DK175382B1 - Terpene flavorings - 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

DK 175382 B1 i

The present invention relates to a process for the preparation of terpene flavors, particularly to flavoring beverages, by a microbiological process using Saccharomyces cerevisiae mutants.

5 Terpenes, considered essentially as products of vegetable origin, are characteristic constituents of several essential oils. v Due to their physiological and sensory properties, the mono · and sesquiterpenes have many applications in the food industry, the perfume industry and the pharmaceutical industry.

In addition, certain monoterpenes (geraniol, nerol, linalol, citronellol, alpha-terpineol) are responsible for the aromatic nature of the nutmeg. The grape varieties with nutmeg flavor and aroma belong to two different classes, mainly defined by the value of the ratio of linalol to geraniol. For the "Muscat" varieties, the ratio of linalol to geraniol is greater than 1, while it is clearly less than 1 for the "Malvoisie" varieties.

Although the vegetable sources of monoterpenes are numerous, for climatic, seasonal, economic and political reasons, they may prove insufficient for an increasingly demanding market, and therefore the microbial production of these aromatics has a considerable interest, which is one of the objects of the present invention.

Unlike filamentous fungi, whose ability to synthesize terpenes has often been described, yeast appears to lack these biosynthetic capabilities. Indeed, the few yeast species for which a production of terpenes (in the form of traces) have been described are all non-cerevisi-4 ae species.

Yeast, especially S. cerevisiae's, low production of terpenes apparently stands in contrast to their presence in cells with enzymatic systems capable of catalyzing the biosynthesis of certain monoterpenes.

DK 175382 B1 I

$. namely, cerevisiae cells synthesize significant amounts (0.5-3Z I

of the dry watch) ergosterol from acetyl-CoA, and the polymerization of I

acetyl * CoA occurs as in higher plants via mevalonate, geranyl · B

pyrophosphate and farnesyl pyrophosphate. Thus, S. cerevisiae has all I

5 the enzymes necessary for the biosynthesis of terpenes, and the reason B

to which these latter are not excreted, it is probable that enzy * I

more from the ergosterol synthesis pathway has a strong affinity for terpene-B

the pyrophosphates, which are thus substantially polymerized to 1 B

ergosterol. IN

The present invention is based on the recognition that certain S. B

cerevisiae mutants are capable of secreting terpenes in opposition B

to that which has hitherto been recognized, and these strains may additionally B

used for the direct manufacture of fermented or non-fermented I

aromatic beverages. B

The present invention specifically relates to a method for I

preparation of terpene flavorings by which process a S. I

cerevisiae mutant which is blocked in the ergosterol synthesis pathway and which B

secretes aromatic terpenes, is grown on an appropriate cultivar

up medium. IN

20 Thanks to the studies that have led to the present op

In fact, it has been shown that the biosynthesis of ergot I

role blocked by mutations in certain steps, for certain strains (in I

hereinafter termed ergosterol auxotrophic mutants) appears at B

accumulation of terpene intermediates. The mutations affecting I

The pathway of synthesis for sterols in the yeast strain S. cerevisiae, and most often B

shown by the accumulation of terpene pyrophosphates, corresponds to bio-B

in the steps catalyzed by squalene synthetase and famesyl-B

diphosphate synthase (EC 2.5.1.10). v B

Strains of this type can be selected by Saccharomyces-B mutation

30 strains and selection on a medium containing a sterol, especially B

ergosterol, and nystatin. fl

Due to the ergosterol auxotrophy, some of these strains exhibit, e.g., B

strain 134, which is further described in the Examples, however, in certain B

DK 175382 B1 3 cases a relatively weak growth due to a generally very low sterol content in the fruit juice. In order to alleviate the often significant qualitative and quantitative fluctuations observed when this yeast type is fermented, strains can be obtained, in particular, by subcloning, which, in addition to the mutations described above, comprises a suppressor mutation which appears in growth in the absence of ergosterol.

v

The partial suppression of the ergosterol auxotrophy leads to restoration of the wild-type phenotype.

By tetrade analysis, it has been established that the 10 - mutation segregates independently of ergosterol auxotrophy; the mutation can be isolated from recombinant spores which are ergoste-role prototrophs; - the mutation is recessive.

However, despite their relative independence of exogenous ergosterol, these clones produce significant amounts of monoterpenes.

