FR2728913A1 - Prepn. of di-methyl-pyrazine, tri-methyl-pyrazine and tetra-methyl-pyrazine - Google Patents

Abstract

Simultaneous prepn. of di-, tri- and tetra-methylpyrazines comprises growing a Bacillus subtilis or Brevibacterium linens microorganism on a soya medium enriched with Thr, acetoin and ammonium ions.

Description

 The present invention relates to a process for the joint preparation of di-, tri- and tetramethylpyrazines, by culture of at least one microorganism of the species Bacillus subtilis or Brevibacterium linas, sm an enriched soy-based medium.

 These pyrazines, in particular 2,5diméthylpyraaine and tétraméthylpyrazine are known flavors which one seeks for years to produce industrially.

 We have in fact long studied their production by bioconversion in soy-based media, but the yields obtained, always low, have not made it possible to consider moving to the industrial stage.

 In particular, it was assumed in 1964 that acetone could be a precursor of tetramethylpyrazine (Pharm. Soc. Japan, 84, 545-548 (1964)).

In particular, the impact of amino acids added to a culture of Bacillus natto on the formation of pyrazines has been studied. 2,5-dimethylpyram was mainly obtained with L-threonine, tetra- and trimethylpyrazines mainly obtained with L-serine. The results of this study were published in Nippon Shokuhin Kogyo Gakkaishi,
Flight. 36, No. 9, 762 - 764 (1989).

However, to date, to the knowledge of the Applicant, these studies have not led to the development of a process ensuring an attractive yield. It is with a view to such a development that the
Applicant has carried out research work. The result of this work constitutes the invention currently described and claimed.

Said invention consists of a process for the production of pyrazines by bioconversion in enriched soy-based media; said bioconversion being carried out under optimal conditions,
The optimization having focused, in the first place, on the choice of the product precursors added with a view to enriching said soy-based media -.

 The presently claimed invention therefore relates to a process for the joint preparation of di-, tri-, tetramethylpyrazines by culture of at least one microorganism of the species Bacillus subtilis or Brevibacteriwx linens, on a medium based on enriched soybeans. Typically, said medium is enriched in threonine, acetone and ammonium ions.

 The method of the invention is based on the following three points: - the culture of microorganism (s) on a soy-based medium: a microorganism is generally cultivated; - the enrichment of this medium with precursors of pyrazines; - joint production on said medium of di-, tri- and tetramethylpyrazines: in the same fermenter, two metabolic pathways are used.

 The precursors involved are therefore on the one hand threonine and on the other hand acetone and ammonium ions.

Threonine generally occurs in its natural form of
L-threonine. It essentially contributes to the production of 2,5-dimethylpyrazine.

 Acetoiie and ammonium ions contribute, them, primarily to the production of tétraméthylpyrazine. According to the invention, the intervention of such ammonium ions has proved, surprisingly, very interesting.

Said ammonium ions can be brought into the culture medium, in different forms, obviously compatible with said medium. They can in particular be provided in the form of chloride and / or ammonium acetate. According to the invention, the intervention of ammonium acetate is recommended. It is with this product that we obtained the best yields.

 Said ammonium ions, according to the process of the invention, generally intervene at a rate of 0.1 to 1M.

 Advantageously, they are used at a rate of 0.1 to 0.7 M and preferably at a rate of about 0.35 M (-5 g N / 1).

 With regard to the intervention of threonine and acetome, the following quantities are recommended according to the invention.

 Threonine is generally added at a rate of 10 to 100 gfl, advantageously at a rate of about 40 g / l.

 Acetic acid is generally added at a rate, also, of 10 to 100 g / l; advantageously at a rate of 40 to 70 g / l and preferably at a rate of about 60 g / l (- 60 ml / 1).

 In general, it is advisable to add, according to the invention, the precursors, in a minimum quantity, to obtain the escompbE effect; namely the increase in yield. Adding them in large quantities often generates an inhibiting effect. It is therefore advantageous to stick to the intervention ranges recommended above.

