IE62461B1 - Process for obtaining terpene flavourings by a microbiological process - Google Patents

Abstract

Process for obtaining terpenic aromas, characterised in that a mutant of S. cerevisiae blocked in the route for the synthesis of the ergosterol which secretes aromatic terpenes is cultured on an appropriate culture medium. This process can be employed more particularly for the production of aromatised beverages.

Description

The present invention relates to a process for obtaining terpene flavourings, in particular for the flavouring of drinks, by a microbiological process employing Saccharomyces cerevisiae mutants.

Terpenes, essentially regarded as products of plant origin, are compounds characteristic of many essential oils. As a result of their physiological and sensorial properties, mono- and sesquiterpenes have a multiplicity of applications in the food industry, in perfumery and in the pharmaceutical industry.

Moreover, some monoterpenes (geraniol, nerol, linalool, citronellol, alpha-terpineol) are responsible for the aromatic nature of the muscat grape. Varieties of grape having the muscat taste and flavour belong to two distinct classes, defined mainly by the value of the linalcol/geraniol ratio. For the muscat variety, the linalool/geraniol ratio is greater than 1, whereas for the malvasia" varieties, it is markedly lower than 1.

The plant sources of monoterpenes, although numerous, could, for climatic, seasonal, economic and political reasons, prove insufficient in the face of an increasingly demanding market; for this reason, the microbial production of these aromatic principles is of consider-able importance, and this is one of the objects of the present invention.

In contrast to filamentous fungi, whose capacity for synthesizing terpenes has often been reported, yeasts appear to be devoid of these biosynthetic capabilities. The aricle of Z. Lebensm. Unter. Forsch. (1984), 179, 450-452, discloses forming traces of Farnesol by S. Cerevisiae.

The small production of terpenes by yeasts, and more especially by S. cerevisiae, is in apparent contradiction with the presence in the cells of enzyme ayatoma capable of catalysing the biosynthesis of certain monoterpenes.

In effect, S. cerevisiae cells synthesize significant amounts (0.5 - 3% of the dry weight) of ergosterol from acetyl-CoA, and the polymerization of aceyl-CoA, as in higher plants, takes place via mevalonate, geranyl pyrophosphate and farnesyl pyrophosphate. S. cerevisiae hence possesses all the enzymes necessary for the biosynthesis of terpenes and, if the latter are not secreted, it is probable that the enzymes of the ergosterol pathway have a high affinity for terpene pyrophosphates, which are thus essentially polymerized to ergosterol.

The present invention is based on the demonstra10 tion that certain S. cerevisiae mutants are capable of secreting terpenes, in contrast to what was hitherto accepted, it being possible for the said strains to be used, in addition, for the direct preparation of fermented or unfermented aromatic drinks.

More especially, the present invention relates to :'a process for obtaining terpene flavourings, characterized in that an S. cerevisiae mutant, blocked in the pathway of ergosterol synthesis and which secretes aromatic terpenes, is cultured on a suitable culture medium.

It has been possible, in effect, to demonstrate, by means of the work on which the present invention is based, that ergosterol biosynthesis blocked by mutations in certain steps for certain strains (hereinafter refer25 red to as mutants auxotrophic for ergosterol) results in accumulations of terpene intermediates. The mutations affecting the sterol pathway in S. cerevisiae yeast which are most likely to result in accumulations of terpene pyrophosphates correspond to blocks in the steps cataly30 zed by squalene synthetase and farnesyl-diphosphate synthase (EC 2.5.1.10).

Strains of this type may be selected by mutation of Saccharomyces strains and selection on a medium containing a sterol, in particular ergosterol, and nystatin.

However, because of the auxotrophy for ergosterol, some of these strains, for example the strain 134 which will be described more fully in the examples, exhibit, in some cases, relatively poor growth on account of the generally low sterol contents of fruit juices. In order to overcome the often large qualitative and quantitative fluctuations which are observed when this type of strain is fermented, it is possible, in particular by subcloning, to obtain strains containing, in addition to » the mutations described above, a suppressive mutation which results in growth in the absence of ergosterol. · The partial suppression of the auxotrophy for ergosterol leads to the restoration of the wild-type phenotype.

By tetrad analysis, it has been possible to establish that: the mutation segretates independently of the auxotrophy for ergosterol, the mutation may be recovered from the recombinant spores which are prototrophic for ergosterol, and the mutation is recessive.