For example, this type of strains may be selected by subcloning the preceding strains onto a medium without ergosterol.

It should be noted that these strains synthesize ergosterol to a limited extent and that they may grow in the absence of ergosterol, but that this mutation does not affect blockage in the sterol synthesis pathway and does not manifest phenotypically in the erg + progeny.

To ensure genetic stability of the erg * mutants, they are conserved in heterozygous state in the form of erg / erg + diploids. Before use iso- \ t.

the erg 'traces are learned after sporulation and dissection of the trace sacs. 1

Using this method, it has been found that certain erg * strains which, after sporulation, emerged from the erg * / erg + diploids, no longer excreted terpenes in the medium. This has led to the detection of a further recessive mutation which, in connection with the blocking of the sterol synthesis pathway, allows the excretion of terpenes.

I DK 175382 B1 I

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Thus, the most interesting mutants according to the invention are for I

In addition to the above mentioned mutations, you carry a mutation which I

You are referred to as ter. and which are either implicated in the permeabilization of mem · I

the brane or in the dephosphorylation of geranyl pyrophosphate. IN

In the recombinant erg + haploids, the ter mutation does not manifest itself

In phenotypic. The erg * spores derived from the diploid erg + ter / erg * I

In ter, still produce terpenes. «I

Mutants producing terpenes should be retained in heterozygous state I

with a strain carrying the ter mutation. as erg + parents. IN

Among the preferred mutants of the invention, mention should be made of the mouse

aunts carrying the following mutations:

I - irritation, blockage in the synthesis steps of sterols, which steps are categorized

You are lysed by farnesyl pyrophosphate synthetase and squalene synthetase, I

I - a recessive suppressor which is not associated with erg * mutation- I

In the nene, and which indicates a relative independence of exogenous ergosterol I

Without affecting the blockage of the synthesis pathway for sterols,

I - ter. a recessive mutation that allows the production of terpenes, I

without which the geranyl pyrophosphate accumulated due to I

I of the erg * blocker, is not excreted in the medium in the form of geraniol. IN

The strains thus obtained secrete aromatic terpenes into considerable I

quantities, but this applicant has been able to demonstrate the possibility that you

You further increase this separation. IN

The studies carried out have shown that alcohol dehydrogenase I

In seme (ADH), an increased production of acetyl-CoA exhibits, as do I

It is possible to increase the activity of the synthesis pathway for sterols, with * I

A larger amount of acetyl-CoA is available. "I

In particular, the following mutations are interesting:

I - ADH I mutants, viz. mutants lacking the fermentation iso- I

In zym and which are unable to form ethanol from acetal-I

In 30 dehydes and thus exhibits increased production of acetyl-CoA; IN

• ADH Ile mutants, ie. constitutive mutants for oxidative isoenzyme which oxidize ethanol to acetaldehyde, even in the presence of large amounts of sugar (a typical condition of fruit juice and must), while the wild-type strain only oxidizes ethanol in the absence of 5 sugars.

in

In particular, a combination of these different mutations makes it possible to obtain mutants producing terpenes in large quantities, especially terpenes of aromatic interest such as farnesol and geraniol.

The mutants of particular interest are the mutants which are ergo-sterol auxotrophs and ADH Ile, the mutants that are ergosterol auxotrophs and ADH I ', and finally the mutants ADH 1' and ADH 11c.

The method of the present invention can be practiced in various ways.

First of all, it is possible to use a culture of this mutant type to produce flavors such as natural Muscat flavors which are purified from the culture and which can be used to flavor food substrates, especially beverages.

In most cases, however, the terpenic flavors are produced directly in situ by growing food substrates, especially food liquids or food products, which can be liquified such as, for example, fruit purees. In this case, these substrates are fermented directly by the S. cerevisiae mutant strains, thus providing for the aromatization of the substrates.

In the case where the mutant used is an ADH I mutant, i.e. a mutant which does not contain fermentative alcohol dehydrogenase, the obtained fermentation products are non-alcoholic products or products with a very low alcoholic strength.

