NL8700783A - Prodn. of fat equiv. to cocoa butter - by culture of mutant strains of fat producing yeast with genetically blocked desaturase enzyme system - Google Patents

Abstract

Fats for use as equiv. to cocoa butter or component of this equiv. are prepd. by cultivating mutant strains of one of the genera Apiotrichum, Candida, Cryptococcus, Endomyces, Hansenula, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Sporobolomyces, Torulopsis, Trichosporon or Yarrowia, in which the enzyme system responsible for conversion of stearic acid to oleic acid has been (partially) blocked genetically. The fat is then recovered.

Description

. N.0. 34260 1 *%

Process for preparing fats to be used as cocoa butter equivalent or as component of cocoa butter equivalent.

As inventors are mentioned; E.C. Verbree, A Ykema and H. Smit.

5

The invention relates to a process for preparing fats to be used as cocoa butter equivalent or as component of a cocoa butter equivalent by cultivating a fat-producing microorganism in a medium and subsequently recovering the fats accumulated therein from the microorganism.

One of the most appreciated vegetable fats is cocoa butter. This is a naturally occurring product, which is extracted from seeds of the cocoa tree (Theobroma cocoa). More specifically, cocoa butter is a product composed largely of 1,3-saturated-2-unsaturated-triglycerides containing stearic, oil and palmitic acids as fatty acid components. In view of the generally recognized unique properties of cocoa butter, the supply, quality and price of which fluctuate strongly, the need for this product is still growing and the natural production of this product can hardly meet the demand. Although the increasing need for cocoa butter can be partially met by analogues with a similar triglyceride build-up (cocoa butter equivalents), they do not make a real contribution to solving the problem as these analogues, in addition to a palmitic, oleic, and palmitic acid rich ( POP-rich palm oil component 25 are largely based on stearic-oleic-stearic acid-rich (SOS-rich) exotic fats such as Sheavet and Tengkawan, whose properties, price and availability clearly limit. An alternative method of preparation for cocoa butter equivalents, resp. for the exotic SOS-rich compo-30 nent thereof, which have both a good and constant quality and a low cost price.

It is known that fats and oils can be prepared using oil / fat producing microorganisms such as algae, bacteria, fungi and yeasts. Such microorganisms synthesize the lipids during the normal course of cellular metabolism. Attempts have now been made to find those microorganisms as well as media and process conditions which enable the production of such cocoa butter equivalents or components thereof on an economic scale.

Many proposals have been put forward in the prior art regarding the preparation of cocoa butter equivalents. For example, from 8 ”ï /» - ·> · J i t. Dutch patent application 78.04549 discloses a method for the microbial preparation of fats to be used as cocoa butter surrogate, wherein the medium for the fat-producing microorganism contains a mixed carbon source consisting of at least a stearyl derivative and at least 5 a palmityl derivative, an oleyl derivative and a saccharide exist. Strains of the genera Candida, Torulopsis, Trichosporon, Pichia and Sporobolomyces are mentioned as suitable microorganisms for conversion. However, such a method has the drawback that a medium must be used which must meet certain high requirements. However, such media are expensive, so that the method according to this Dutch patent application 78.04549 can hardly be used on an economic scale.

European patent specification 0.005.277 discloses a microbiological preparation method of oils and fats, which is based on: a) preparing a growth medium in which the carbon source contains fatty acids with 10-20 carbon atoms, b) inoculating the growth medium with oil-producing yeast cells, such as strains of the genera Rhodosporidium, Lipomyces, Candida, Saccharomyces, Endomyces and Rhodotorula, 20 c) aerobically cultivating the yeast cells at a pH between 4.0 and 6.0 and a temperature of 20-40 ° C, d) separating the cultivated cells from the growth medium, and e) recovering the oil from the cultivated cells.

Palmitic, oleic and stearic acids and mixtures thereof are mentioned as examples of such fatty acids to be used as carbon source. This latter method also has the drawback that it cannot be economically carried out for the preparation of a cocoa butter equivalent because of the substrate required. In this connection it is also pointed out to the phrase stated in the main claim, that the composition of the synthesized oil depends on the ratio between the saturated fatty acid present in the medium and the unsaturated fatty acid present therein.

US Pat. Nos. 4,485,172 and 4,485,173 also disclose processes for preparing fats and oils using fat-producing microorganisms, wherein at least one fatty acid of 10-20 carbon atoms is to be used in the substrate as the carbon source. The carbon source is advantageously used as palmitic, oil and stearic acid or mixtures thereof, but the relative concentration of these acids is adjusted such that 40 cocoa butter equivalents can be prepared. Furthermore, in the 6 S U 0/8> 3 Λ *

U.S. Patent 4,485,173 also discloses the use of a desaturase enzyme inhibitor which promotes a higher ratio of stearic acid to oleic acid in the prepared triglycerides. It is indicated that this inhibitor greatly reduces the desaturation of stearic acid in oleic acid. Strains of the genera Rhodosporidium, Lipomyces, Candida »Endomyces» Saccharomyces »Rhodo-torula, Trichosporon and Torulopsis are mentioned as suitable yeasts. Since substrates in which a carbon source having the above-mentioned specific composition is to be used are to be used for the preparation of cocoa butter equivalents, it has been suggested that the use of the methods known from these US patents is not considered economically feasible. .