 The culture process according to the invention - with the addition of threonine, acetoin and ammonium ions - is carried out as indicated above, on a soy-based medium. This medium can be solid or liquid.

 It can in particular consist of soya beans. In this hypothesis, said beans are previously impregnated with an aqueous solution which contains the precursors of the invention.

 It can also consist of an aqueous solution of ground grains, a suspension of soya flour, or soya milk, more or less diluted.

In this hypothesis of liquid medium, said medium contains the precursors of the invention; preferably in the quantities indicated above
As regards soybeans, said liquid culture medium advantageously contains from 20 to 300 g / l, and preferably from 50 to 100 g / l.

 With the above ingredients, which are advantageously involved in the recommended amounts, it is possible, by culture of a microorganism of one of the species also specified above, under conventional culture conditions, to obtain, according to the invention, pyrazines , in interesting quantities ...

 However, it is still possible to improve the yield of the process of the invention, by implementing it according to preferred variants, explained below. These variants are not mutually exclusive.

 According to a first variant, the culture medium is enriched firstly with threonine only and secondly, advantageously after the consumption of said threonine (mainly the synthesis of 2,5dimethylpyrazine), in acetony and ammonium ions. It is of course possible, according to the invention, to carry out the global enrichment (threonine + acetoin + ammonium ions) from the start of the culture. It is however much more advantageous to implement the above variant; that is to say to offset in time the contributions of threonine on the one hand and acetone and ammonium ions on the other hand. Said lag, assuming that the synthesis of 2,5dimethylpyrazine is awaited, is from 30 hours to 50 hours.

 According to a second variant, the addition of threonine to the culture medium is carried out sequentially. Said medium, enriched or not with acetome and ammonium ions (hypothesis of the first variant described d-dessas) is enriched by successive additions, spaced over time, of threonine. The desired concentration of threonine can, for example, be restored every 6 hours.

 According to a third variant, the culture is carried out at a controlled pH. When this parameter is not controlled, it varies from around 6.5 at the start of the process to around 9 at the end of the process. Advantageously, it is controlled, throughout the culture, at a value around 7.5.

 As indicated above, all of these variants of the invention can advantageously be implemented, jointly or successively, during the course of the process of the invention.

 Furthermore, conventionally, the culture medium can be sterilized before said culture. Sterilization (or initial autoclaving) is advantageously carried out under the following conditions: at 132in for 30 minutes. Before such sterilization and before inoculation, the culture medium can advantageously be acidified, for example at a pH close to 4.

 Conventionally, it is also possible, at the end of culture, to sterilize the medium.

 With regard to the other parameters of the culture (temperature, duration, oxygen pressure), those skilled in the art are able to optimize them.

The culture, in a conventional manner, is generally carried out between 25 and 45 ° C., advantageously at around 40 μl. Regarding the oxygen pressure, there is no particular constraint. However, care should be taken to avoid being limited by oxygen insofar as the aim is to achieve an attractive yield.

Finally, it was previously indicated that the microorganisms involved in the culture method according to the invention are chosen from the species Bacillus subtilis and Brevibacterium linens. Advantageously, the microorganisms identified below are used:
- Bacillus subtilis ATCC 7058
- Bacillus subtilis ATCC 7059
- Bacillus subtilis ATCC 15245
- Bacillus subtilis Ifs 3009
- Bacillus subtilis IFO 3013
- Bacillus subrilis DFO 3335
- Bacillus subtilis IFO 3336
- Bacillus subtilis IFO 3936
- Brevibacterium linens CNCM I-1416
-Brevibacterluna linens CNCM I-1417; (ATCC: American Type Culture Collection;
IFO: Institute of Fermentation of OSAKA;
CNCM: National Collection of Cultures of Microorganisms
from the Pasteur Institute).

 The process of the invention is illustrated in Examples 4, 5 and 6 below. Examples 1 to 3 are given for comparison.