Of course, in some cases it may be interesting to allow this type of strain to ferment media that is already alcoholic to give them certain aromas, and in this case successive fermentations may be carried out with a yeast producing alcohol and then with

I DK 175382 B1 I

I I

In mutants of the present invention, or whenever possible I

In or compatible with the growth of yeast strains, to perform a cofermentation

In with the two gerbils. These yeast strains can also be used for export

see of bottling, for example in sparkling wines. IN

In 51 many cases, the preparation of alcoholic and aromatized I

the beverages are of course carried out using a mutant of I

In the invention, however, which contains a fermentative alcohol dehy- <1

In drugase (ADH I +). IN

In the strains of the present invention for some people * I

When they are ergosterol auxotrophic, it is when the culture medium is a full I

In constant synthetic medium, it is necessary to provide the addition of ergo- I

In sterol to ensure the growth of gerber strains. In the case of substrates of I

In vegetable origin, such an additive is generally not necessary

you, because these compounds exist as trace elements in the differences I

In 15 equal vegetable extracts. IN

In the experiments performed using the mutants of the present invention

The present invention has shown that it was thus possible, for example, to obtain I

In non-alcoholic fruit juices which had it for Muscater character

In teristic aroma, or alcoholic products of the wine type or I

In 20 the sparkling wine type they had for Muscat or Kalvoisie I

In distinctive aromas. IN

In the mutants ADH I * or ADH Ile, in particular, are I

Of interest in the preparation of sterols, especially ergosterol, and gene I

In the case of products containing an increased amount of acetyl-CoA. IN

The invention is further illustrated by the following examples. IN

I I

In Example 1 I

In Preparation of an ADH I ~ Mutant I

In the mutant FL 100 ADH I 'is selected from the domestic I of the haplolled wild type

In FL 100 (ATCC 28383) by resistance to allyl alcohol in presence I

DK 175382 B1 7 of glucose. This mutant, which is ergosterol prototrophic, does not accumulate terpene intermediates, as all steps in the synthesis pathway for sterols are functional.

In contrast, the increased amount of acetyl-CoA due to the ADH I * 5 mutation is shown by a significant increase in the amount of synthesized ergosterol (Table 1). This type of mutant forms strains that hyper * produce ergosterol.

TABLE 1

Ergosterol 10 (X of the wheat gun ^ FL 100 0.75 ± 0.06 FL 100 ADH 1 '1.06 ± 0.05 EXAMPLE 2

Preparation of a mutant that is ergosterol auxotroph 15 Mutagenesis is performed by ultraviolet rays of the wild-type strain FL 100.

Next, the strains resistant to nystatin in the presence of ergosterol are selected. Among the resistant mutants, one is selected, termed "mutant erg 9".

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

Measurement of squalene synthetase activity is performed in the microsomal fraction by the method of Agnew and Popjak (WS Agnew, G. Popjak 25 (1978) J. Bio. Cheat. 253, 4566-4573), demonstrating that this mutant erg 9 lacks squalene synthetase activity.

I DK 175382 B1 I

i 8 I

In the mutant erg 9 is also characteristic of the absence of ADH I (rings I

In the production of ethanol) and by constitutivity for ADH II. Its fzno- I

type is given in Table 2. I

I TABLE 2 I

IN

I 5 Growth Squalene synthetase ADH I ADH II Ethanol I

I YPG + erg YPG (s.a.) (s.a.) (s.a.) (g / 1) I

In FL 100 +++ +++ 0.24 16.2 12.3 36.2 I

I very 9 ++ 0 0 10.8 (*) 3.7 I

10 _____ I

I s.a. - specific activity I

I (*) - in the presence of 90 g / l glucose I

In Example 3 I

I Preparation of a mutant which is ergosterol auxotroph I

I This mutant was obtained from a heat sensitive mutant which was gly-I

In cin auxotroph (aux 32 ts), after UV mutagenesis on medium with nystatin. IN

I This mutant accumulates dimethyl-allyl pyrophosphate in vitro and I

You do not produce farnesyl pyrophosphate in the presence of isopentenyl-I

In pyrophate (labeled with carbon-14) and geranyl pyrophosphate, suggesting I

I 20 in the absence of farnesyl diphosphate synthase activity. IN

In addition, mutant 134 exhibits the characteristic of strain 9 strains I

In the phenomenon; the phenotypic allelism suggests the presence of an I

In allele of erg 9; it is therefore a double farnesyl diphos- I

In phat synthase mutant. t I

Like the mutant erg 9, the mutant 134 is constitutive ADH I * and I

In ADH II. The results for the secretion of geraniol in vivo show that I

In the mutation farnesyl diphosphate synthase is bradytrof. IN

EXAMPLE 4 DK 175382 B1

The two mutants erg 9 and 134 are grown 1 shake culture (24 h) or stations culture (4 days) in complete medium (IX yeast extract, IX bac-topeptone, 2 h glucose, 0.0081 ergosterol) at 28 ° C. These two mutants • 5 efficiently secrete famesol and geraniol, as shown in Table 3.