US-A-4,235,933 discloses a process for the preparation of oils using an oil-producing strain of Candida curvata, wherein a whey permeate obtained by ultrafiltration is used as the inexpensive substrate. As examples of microorganisms to be used, the strains Candida curvata strain D, ATCC No. 20509 and Candida curvata strain R »ATCC No. 20508 applied. However, the product obtained in this method has an insufficient quality compared to cocoa butter due to an excessive content of unsaturated fatty acids and is therefore processed in animal feed.

The invention relates to a method for economically profitable preparation of cocoa butter equivalents or components thereof by using as a microorganism a mutant of a fat-producing yeast belonging to one of the genera Apiotrichum »Candida, Cryptococcus, Endomyces, Hansenula, Lipomyces, Pichia , Rhodosporidium, Rhodo-torula, Saccharomyces, Sporobolomyces, Thorulopsis, Trichosporon and Yarrowia, in which the enzyme system responsible for the conversion of stearic acid to oleic acid is genetically partially or partially blocked ·

For the purposes of the invention, the term "cocoa butter equivalent" is understood to mean a fat blend composed of more than 25%, preferably more than 50%, of 1,3-saturated-2-unsaturated triglycerides, whether or not contain unsaturated forms of C 1 g and C 3 g fatty acids as fatty acid components. Furthermore, the term "cocoa butter equivalent component" means a fat blend composed of greater than 25%, preferably greater than 50%, of 1,3-di-saturated-2-unsaturated triglycerides, which contain saturated and unsaturated forms of Cjg fatty acids as fatty acid components.

The mutants to be used in the method according to the invention of fat-producing yeasts such as advantageously Apiotrichum curvatum, also referred to as desaturase mutants, are obtained by mutagenization. This mutagenization can be carried out, for example, by means of chemical mutagens such as N-methyl-N'-nitrose-guanidine, ethylmethane-5 sulfonate or by irradiation with UV light. The desaturase mutant can be screened using culture media, to which oleic acid has been added or not.

Within the scope of the invention, first those mutants are isolated in which the enzyme desaturase, which catalyzes the conversion of stearic acid to oleic acid, is not active. The desaturase mutants isolated in this way depend for their growth on the addition of oleic acid to the growth medium. In this connection it is also pointed out that, starting from the isolated absolute desaturase mutants in which the desaturase is completely inhibited, possibly after induction with a mutagen, partial desaturase mutants in which the desaturase is partially inhibited have been isolated, which no longer have oleic acid requirement.

The morphology of the isolated absolute desaturase mutants, seen under a phase contrast microscope, is markedly different from that of the wild type Apiotrichum curvatum (ATCC 20509). Some mutant cells are larger and have a marked tendency to form pseudomycelia. Under fat-producing conditions, these desaturase mutants are not observed in the presence of limited amounts of oleic acid as in the wild type intracellular oil droplets, but a granular structure indicating solid fat at the culture temperature of 30 ° C employed.

The results in shaking cultures and fermenter cultures indicate that the absolute desaturase mutants in the presence of small amounts of oleic acid and the partial desaturase mutants also pass through a fairly normal growth phase and fat production phase in the absence of oleic acid.

One of the absolute desaturase mutants of Apiotrichum curvatum ATCC 20509 has been deposited with the Central Bureau of Fungal Cultures in Baarn under number CBS 199.87.

Since desaturase mutants of Apiotrichum curvatum require oleic acid for the synthesis of membrane lipids, partial revertants of desaturase mutants are preferred since they are only partially inhibited in the desaturase activity and are therefore capable of growing in the absence of oleic acid. This advantage manifests itself in a favorable influence on the cost price of the growing medium.

In addition to the above, in the method according to the invention the invention can then be used as substrate cheap whey or whey sizes or molasses as a substrate. For example, to obtain a proper C / N ratio, NH4 + can be added to the growth medium. By using fed batch cultures and, in particular, partial recycle cultures of Apiotrichum curvatum in whey permeate, very high fat production rates were achieved, whereby an economically viable production of yeast lipids has been achieved.

From the analysis results it can be deduced that with the aid of the mutants according to the invention it is possible to prepare triglycerides which have a percentage of saturated fatty acids, which is comparable to that of cocoa butter.

To further illustrate the invention, the examples below are set forth; these examples should not be interpreted restrictively.

15

Example I

A) Isolation procedure used to obtain desaturase mutants of the fat-producing yeast Apiotrichum 20 curvatum ATCC 20509.

