6 44 6 4 4 4 4 44 4 4 4 444 4.

444444 4 4 444* 4.444 .64444 6 4 4 44 4 4 4 4 44 4 44 4 4 4 4 44 4444 4444 medium mg oleic acid/g glucose mg Iween 80/g glucose fatty acid composition: do0 C12 C14 C1 6 C16 .1 018 C18 .1 C18.2 C18.3

ATC

25657 IL5* 0 0 1 .34 4.88 6.19 17.98 35 .43 8.55 19.63 4.52 1.49 M,5 0.1 2.5 1.31 6.01 8.20 39.18 0.00 10.79 21.26 6.54 7.03 M5 0.5 12.5 4.46 7.63 11.33 31.24 0.00 18.93 18.12 7.42 0.85 t5 1.0 25.0 1.90 11.88 8.05 21-41 6.96 8.67 20.36 12.01 8.74 M45 250 3.38 3.32 4.22 10.39 11.74 4.01 54.81 1.99 2.41 UFA3 UFA3 UFA3 UFA3 %SFA** 38.94 65.50 73.59 51.91 25.32 M 15 medium has been described in Example 2 *%SFA saturated fatty acids 4 44 I a i; 17 Example The following Table H shows a review of the parameters applied for the production of fusants based on an amino acid auxotrophic Apiotrichum curvatum-mutant obtained by means of mutagenation with the help of nitroso-guanidine and the desaturase-mutant of Apiotrichum curvatum having the deposition number CBS 199.87.

TABLE Isolation method for the production of fusants (hybrides) derived from CBS 199.87 and an amino acid auxotrophic mutant of A. curvatum ATCC 20509.

I. Isolation of protoplasts a) cultures of CBS 199.87 and an amino acid auxotrophic mutant of ATCC 20509 were harvested in the logarithmic growth phase by means of centrifugation.

b) the cell pellets were washed once with sterile bidest.

c) after centrifugation the pellets were introduced in a buffer of 1 M sorbitol, 25 mM EDTA 50 mM d) the suspensions were incubated for 30 minutes in a shaking water bath at 30 0

C.

e) after centrifugation the pellets were introduced in 1.2 M sorbitol.

f) after centrifugation the pellets were introduced in a buffer of 1.2 M sorbitol, 100 mM sodium citrate, 5 mM DTT 2 Smg/ml Novozym 234, pH=5.8.

g) the suspensions were incubated for 15-30 minutes in a shaking water bath at 30 0

C.

h) the formed protoplasts suspensions were washed three times in a buffer of 1.2 M sorbitol 10 mM Tris-HCl, i) after centrifugation the pellets were introduced in a buffer of 1.2 M sorbitol 10 mM Tris-HCl, (II) Fusion of protoplasts and isolation of fusants (hybrides).

a) about 5.108 protoplasts of each fusion partner were combined and well mixed.

18 b) after centrifugation a PEG-solution (35% of polyethylene glycol 4000 10 mM CaC12 10 mM Tris-HCl, pH=8.0) was poured on the pellet and subsequently mixed therewith.

c) the fusion mixture was incubated for 30 minutes in a shaking water bath at 30 0

C.

d) after centrifugation the pellet was washed once in a buffer of 1.2 M sorbitol 10 mM Tris-HCl, pH=8.0 and suspended again in the same b. ffer.

e) this suspension was spooned out on a selective medium consisting of 0.67% of bacto yeast nitrogen base without amino acids, 2% dextrose, 1.2 M sorbitol 1.5-3% agar.

f) colonies of fusants (hybrides) appeared after an incubation time of 4-7 days at 30 0

C.

the original fusion partners cannot grow on this medium).

A fusant obtained in the above described way has been deposited according to the Budapest treaty under number CBS 159.88 with the Centraal Bureau voor Schimmelcultures, The Netherlands.

Example 6 Application of several fusants obtained according to the process j i mentioned in Example 5 as well as the analysis of the products obtained.

In a continuously stirred fermentor, provided with an aeration pipe and a pH controlling device a pure culture of three fusants obtained according to the methods described in Example 5 as well as a strain of A.curvatum ATCC 20509 was inoculated on a medium of whey permeate in which the carbon/nitrogen-ratio had been adjusted to 30 by means of NH 4 C1.

The cells were cultivated at a pH of 5.0 and a temperature of and were harvested after that the lactose in the growth medium had been consumed. The lipids were extracted with organic solvents and analysed for triglyceride-composition and fatty acid distribution (according to methods generally known in the art).

A

Table I shows the test results obtained with three fusants obtained according to the process described in Example 5 and with the wild type (Apiotrichum curvatum ATCC 20509).

TABLE I Strain C14 C16 C16-1 C18 C18-1 C18-2 C18-3 C22 C24

%SFA*

%TGL**

CBS 159.88 F 33.54 F 33.05 ATCC 20509 Cacao butter 1.72 23.75 0.35 31.21 29.58 5.61 1.07 1,54 0.16 3.64 62.57 95 1.61 27.85 24.13 31.64 7.33 0.92 1,29 0.62 3.38 59.05 95 1.52 32.39 0.39 25.42 28.81 6.41 0.52 1,11 0.50 1.69 62.63 95 0.53 28.10 0.39 13.90 43.70 9.57 1.74 0,31 0.30 1.05 44.25 95 23 32 37 30 37 2 4 0 0.3 0 1 0 0.2 trace 60 3 96 %SFA saturated fatty acids %TGL triglycerides Example 7 In this example the fatty acid composition of the fats produced by some intergeneric fusants are compared with the fatty acid composition o~f the fats produced by their parental strains.