TABLE 3

Famesol Geraniol (Mg / 100 ml) (Mg / 100 ml) 10 Stations Shaking Stations Rvste FL 100 n.d. n.d. n.d. n.d.

bad 9 24 138 n.d. n.d.

134 17 40 - 22 15 n.d. «· Not detectable

In comparison, the wild-type strain FL 100 produces neither farnesol nor geraniol, neither in shake culture nor in station culture.

EXAMPLE 5

Aromatization of fruit juices with the mutants erg 9 and 134 20 Fruit concentrates (white grapes, red grapes, raspberries, peaches, pear, banana) diluted with 120 g sugar / 1, smeared with 10 ^ cpm and incubated under agitation (150 rpm) at 30®C.

The sensory analysis is performed after 4 days on juices released for cells. Odor analyzes systematically show signs of terpenes in 25 cases of the mutants erg 9 and 134 as opposed to the products obtained by the wild-type strain FL 100. The observed results are reported in Table 4.

In DK 175382 B1

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In Table 4 I

I FL 100 Erg 9 134 I

White grape wine-like muscat-type muscat-type I

Red grape winey floral scent floral scent I

5 Peach "yeast" flower fragrance fruity I

In the ferment "yeast * fruity, fruity, I

floral floral I

Banana "yeast" muscat fragrance, muscat fragrance I

In the "fame sun" I

In 10 Raspberries "yeast", very "famine-like" fragrance I

solvents-like I

EXAMPLE 6 I

H From strain 134, a new strain capable of subcloning is obtained

15 to grow on a medium without exogenous ergosterol and having the following I

Characteristics: I

- relatively good growth without ergosterol (intermediate level between I

I 134 and the wild type strain FL 100), I

- biosynthesis of ergosterol (10X of the wild-type strain), I

H 20 block in the step catalyzed by famesyl pyrophosphate-I

H synthetase, with accumulation of geranyl pyrophosphate, I

excretion in the culture medium of monoterpenes, especially geraniol and I

In linalol. IN

This strain provides much more in industrial terms and enables you to

In 25 to achieve stable productions, both in terms of quantity and quality

In tet. IN

Claims (13)

A process for the preparation of terpene flavorings, characterized in that a S. cerevisiae mutant is grown on an appropriate culture medium which is blocked in the synthesis pathway of ergosterol and secretes aromatic terpenes. Process according to claim 1, characterized in that the suitable culture medium comprises a liquid liquid or a liquid liquid food product. Method according to claim 1 or 2, characterized in that the mutant has a very low squalene synthetase activity and / or farnesyl diphosphate synthase activity. Method according to any one of claims 1-3, characterized in that the S. cerevisiae mutant comprises a suppressor mutation which is shown by growth in the absence of ergosterol. Method according to any one of claims 1-A, characterized in that the mutant is a ter mutant. Method according to claim 5, characterized in that the preservation of the mutants used in the process is carried out in heterozygous state with a strain carrying the ter mutation, as erg + progenitor. Process according to any one of claims 1-6, characterized in that the mutant is constitutive of oxidative alcohol dehydrogenase. Process according to any one of claims 1-7, characterized in that the mutant lacks fermentative alcohol dehydrogenase. Method according to any of claims 1-8, characterized in that the mutant is ADH I +. I DK 175382 B1 I I I Process according to any one of claims 1-9, characterized in that the terpene aromas are extracted from the medium after cultivation. IN Process according to any one of claims 1-10, characterized in that the food wash is selected from 1 H of fruit juices, milk or milk derivatives and cereals. «I Process according to any one of claims 1 to 10, characterized in that beverages or aromatized products are produced without alcohol or with a low alcohol content by fermentation of starting products with an ADH I mutant of S In cerevisiae blocked in the biosynthetic pathway of ergosterol. IN The method of claim 12, characterized in that the mutant is ADH Ile. IN Process according to Claim 13, 15, characterized in that the S. cerevis iae mutant is used as a yeast in bottle ring to produce sparkling wines. I I I