Example 1
60 g of soybeans are impregnated for 15 hours at 4 ° C. in 300 ml of an aqueous solution containing 15 g (50 g / l) of L-threonine and 18 ml of acetoin (60 ml / l). The excess solution is then removed, then the grains are introduced into a Pyrex column reactor with a volume of 0.5 l; the whole is sterilized in an autoclave for 20 minutes at 12C.

 After cooling, the medium is inoculated using 20 ml of a preculture of Bacillus subtilis IFO 3013 carried out in a 250 ml Erlenmeyer flask containing 50 ml of nutritive solution incubated for 20 hours at 2C. This preculture is itself seeded from the colonies obtained on an agar tryptone-soy agar medium in a petri dish, by stirring in 2 ml of sterile distilled water.

The nutrient solution is obtained by adding 1 ml of solution B to 49 ml of solution A, the two being sterilized separately at 12 (rC for 20 minutes. Solution A contains 1.36 g "of K2HPO4; 16 g / l Na2HPO4, 2H20; 0.5 g / l NaCl, and 25 g / l yeast extract.
B consists of 250 g / l of glucose.

 The culture is carried out at 2rc with an aeration rate of 1 l / h (0.033 VVM).

 2.5 g of wet solid substrate, corresponding to 1 g of dry matter, are suspended in 20 ml of distilled water containing 5 ssd of butanol-1 (internal standard). The whole is placed in a steam training device of the Likens-Nickerson type, and the volatile metabolites are recovered in 2 ml of dichloromethane. This solution is then injected into a gas chromatograph equipped with a polar type capillary column (for example Carbowax). The identification of the compounds is carried out by comparison of the retention times with authentic samples.

 0.02 g / l of 2,5-dimethylpyrazine, 0.10 g / l of trimethylpyrazine and 0.32 g / l of tetramethylpyrazine are obtained after 6 days, for a total of 0.54 g / l of pyrazines .

Example2
100 g of skinned yellow soybeans are impregnated in 500 ml of distilled water for 15 hours at 4 ° C. The excess water is removed, then the solid is resuspended in 1 1 of water. The whole is ground, and the medium obtained is supplemented with 40 g of L-threonine. LrmocUiaLion is carried out after autoclaving at 12 (rC for 20 minutes, using 20 ml of inoculum prepared as in Example 1.

The culture is carried out at 40 ° C. with an aeration flow of 12 Vh (0.2
VVM), without pH regulation, and 60 ml of acetome are added after 30 hours.

 The extraction and the metering of the metabolites are carried out as in Example 1, replacing the aqueous suspension of the grains with 5 ml of culture medium.

 0.65 g / l of 2,5-dimethylpyrazine, 0.27 g / l of trimethylpyrazine and 0.50 g / l of tetramethylpyran are obtained after 72 hours, for a total of 1.42 g / l of pyrazines.

Example 3
100 g of soy flour (Soyamin 50I) are suspended in 1 I of water containing 40 g of L-threonine. The inoculation is carried out after autoclaving as in Example 2.

 The culture is carried out as in Example 2, but the pH of the culture medium is kept constant at a value of 7.5.

 After 72 hours, 1.42 g / l of 2,5-dimethylpyrazine, 0.22 g / l of trimethylpyrazine and 0.10 g / l of tetramethylpymne are obtained, for a total of 1.74 g / l of pyrazines.

Example 4
The procedure is as in Example 3, but 27.5 g of ammonium acetate are added, with acetone (60 ml), after 30 hours of culture.

 1.32 g / l of 2,5-dimethylpyras, 0.21 g / l of trimethypyrazine and 1.08 g / l of tetramethylpyrazine are obtained after 72 hours, giving a total of 2.61 g / l of pyrazines.

