NZ224080A - Cacao butter substitutes and formentation process for production - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £24080 r* i f- *■ i d 7 J0** ? <w ^'il* SV NO DRAWINGS Priority Datefs}: Complete Specification Filed: CtasK ... /r\??.w'.0 }^£F- Fubfication Date: l!?',^,.^!.

P.O. Journal, Nc: 22 408 N.Z. NO.

NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION PROCESS FOR THE PREPARATION OF FATS USABLE AS CACAO BUTTER SUBSTITUTE OR AS A COMPONENT OF A CACAO BUTTER SUBSTITUTE We, WESSANEN NEDERLAND B.V. of Prof E.M. Meyerslaan 2, 1183 AV Amstelveen, The Netherlands, a *k.lV*»WivAs cocf• do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- lA 22 408 0 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 microorgani sm.

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 product mainly consisting of 1.3-disaturated-2-unsaturated triglycerides, containing stearic acid, oleic acid and palmitic acid as fatty acid components. In the following Table A the average fatty acid composition of cacao butter is illustrated (Fincke A., (1965), Handbuch der Kakaoerzeugnisse, page 333, Springer Verlag).

TABLE A Palmitic acid (C 16) Stearic acid (C 18) Oleic acid (C'18) Linoleic acid (C"18) Linolenic acid (C'''18) Arachic acid (C 20) Behenic acid (C 22) Lignoceric acid (C 24) 23 - 30 % by weight 32 - 37 Z by weight 30 - 37 % by weight 2 - 4 % by weight 0 - 0.3 % by weight 0 - 1 % by weight 0 - 0.2 % by weight trace In view of the strongly fluctuating offer, quality 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 for 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 224080 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 acid/stearic acid-rich (SOS-rich) exotic fats like Sheafat and 5 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 5 respectively for the exotic SOS-rich component thereof which have both a good and constant quality and a low cost price. 10 It is known that fats and oils can be produced by cultivating : C oil/fat-producing microorganisms like algae, bacteria, moulds and ; yeasts. Such microorganisms synthesize the lipids in the ordinary 7;* course of their cellular metabolism. Extensive research has been i carried out in an effort to find the microorganisms, media and conditions which would permit an economic production of cacao butter substitutes or components thereof.

All kinds of propositions concerning the preparation of cacao J i * ' butter equivalents have been disclosed in the prior art. For instance, from United States Patent No. 4,308,350 eprocess 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, Toru-C 25 lopsis, Trichosporon, Pichia and Sporobolomyces are mentioned as microorganisms 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 30 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 35 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, ?! r* 22 4 0 8 1 c) cultivating said yeast cells under aerobic conditions, a pH between 4.0 and 6.0 and a temperature between 20 — 40°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 in an economic way in view of the required expensive substrate. In 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 acid present in the growth medium.

Further processes for the production of fats and oils with the help of fat-producing microorganisms requiring at least one fatty acid having 10-20 carbon atoms as carbon nutrient in the growth medium are known from D.S. Patent Specifications 4,485,172 and 4,485,173. Preferably palmitic acid, oleic acid and stearic acid as well as mixtures thereof are used as carbon nutrient, whereas the relative concentration of these acids is adjusted in such a way that cacao butter substitutes can be produced. Strains of the genera Khodosporidium, Lipomyces» Candida, Endomyces, Saccharo-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 butte 4 22408 0 substitutes. With 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) 9676c, 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, Crypto-coccus, Endomyces, Hansenula, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Sporobolomyces, Torulopsis, Tricho-sporon 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 for a fatty mixture comprising for more than 25%, perferably more than 50%, of 1.3-disaturated-2-unsaturated triglycerides containing saturated or unsaturated C^g- and r>' V X.

C^g-fatty acids as fatty acid components. Further the term ,;' V 22 4 0 8 f t. "component of a cacao butter substitute" stands fora fatty mixture J comprising for more than 25%, preferably more than 50%, of 1.3—di- V saturated-2-unsaturated triglycerides, containing saturated and un- saturated forms of C^g-fatty acids as fatty acid components.