In spite of their relative independence of exogenous ergosterol, these clones always produce large amounts of monoterpenes.

This type of strain may be selected by subcloning from the above strains on medium devoid of ergosterol, for example.

It is appropriate to note that these strains synthesize ergosterol to a limited extent, and can grow in the absence of ergosterol, but that this mutation does not affect the block in the sterol pathway and, in the erg* progeny, is not manifested phenotypically.

In order to provide for the genetic stability of the erg- mutants, they are stored in the heterozygous state in the form of erg-/erg+ diploids. Before use, the erg- spores are recovered after sporulation and dissection of asci. .

When this process was carried out, it was noticed that some erg- strains derived, after sporulation, from , erg-/erg+ diploids, no longer excreted terpenes into the medium. This led to the demonstration of an additional recessive mutation which, associated with the block in the sterol pathway, permits the excretion of terpenes.

Thus, the most advantageous mutants according to the invention must preferably bear, in addition to the above mutations, a mutation designated ter. which is involved either in rendering the membrane permeable, or in the dephosphorylation of geranyl pyrophosphate.

In erg+ recombinant haploids, the ter mutation is not manifested phototypically. However, the erg- spores derived from erg* ter/erg- ter diploids always produce terpenes.

The storage of the mutants that produce terpenes must be carried out in the heterozygous state with a strain bearing the ter mutation as erg* parent.

Among the preferred mutants according to the invention, the mutants bearing the following mutations must be mentioned: - ero-. block in the steps of the sterol pathway that are catalysed by farnesyl-pyrophosphate synthetase and sgualene synthetase, - a suppressor, recessive, not linked to the ergmutation, which confers a relative independence with respect to exogenous ergosterol without affecting the block in the sterol pathway, ter, a recessive mutation permitting the production of terpenes, without which the geranyl pyrophosphate accumulated as a result of the erg- block is not excreted into the medium in the form of geraniol.

The strains thereby obtained secrete appreciable amounts of aromatic terpenes, but the Applicant Company has been able to demonstrate the possibility of further increasing this secretion.

In effect, the studies performed have shown that the operation of alcohol dehydrogenases (ADH), turned in the direction of an increase in the acetyl-CoA content, enables the activity of the sterol pathway to be increased by a greater availability of acetyl-CoA.

In particular, the following mutations are advantageous: ADH I mutants, that is to say mutants devoid of a fermentative isoenzyme, which are incapable of forming ethanol from acetaldehyde and hence exhibit an increased production of acetyl-CoA; ADH lie mutants, that is to say mutants constitutive for the oxidative isoenzyme, which oxidize ethanol to acetaldehyde even in the presence of large amounts of sugar (a typical condition for fruit juices and musts), whereas the wild-type strain oxidizes ethanol only in the absence of sugar.

The combination of these different mutations makes it possible to obtain, in particular, mutants that produce large amounts of terpenes, in particular terpenes of aromatic importance such as farnesol and geraniol.

Thus, the mutants which are especially advantageous are the mutants auxotrophic for ergosterol and which are ADH lie, the mutants auxotrophic for ergosterol and which are ADH 1", and finally the ADH I’ and ADH lie mutants.

The process according to the present invention may be carried out in a variety of ways.

In the first place, it is possible to use the culture of this type of mutant for the preparation of flavourings such as natural muscat flavours, which will be purified from the culture and which may be used for the flavouring of food substrates, in particular drinks.

In most cases, however, the terpene flavourings will be prepared directly in situ by culturing food substrates, in particular liquid foodstuffs or liquidizable food products such as fruit purges, for example. In this case, these substrates will be directly fermented by the mutant S. cerevisiae strains which will hence provide for the flavouring of the substrates.

In the case where the mutant used is an ADH I* mutant, that is to say a mutant not having a fermentative alcohol dehydrogenase at its disposal, the fermentation products obtained will be non-alcoholic products, or products having a very low alcohol content.

Naturally, in some cases, it may be advantageous to ferment, with this type of strain, media which are already alcoholic, in order to impart certain flavours to them; in this case, successive fermentations may be performed, with a yeast producing alcohol and then with mutants according to the present invention, or alternatively, when possible and compatible with the growth of yeasts, a cofermentation with the two yeasts may be performed. These yeasts may also be used for carrying out the process of secondary fermentation, for example in sparkling wines.