Example 5
50 g of soy flour are suspended in 1 I of water, then the whole is autoclaved and inoculated as in Example 2. The culture is carried out at a temperature of 40 C, with an aeration flow rate of 121h (0.2 VVM) and the pH of the culture medium is kept constant at a value of 7.5. No addition of acetoin is made. 20 g of L-threonine are added after 6 hours. The residual amino acid concentration is followed by injection of the trimethylsilyl derivative (TMS) into a gas chromatograph equipped with an apolar type capillary column (for example SPB-5 from Supelco). The concentration of L-threonine is restored at 20 g / l every 24 hours, adding the necessary volume of aqueous solution containing 400 g / l of this compound.

50 g / l of L-threonine are consumed after 96 hours of culture. At this time, 3.80 g / l of 2.5dimethylpyrne is obtained, ie a total of 4.10 g / l of pyrazines (These concentrations are expressed relative to the initial undiluted culture medium.)
At the end of these 96 hours, 60 ml of acetic acid and 27.5 g of ammonium acetate are added to the culture medium. 72 hours later, 3.80 g / l of 2,5-dimethylpyrazine, 0.30 g / l of trimethylpyrazine and 1.08 g / l of tetramethylpyrazine are obtained, ie a total of 5.18 g / l of pyrazines.

Example 6
50 g of soy flour and 40 g of threonine are suspended in 1 liter of water, then the whole is acidified to pH 4 with hydrochloric acid, then autoclaved at 132 C for 30 minutes. It is then inoculated using a preculture of Brevibacterium linens CNCM I-1417, carried out as in Example 1. The culture is carried out as in Example 4, but the ammonium acetate and acetoin are added after 48 hours of culture.

 After 96 hours, 1.30 g / l of 2.5 dimethylpyrazine, 0.30 g / l of trimethylpyrazine and 1.85 g / l of tetramethylpyrazine are obtained, for a total of 3 * 45 g / l of pyrazines. .

Claims (11)

- Claims 1. Process for the joint preparation of di-, tri-, tetramethylpyrazijies by culture of at least one microorganism of the species Bacillus sub or Brevibacterium linens, on an enriched soy-based medium, characterized in that said medium is enriched in threonine, acetoin and ammonium ions. 2. Method according to claim 1, characterized in that the ammonium ions are added in the form of chloride and / or ammonium acetate, advantageously in the form of ammonium acetate. 3. Method according to one of claims 1 or 2, characterized in that the threonine is added in an amount of 10 to 100g / l, advantageously in an amount of about 40g / l. 4. Method according to any one of claims 1 to 3, characterized in that the acetoin is added in an amount of 10 to 100g (l, advantageously in an amount of 40 to 70g / l and preferably in an amount of about 60g / l. 5. Method according to any one of claims 1 to 4, characterized in that the ammonium ions are added in an amount of 0.1 to 1M, advantageously in an amount of 0.1 to 0.7 M and preferably in an amount of 'about 0.35M. 6. Method according to any one of claims 1 to 5, characterized in that it is implemented in solid or liquid medium; said liquid medium advantageously containing from 20 to 300 g / l of soya and preferably from 50 to lOOg / l of soya. 7. Method according to any one of claims 1 to 6, characterized in that the medium is enriched firstly with threonine for the synthesis, mainly, of 2,5-dimethylpyrazine and only secondly, advantageously after said synthesis, in acetoin and ammonium ions. 8. Method according to any one of claims 1 to 7, characterized in that the threonine is added to the medium sequentially. 9. Method according to any one of claims 1 to 8, characterized in that the culture is carried out at controlled pH, advantageously at a pH close to 7.5. 10. Method according to any one of claims 1 to 9, characterized in that the culture medium is initially sterilized at a pH close to 4. 11. Method according to any one of claims 1 to 10, characterized in that it is implemented by culture of at least one microorganism chosen from:  - Bacillus subtilis ATCC 7058  - Bacillus subtilis ATCC 7059  - Bacillus subtilis ATCC 15245  - Bacillus subtilis IFO 3009  - Bacillus subtilis IFO 3013  - Bacillus subtilis IFO 3335  - Bacillus subtilis' F0 3336  - Bacillus subtilis IFO 3936  - Brevibacterium linens CNCM I-1416  -Brevibacterium linens CNCM 1-1417.