The mutants of fat-producing yeast genera used in the process f—\ according to the invention like preferably Apiotrichum curvatum, also called desaturase-mutants, are obtained by means of mutagena-tion. This mutagenation can be carried out with for instance chemical mutagentia like N-methyl-N'-nitroso-guanidine, ethyl-10 methane sulphonate or by means of radiation with UV—light. The screening of the desaturase-mutant can be performed by the application of nutrient substrates to which oleic acid has been added or not.

A v ' - Within the scope of the invention ac first those mutants are j- 15 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 J the growth medium. In this respect it is stated that starting from the isolated "absolute" desaturase-mutants wherein the enzyme 20 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 25 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 30 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 35 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.

V c ■P- " ■ 22408 0 / • n r> 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 10 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 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 nitrogen nutrient, however, use can also be made of other conventional nitrogen-containing compounds like urea, asparagine, glutamine, 20 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 25 carbon—to-nitrogen-ratio in the growth medium is between 10 and 100 (g carbon per g nitrogen) and preferably between 20 and 70.

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 30 elements are added in the salt form. yjy 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 2.5 to 9.

The temperature at which the fermentation is carried out is 35 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• E *^ 7,*- i-- 1' I ■/.I r> 22408 0 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 NH4+ to the growth medium. By using fed batch cultivations and in particu-5 lar partial recycle cultivations of Apiotrichum curvatum in whey-permeate 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 10 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 V"""\ 15 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 20 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 25 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 30 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 35 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, Pichijgffcfc'f3^ /-"v.

T if V 8 22 40 8 Rhodosporidium, Rhodotorula, Saccharomyces, Sporobolomyces, Toru-lopsis, 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 (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" desaturase-mutants derived from the fat-producing yeast strain Apiotrichum curvatum ATCC 20509.

The following Table B lists the parameters ap production of desaturase-mutants of Apiotrichum 22 408 ( 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.

C TABLE B Isolation method of absolute desaturase-mutants r-N 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: 20 mutant frequence: Apiotrichum curvatum ATCC 20509 N-methyl—N'-nitroso-guanidine (NG) 500-1000 microgram NG/ml 0.1 M of phosphate buffer, pH 7.0 logarithmic E660 = 10 30-180 min.

°C 2.109 0.0014 - 0.025% 6 1 per 16.000 a 1 per 80.000 B) Isolation procedure used for obtaining "partial" desaturase-mutants starting from an absolute desaturase-mutant of the fat-25 producing yeast strain Apiotrichum curvatum ATCC 20509.

The following Table C lists the parameters applied for obtaining the partial desaturase-mutants. The absolute desaturase-mutant of Apiotrichum curvatum ATCC 20509 deposited under number 30 CBS 199.87 has been used as starting strain. 22 408 TABLE C Isolation method of partial desaturase-mutants Mutagens: dose: incubation medium: growth phase: density of suspension: incubation time: incuabtion temperature: % survival: (double) mutant frequence: Starting strain: Absolute desaturase-mutant CBS 199.87 N-methyl—N'-nitroso-guanidine (NG) 200—500/ug NG/ml 0.1 M phosphate buffer pH 7.0 logarithmic e660 = 10 —120 min.

°C 0.00008 - 2.5% 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: Glucose Oleic acid Tween 80 NH4C1 KH2P04 Na2HP04 MgS04 g 0-0.12 g CaCl2 FeCl3 ZnS04 Water Yeast extract 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 22408 0 The cells were cultivated at a pH of 5.0 and a temperature of 30°C 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 The following Table D shows the test results of an absolute desaturase-mutant according to the invention, cultivated in the 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 20509). art).

X/xOL,L L CO *r CsJ C\4 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 20509 CBS 199.87 CBS 199.87 CBS 199.87 R22.7 R22.14 R22.50 R22.102 Cacao 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 (%) 40 53 58 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 .10 22.60 .80 23-30 C16.1 0.39 0.63 1.16 1.91 - - - - C18 13.90 52.13 45.40 .70 .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 -37 C18.2 9.57 7.17 7.74 8.58 .45 .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 C20 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 o o o o :rV ■; X' 22 4 0 8 Example 3 In this example further details are given concerning the distribution of the saturated an unsaturated fatty acids on the 1-and 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 desaturase-mutants i.e. the revertants R33.192 and R38.ll 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.ll the unsaturated fatty acids do preferably have the 2-position of the glycerol molecule which is also the case in cacao butter.