TABLE J Strain C1 4 C1 6 C16-1 C1 8 C18-1 C18-2 C18-3 C2 2 C2 4

SFA*

0.53 28.10 0.39 13 .90 43.70 9.57 1.74 0.31 0.30 1.05 44.25 8.36 13 .54 28.11 5 .35 29 .69 3.69 4.66 0.24 17 .74 9.70 7 .45 47.12 13.10 0.23 0.40 2.1 1.11 20.48 1.87 11 .46 32.95 23.42 2.30 0.38 0.63 1.94 2.09 23.26 0.30 6.35 24 .04 33.51 5 .03 0.11 2.14 2.13 1.54 29 .04 0.24 13.74 26.48 16.18 2.05 0.62 4.67 2.18 1.24 13.14 6.61 6 36 .94 12.31 1.02 1.71 2.72 1.56 31.44 27.93 36.50 37.62 52.40 26.42 *%SFA saturated fatty acid 1Apiotrichum. curvatum ATCC 20509 2 Saccharomyces cereviseae ATCC 25657 3 Yarrowia lipolytica CBS 599 4 fusant ASi (A.curvatum ATCC 20509 x S-cerevisiae ATCC 25657) =fusant Aufa S2 (A.curvatun CBS 199.87 x S-cereviseae ATCC 25657) 6 fusant Aufa S5 (A.curvatum CBS 199.87 x S-cerevisiae ATCC 25657) 7 fusant AufaY3 (A-curvatum CBS 199.87 x Y-lypolytica CBS 599)

Claims (14)

1. A process for the production of fats usable as cacao butter substitute or as a component of a cacao butter substitute comprising cultivating a fat-producing micro-organism in a medium and subsequently recovering the produced fats from the micro-organism, 1 0 characterized by using as a micro-organism a genetically mutagenated strain of the genera Apiotrichum, Candida, Cryptococcus, Endomyces, Hansenula, Lipomyces, eichia, Rhodosporidium, Rhodotorula. Saccharomyces, Sporo- bolomyces, Torulopsis, Trichosporon and Yarrowia, wherein the '1 5 enzyme system responsible for the conversion of stearic acid into oleic acid is totally or partially blocked by the genetic mutagenation or S- a fusant derived from in total two, three or four genetically different genetically mutagenated strains, all belonging to 20 the above mentioned genera, wherein the enzyme responsible for the conversion of stearic acid into oleic acid is totally or partially blocked by the genetic mutagenation.

2. The process according to Claim 1, characterized by using as a micro-organism a mutant of Apiotrichum, Candida, Cryptococcus, 0o° Endomyces, Hansenula, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Sporobolomvces, Torulopsis, Trichosporon and Yarrowia wherein the enzyme system responsible for the conversion of stearic acid into oleic acid is partially blocked.

3. A process according to Claim 1 or 2, characterized by 0 using a mutant of the species Apiotrichum curvatum as a micro- S organism.

4. A process according to Claim 3, characterized by using a mutant of the strain Apiotrichum curvatum ATCC 20509 as a micro- organism. A process according to Claim 4, characterized by using a mutant of Apiotrichum curvatum as a micro-organism, deposited under number CBS 199.87 with the Centraal Bureau voor Schimmelcultures, The Netherlands. PflFFiiI~tIinAE!IIII III mmuwiummi iii' ~iTi mu. 21 a

6. The process according to Claim 5, characterized by using a partial desatu rase- mutant as a microorganism derived from the absolute desatu rase- mutan t, deposited under number CBS 199.87 with the Centraal Bureau voor Schimmelcultures, The Netherlands. 441111 I 4001 I I I 04 0 64 o a 404 4 00 6 o 0 00 0 0 0 00 2~ 4146 0

7. The process according to Claim 1, characterized by using a fusant obtained by combining the protoplasts of two genetically different yeast cells of the species Apiotrichum curvatum as a microorganism.

8. The process according to Claim 7, characterized by using a fusant obtained by combining the protoplasts of two genetically different yeast cells derived from the strain Apiotrichum curvatum ATCC 20509 respectively by combining the strain ATCC 20509 and a yeast cell derived thereof as the microorganism.

9. The process according to Claim 8, characterized by using a fusant obtained by combining the protoplasts of on the one hand an amino acid auxotrophic mutant of the strain ATCC 20509 and on the other hand the strain CBS 199.87 as the microorganism. The process according to Claim 9, characterized by using the fusant deposited under number CBS 159.88 with the Centraal Bureau voor Schimmelcultures, The Netherlands, as the microorga- nism.

11. The process according to one or more of Claims 1-10, characterized by using a medium comprising whey, whey permeate or molasses and possibly oleic acid as carbon nutrient and a water soluble nitrogen nutrient.

12. Cacao butter substitute or a component thereof, obtained according to the process disclosed in one or more of the claims 1-11.

13. Luxuries and food products containing the cacao butter substitute or a component thereof defined in Claim 12.

14. The mutant of the species Apiotrichum curvatum deposited under number CBS 199.87 with the Centraal Bureau voor Schimmelcul- tures, The Netherlands.

15. Fusants, obtained by combining the protoplasts of on the one hand the strain ATCC 20509 and on the other hand the strain CBS

199.87. 16. Fusant according to Claim 15, deposited under number CBS 159.88 with the Centraal Bureau voor Schimmelcultures, The Nether- lands. 23 17. A process for the production of fats usable as cacao butter substitute substantially as hereinbefore described with reference to the examples. Dated this 5th day of April 1988 WESSANEN NEDERLAND B.V.; By their Patent Attorneys, COLLISON CO.