Naturally, in many cases, the preparation of alcoholic and flavoured drinks may be carried out using a mutant according to the invention but which has at its disposal a fermentative alcohol dehydrogenase (ADH I’) .

Since some of the strains according to the present invention are auxotrophic for ergosterol, it will be necessary, when the culture medium is a purely synthetic medium, to arrange for the addition of,; ergosterol in order to provide for the growth of the yeasts. In the case of substrates of plant origin, an addition of this kind is not generally necessary, on account of the existence of these compounds in trace state in different plant extracts.

The tests performed using the mutants according to the present invention showed that it was possible to obtain, for example, non-alcoholic fruit juices possessing the characteristic flavour of muscats, or alternatively alcoholic products of the wine or sparkling type possessing characteristic flavours of muscat or malvasia.

The ADH I' or ADH lie mutants are, for their part, especially advantageous in the production of sterols, in particular ergosterol, and, generally speaking, of all products involving an increase in the presence of acetyl-CoA.

The examples which follow, illustrated by figures, will enable other characteristics and advantages of the present invention to be demonstrated.

PRODUCTION OF AN ADH I~ MUTANT The mutant FL 100 ADH I- is selected from the haploid wild-type strain FL 100 (ATCC 28383) by resistance to allyl alcohol in the presence of glucose. This mutant, which is prototrophic for ergosterol, does not accumulate terpene intermediates, all the steps of the sterol pathway being operational.

On the other hand, the increased availability of acetyl-CoA due to the ADH I mutation result in a significant increase in the amount of ergosterol synthesized (Table 1). This type of mutant represents strains that are hyperproductive of ergosterol.

Table 1 Ergosterol (% of the dry weight) FL 100 0.75 ♦ 0.06 FL 100 ADH I' 1.06+0.05 EXAMPLE 2 PRODUCTION OF A MUTANT WHICH IS AUXOTROPHIC FOR ERGOSTEROL A mutagenesis of the wild-type strain FL 100 is performed using ultraviolet rays.

Strains resistant to nystatin are then selected in the presence of ergosterol. Among the resistant mutants, one is selected, designated mutant erg 9.

The auxotrophy of this mutant is confirmed on a complete medium (1% yeast extract, 1% bactopeptone, 2% glucose) with or without ergosterol (80 Mg/ml).

The measurement of the squalene synthetase activity is performed in the microsomal fraction by the method of Agnew and Popjak (Agnew W.S., Popjak G. ( 1978) J. Bio. Chem. 253. 4566-4573) which demonstrates the fact that this mutant erg 9 is devoid of squalene synthetase activity.

The mutant erg 9 is also characterized by the absence of ADH I (low production of ethanol), and by being constitutive for ADH II. Its phenotype is summarized in Table 2.

Growth YPGjerg YPS FL 100 +++ +++ erg 9 ++ (*) in the presence Table 2 Soualena AEH I ΑΓΗ II Ethanol synthetase (Sp act) (Sp act) (g/l) (specific activity) 0.24 16.2 12.3 0 10.8(*) of 90 g/l of glucose exam 3 36.2 3.7 PRODUCTION OF A MUTANT WHICH IS AUXOTROPHIC FOR ERGOSTEROL This mutant was obtained from a temperaturesensitive mutant which is auxotrophic for glycine (aux 32 ts) after UV mutagenesis on medium containing nystatin. This mutant accumulates dimethyl allyl pyrophosphate in vitro, an$ does not produce farnesyl pyrophosphate in the presence of isopentenyl pyrophosphate (labelled with carbon-14) and geranyl pyrophosphate, which indicates the absence of farnesyl-diphosphate synthase activity.

Moreover, the mutant 134 exhibits the phenomenon characteristic of erg 9 strains; the phenotypic allelism indicates the presence of an erg 9 allele; it is hence a farnesyl-diphosphate synthase double mutant.

Like the mutant erg 9, the mutant 134 is ADH I and ADH II-const!tutive. The results relating to the secretion of geraniol ln vivo show that the farnesyldiphosphate synthase mutation is bradytrophic.

EXAMPLE 4 The two mutants erg 9 and 134 are set up in a stirred culture (24 hours) or stationary culture (4 days) in complete medium (1% yeast extract, 1% bactopeptone, 2% glucose, 0.008% ergosterol) at 28°C. These two mutants effectively secrete farsenol and geraniol, as summarized in Table 3.