Table E-l. fatty acid position determination A.curvatum ATCC 20509 cvj CVJ fatty % by weight In 7. by weight on acid triglycerides the 2-position C16 28.1 2.0 C16.1 - - C18 13.9 1.4 C18.1 43.7 83.7 C18.2 9.6 12.5 C18.3 1.7 0.4 C24 - - Table E-3. fatty acid position determination partial desaturase-mutant R33.192 fatty % by weight in % by weight on acid triglycerides the 2-position C16 16.9 1.2 C16.1 3.3 3.0 C18 27.9 2.0 C18.1 30.3 60.7 C18.2 13.3 28.6 C18.3 2 2.4 C24 2.6 0.3 Q O O Table E-2. fatty acid position determi CBS 199.87 W^u l\uJ V, »"* *V// fatty % by weight in % by weighV^n _ acid triglycerides the 2-positi£ffe^?. v C16 19.2 .7 C16.1 1.9 3.1 CI 8 .7 6.4 C18.1 28.7 56.3 C18.2 8.6 21.9 C18.3 1.0 1.1 C24 1.9 1.0 Table E-4. fatty acid position determination partial desaturase-mutant R38.ll fatty % by weight in % by weight on acid triglycerides the 2-position C16 33.6 3.2 C16.1 0.2 0.3 C18 17.4 1.8 C18.1 34.4 74.2 C18.2 9.5 16.1 C18.3 1.5 1.1 C24 1.2 0.6 22408 Example 4 A) Isolation procedure used for obtaining desaturase-mutants of the yeast strain Saccharomyces cerevisiae ATCC 25657. n 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 4 O starting strain: mutagens: dose: incubation medium: growth phase: density of suspension: incubation time: 20 incubation temperature: inactivation of EMS: inactivation time: inactivation temperature: number of treated cells: 25 % survival: number of desaturase-mutants: mutant frequence: Saccharomyces cerevisiae ATCC 25657 ethylmethane sulphonate (EMS) 2.9% (v/v) 0.1 M of phosphate buffer pH 7.0 logarithmic E660 = 10 120 min.

° C % of sodium thiosulphate 30 min. room temperature 250,000 15 % 3 1 per 6250 C B) Fatty acid composition of the fats produced by desaturase-mutants of 30 Saccharomyces cerevisiae ATCC 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 35 type Saccharomyces cerevisiae ATCC 25657. 16 22 4 0 8 TABLE G Strain ATCC DFA3 UFA3 UFA3 DFA3 25657 medium M5* M5* M5* M5* M5* mg oleic acid/g glucose 0 0.1 0.5 1.0 mg Tween 80/g glucose 0 2.5 12.5 .0 250 fatty acid composition: CIO 1.34 1.31 4.46 1.90 3.38 C12 4.88 6.01 7.63 11.88 3.32 C14 6.19 8.20 11.33 8.05 4.22 C16 17.98 39.18 31.24 21.41 .39 C16.1 .43 0.00 0.00 6.96 11.74 C18 8.55 .79 18.93 8.67 4.01 C18.1 19.63 21.26 18.12 .36 54.81 C18.2 4.52 6.54 7.42 12.01 1.99 C18.3 1.49 7.03 0.85 8.74 2.41 %SFA** 38.94 65.50 73.59 51.91 .32 * M5 medium has been described in Example 2 ** XSFA ■ % saturated fatty acids fir i't a. 17 22 40 8 Example 5 The following Table H shows a review of the parameters applied for the production of fusants based on (1) an amino acid auxotrophic Apiotrichum curvatum—mutant obtained by means of mutagenation with the help of nitroso-guanidine and (2) the desaturase-mutant of Apiotrichum curvatum having the deposition number CBS 199.87.