Table A below shows the parameters observed in obtaining desaturase mutants of Apiotrichum curvatum. As strain Apiotrichum curvatum ATCC 20509 has been used, which corresponds to the Candida strain Curvata ATCC 20509 mentioned in the above-mentioned US patent 4,235,933.

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TABLE A

Isolation method of absolute desaturase mutants 5 Starting strain: Apiotrichum curvatum ATCC 20509

Mutagens: N-methyl-N'-nitro-guanidine dose: 500-1000 micrograms NG / ml incubation medium: 0.1 M phosphate buffer pH 7.0 growth phase: bud-forming cells 10 density suspension: E550 = 10 incubation time: 30-180 min.

incubation temperature: 30 ° C

q number of cells treated: 2.10% survival: 0.0014 - 0.025% 15 number of desaturase mutants: 6 mutant frequency: 1 per 16,000 a 1 per 80,000 B) Isolation procedure, which was used to obtain partial desaturase mutants, assuming an absolute 20 desaturase mutant of the fat-producing yeast Apiotrichum curvatum ATCC 20509.

Table B below shows the parameters taken to obtain partial desaturase mutants. The strain used is the absolute desaturase mutant of Apiotrichum curvatum ATCC 20509 deposited with CBS under number CBS 199.87.

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TABLE B

Isolation method of partial desaturase mutants 5 Starting strain: Absolute desaturase mutant CBS 199.87

Mutagenesis: NG

dose: 200-500 / µg / ml incubation medium: 0.1 M phosphate buffer pH 7.0 growth phase: logarithmic density suspension: ^ 660 = incubation time: 30-120 min.

temperature during incubation: 30 ° C

% survival: 0.00008 - 2.5% (double) mutant frequency: 1 per 140,000 to 1 per 1,500,000 15 8 "Γ 'r <3 Η 8 i

Example II

Use of the isolated absolute mutant and partial desaturase mutants isolated therefrom and analysis of the product prepared.

In a continuously stirred fermentation vessel equipped with aeration pipe and pH control equipment, a pure culture of an absolute or partial desaturase mutant of Apiotrichum curvatum ATCC 20509 10 is inoculated on a medium of the following composition:

Glucose 10 g

Oleic acid 0 to 0.12 g

Tween 80 0 to 0.3 g 15 NH4CI 0.25 g KH2PO4 3.5 g

Na2HPO4 1.0 g

MgS04 0.75 g

Yeast extract 0.75 g 20 CaCl2 50 mg

FeClg 5 mg

ZnS04 0.5 mg

Water 1 liter

The cells were grown at pH 5.0, at a temperature of 30 ° C

and were harvested after the glucose was consumed in the growth medium. The lipids were extracted with organic solvents and analyzed for triglyceride composition and fatty acid composition (by generally accepted methods).

Table C below shows the test results of an absolute desaturase mutant grown in the presence of different amounts of oleic acid and of three partial desaturase mutants of the invention compared to those of the wild type (Apiotrichum curvatum ATCC 20509).

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Claims (10)

1. Process for preparing fats to be used as cocoa butter equivalent or as component of cocoa butter equivalent by cultivating a fat-producing micro-organism in a medium and then recovering the prepared fats from the micro-organism, characterized that as a microorganism a mutant of a strain belonging to one of the genera Apiotrichum, Candida, Cryptococcus, Endomyces, Hansenula, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharo-10 myces, Sporobolomyces, Thorulopsis, Trichosporon and Yarrowia is used for the conversion of stearic acid to oleic acid responsible enzyme system is genetically partially or completely blocked. 2. Process according to claim 1, characterized in that as a microorganism a mutant of Apiotrichum, Candida, Cryptococcus, Endomyces, Hansenula, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Sporobolomyces, Thorulopsis, Trichosporon and Yarrowia is used, in which the enzyme system responsible for the conversion of stearic acid to oleic acid is genetically partially blocked. 3. Process according to claim 1 or 2, characterized in that a mutant of the species Apiotrichum curvatum is used as the micro-organism. 4. Process according to claim 3, characterized in that a mutant of the strain Apiotrichum curvatum ATCC 20509 is used as the microorganism. Method according to claim 4, characterized in that the mutant of Apiotrichum curvatum, which has been deposited under the number CBS 199.87 at the Central Bureau for Fungal Cultures in Baarn, is used as the microorganism. 6. A method according to claim 5, characterized in that a microorganism is used as a partial desaturase mutant, which is derived from the absolute desaturase mutant, which has been deposited with the Central Bureau of Mold Cultures in Baarn under the number CBS 199.87. Process according to one or more of Claims 1 to 6, characterized in that the medium used is whey permeate plus optional oleic acid and a water-soluble ammonium salt. A cocoa butter equivalent or a component thereof obtained according to the method of any one of claims 1 to 7. Pleasure and foodstuffs containing the cocoa butter equivalent or a component thereof according to claim 8. 10. The mutant of the species Apiotrichum curvatum, which has been deposited under the 40 number CBS 199.87 at the Central Bureau of Fungal Cultures in Baarn. 870 07 8 ^ ****