Table 3 Farnesol Geraniol (Mg/100 ml) (Mg/100 ml) Stationary Stirred Stationary Stirred FL 100 n.d. n.d. n.d. n.d. «- erg 9 24 138 n.d. n.d. 134 17 40 - 22 n.d. - not detectable By way of comparison, the wild-type strain FL 100 does not produce either farnesol or geraniol, either in a stirred culture or in a stationary culture.

EXAMPLE 5 FLAVOURING OF FRUIT JUICES WITH THE MUTANTS erg 9 AND 134 Fruit concentrates (white grape, red grape, raspberry, peach, pear, banana), diluted on the basis of 120 g/1 of sugar, are seeded with 107 cpm and incubated with stirring (150 rpm) at 30°C.

Sensorial analysis is performed on the juices at the end of 4 days after the removal of cells. The olfactory analyses systematically indicate a terpene note in the case of the mutants erg 9 and 134, in distinction to the products obtained using the wild-type strain FL 100. The results observed are collated in Table 4. Table 4 Fl 100 Fra 9 ill White grape winy muscat type muscat type Red grape winy floral perfume floral perfume Peach yeast floral perfume fruity Pear yeast fruity, floral fruity, floral Banana yeast muscat,farnesol" note muscat note Raspberry yeast, very far- vinic note solvent nesol note EXAMPLE 6 From the strain 134 and by subcloning, a new strain is obtained, capable of growing on medium in the absence of exogenous ergosterol and which possesses the following characteristics s relatively good growth without ergosterol (level intermediate between 134 and the wild-type strain FL 100), biosynthesis of ergosterol (10% of the wild-type 5 strain), block in the step catalysed by farnesyl pyrophosphate synthetase, with accumulation of geranyl pyrophosphate, - excretion of raonoterpenes, more especially geraniol 10 and linalool, into the culture medium.

This strain performs much more effectively from the industrial standpoint, and enables productions to be obtained which are stable as regards both quantity and quality.

Claims (16)

1. Process for obtaining terpene flavourings, characterized in that an S. cerevisiae mutant, blocked in the pathway of ergosterol synthesis and which secretes aromatic terpenes, is cultured on a suitable culture medium.

2. Process according to Claim 1, characterized in that the suitable culture medium contains a liquid foodstuff or a liquidizable food product.

3. Process according to one of Claims 1 and 2, characterized in that the mutant has a very low squalene synthetase and/or farnesyl-diphosphate synthase activity.

4. Process according to one of Claims 1 to 3, characterized in that the S. cerevisiae mutant contains a suppressive mutation which results in growth in the absence of ergosterol.

5. Process according to one of Claims 1 to 4, characterized in that the mutant is a ter mutant.

6. Process according to Claim 5, characterized in that the storage of the mutants used in the process is accomplished in the heterozygous state with a strain bearing the ter mutation as erg* parent.

7. Process according to one of Claims 1 to 6, characterized in that the mutant is constitutive for oxidative alcohol dehydrogenase.

8. Process according to one of Claims 1 to 7, characterized in that the mutant is devoid of fermentative alcohol dehydrogenase.

9. Process according to one of Claims 1 to 7, characterized in that the mutant is ADH I*.

10. Process according to one of Claims 1 to 9, characterized in that the terpene flavourings are extracted from the medium after culturing.

11. Process according to one of Claims 1 to 10, characterized in that the liquid foodstuff is chosen from fruit juices, milk or milk derivatives and cereal musts.

12. Process according to one of Claims 1 to 8 and 10-11, characterized in that drinks or flavoured products without alcohol or with a low alcohol content are 1 2 prepared by fermentation of the primary products with an ADH Γ S. cerevisiae mutant blocked in the pathway of ergosterol biosynthesis.

13. Process according to Claim 12, characterized in 5 that the mutant is ADH lie.

14. Process according to Claim,13, characterized in that the S. cerevisiae mutant Is used as a yeast for the process of secondary fermentation in the production of sparkling wines.

15. A process according to Claim 1 for obtaining terpene flavourings, substantially as hereinbefore described and exemplified.

16. Terpene flavourings whenever obtained by a process claimed in a preceding claim.