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 DTT.pH=8.0 d) the suspensions were incubated for 30 minutes in a shaking water bath at 30°C. e) after centrifugation the pellets were introduced in 1.2 H sorbitol. f) after centrifugation the pellets were introduced in a buffer of 1.2 M sorbitol, 100 mM sodium citrate, 5 mM DTT + 2 mg/ml Novozym 234, pH=5.8. g) the suspensions were incubated for 15-30 minutes in a shaking water bath at 30°C. h) the formed protoplasts suspensions were washed three times in a buffer of 1.2 M sorbitol + 10 mM Tris-HCl, pH=8.0. i) after centrifugation the pellets were introduced in a buffer of 1.2 M sorbitol + 10 mM Tris-HCl, pH=8.0.

(II) Fusion of protoplasts and isolation of fusants (hybrides). o a) about 5.10 protoplasts of each fusion partner were combined and well mixed.

TABLE H a t—\ 4 n 18 ki 2 4 0 8 b) after centrifugation a PEG-solution (35% of polyethylene glycol 4000 + 10 mM CaCl2 + 10 mM Tris—HC1, 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°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 buffer. 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.

I x-' f) colonies of fusants (hybrides) appeared after an incubation time of 4—7 days at 30°C.

(N.B. the original fusion partners cannot grow on this 15 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 mentioned in Example 5 as well as the analysis of the products obtained. ^ 25 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 30 to 30 (g/g) by means of NH4CI.

The cells were cultivated at a pH of 5.0 and a temperature of 30°C 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 distri-35 bution (according to methods generally known in the art). 19 22 40 8 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 CBS 159.88 F 33.54 F 33.05 ATCC 20509 Cacao butter C14 1.72 1.61 1.52 0.53 C16 23.75 27.85 32.39 28.10 23 - 30 C16-1 0.35 - 0.39 0.39 C18 31.21 24.13 .42 13.90 32 - 37 C18-1 29.58 31.64 28.81 43.70 - 37 C18-2 .61 7.33 6.41 9.57 2 - 4 CI 8-3 1.07 0.92 0.52 1.74 0 - 0.3 C20 1,54 1,29 1,11 0,31 0 - 1 C22 0.16 0.62 0.50 0.30 0 - 0.2 C24 3.64 3.38 1.69 1.05 trace %SFA* 62.57 59.05 62.63 44.25 60 ± 3 ZTGL** 95 95 95 95 >y 96 *) %SFA = % saturated fatty acids **) %TGL = % triglycerides 22408 Example 7 In this example the fatty acid composition of the fats produced by some intergeneric fusants are compared with the fatty acid composition of the fats produced by their parental strains.

TABLE J Strain 1 2 3 4 6 7 C14 0.53 8.36 0.24 1.11 2.09 1.54 1.24 CI 6 28.10 13.54 17.74 .48 23.26 29.04 13.14 C16-1 0.39 28.11 9.70 1.87 0.30 0.24 6.61 C18 13.90 .35 7.45 11.46 6.35 13.74 6.05 C18-1 43.70 29.69 47.12 32.95 24.04 26.48 36.94 C18-2 9.57 3.69 13.10 23.42 33.51 16.18 12.31 C18-3 " 1.74 4.66 0.23 2.30 .03 2.05 1.02 C20 0.31 - - 0.38 0.11 0.62 1.71 C22 0.30 - 0.40 0.63 2.14 4.67 2.72 C24 1.05 - 2.1 1.94 2.13 2.18 1.56 %SFA* 44.25 31.44 27.93 36.50 37.62 52.40 26.42 *) %SFA = % saturated fatty acid 1 = Apiotrichum curvatum ATCC 20509 2 = Saccharomyces cereviseae ATCC 25657 3 = Yarrowia lipolytica CBS 599 4 = fusant AS1 (A.curvatum ATCC 20509 x S.cerevisiae ATCC 25657) = fusant Aufa S2 (A.curvatum 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.lypolytlca CBS 599) 224080 The deposited strains CBS 199.87 and CBS 159.88 have both been derived from the oleaginous yeast strain Apiotrichum curvatum ATCC 20509.

This yeast strain was isolated by Moon, N.J. and Hammond E.J. (1978), J.An.Oil Chem.Soc. 55: 683-688, from cheese plant floors and floor drains, and identified as Candida curvata D. Since then the classification on this yeast strain has been changed a few times. By the American Type Culture Collection (Rockville, Md., USA) this yeast has been classified as an Apiotrichum curvatum (ATCC 20509). After re-identification of this yeast by the CBS [Centraal Bureau voor Schimmelcultures, Delft, The Netherlands] it was concluded that this yeast was a Trichosporon beigelii on basis of the ability of this strain to consuae raffinose and melibiose and the ability to grow at 37°C.

Morphology of strain ATCC 20509 The morphology of A.curvtum ATCC 20509 ATCC can vary with culture conditions and growth media and some characteristics are given here: (i) Morphology after growth in whey permeate: After 1 day at 30°C, the yeast cells are spherical to oval (3.5-7)x(3.5-14) ^tm. Budding cells are abundant (provided sufficient oxygen is available). Budding is on a broad base. After prolonged cultivation intracellular oil droplets appear that eventually can fuse to one big droplet, filling the whole cell. At sore elevated temperatures, pseudomycelia may occur. (ii) Morphology after growth on glucose-yeast extract-peptone After 2-5 days at 30°C yeast colonies are white to cres smooth and shiny. No mycelia are observed. :n r-7 MAR 1990 Morphology of strain CBS 199.87 This strain is an absolute desaturase mutant (oleic acid auxotrophic mutant) of strain ATCC 20509. There are some differences in' BOrpholugy ' '—" 224080 between CBS 199.87 and ATCC 20509. Strain CBS 199.87 is dependent for growth on the presence of snail aaounts of some source of oleic acid in the growth medium. The cells of this strain can be somewhat larger than that of ATCC 20509 and have a clear tendency to pseudomycelia formation especially at low oleic acid concentrations in the growth medium. Under fat producing conditions one may observe a granular structure inside the cells, indicating solid fat at the used cultivation temperature of 30°C. This structure is in contrast to that of wild type showing intracellular oil droplets. At high oleic acid concentrations in the growth medium, CBS 199.87 also forms oil droplets.

On glucose-yeast extract-peptone agar supplemented with oleic acid, colonies of CBS 199.87 are more brownish and much smaller than that of ATCC 20509. In contrast to ATCC 20509 colonies of CBS 199.87 are not shiny.

Morphology of strain CBS 159.88 This strain has been obtained after protoplast fusion of an amino acid auxotrophic mutant of ATCC 20509 and strain CBS 199.87. The morphology of strain CBS 159.88 only differs in that cells can be somewhat larger and that granular lipid is formed instead of oil droplets. 224080

Claims (19)

WHAT WE CLAIM IS:

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 microorganism in a medium and subsequently recovering the produced fats from the microorganism characterized by using as a microorganism - a mutant of a strain 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 or four, genetically different mutants and/or yeast cells, all belonging to the above mentioned genera, wherein the enzyme system responsible for the conversion of stearic acid into oleic acid is totally or partially blocked*

2. The process according to Claim 1, characterized by using as a microorganism a mutant of Apiotrichum, Candida, Cryptococcus, Endomyces, Hansenula, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces. Sporobolomyces, 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 using a mutant of the species Apiotrichum curvatum as a microorganism*

4. A process according to Claim 3, characterized by using a mutant of the strain Apiotrichum curvatum ATCC 20509 as a microorganism.

5* A process according to Claim 4, characterized by using a mutant of Apiotrichum curvatum as a microorganism, deposited under number CBS 199.87 with the Centraal Bureau voor Schlmmelcultures, The Netherlands.

6. The process according to Claim 5, characterized by using,a partial desaturase-mutant as a microorganism derived from the absolute desaturase-mutant, deposited under number CBS 199.87 with the Centraal Bureau voor Schlmmelcultures, The Netherlands* "7 MAR 1990* 22408' -24-

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.

10. 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 microorganism.

11. The process according to one or more of Claims 1—10, characterized by using a medium comprising whey, whey permeate or molasses amI optionally acidas 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. 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 Schimmelcultures, 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 Netherlands . S-7 MAR 1990 V pg/, 224080 •25-

17. A process according to claim 1 substantially as herein described or exemplified.

18. A microrganism according to claim 14 substantially as herein described or exemplified.

19. A fusant according to claim 15 substantially as herein described or exemplified. WESSANEN NEDERLAND B.V By Their Attorneys HENRY HUGHES LTD By: