FR2733763A1 - PROCESS FOR THE PRODUCTION OF VANILLIN BY BIOCONVERSION OF BENZENIC PRECURSORS - Google Patents

Abstract

Method for producing vanillin using the bioconversion of benzene precursors of vanillin, particularly ferulic acid or sodium ferulate, characterised in that an immobilised culture of Actinomycetes micro-organisms, for example of the Streptomyces genus, is provided in a medium to which the benzene precursor of vanillin has been added and which further contains an organic phosphate, and the vanillin resulting from the bioconversion of said precursor is recovered, the Streptomyces strain being selected for its metabolic capacity to store vanillin in the medium from the benzene precursor-containing medium.

Description

 The invention relates to a process for the production of vanillin from benzene precursors by bioconversion.

It is already known to produce vanillin microbially. By way of example, patent EP 0 405 197 describes a process for the production of vanillin by microbial fermentation of eugenol or isoeugenol. For this, a free microorganism of the Serratia species,
Enterobacter or Klebsiella is used in a medium comprising in particular a carbon source as well as, where appropriate, various inorganic salts, organo-elements and vitamins.

 However, this method has certain drawbacks. On the one hand, eugenol and isoeugenol are difficult to handle substrates since they are hydrophobic and, on the other hand, the use of a complex medium is not desirable, both in terms of preparation that of the isolation of vanillin. Finally, the yield of this process is low, of the order of 20%.

 Patent EP 0 453 368 describes a process for the production of vanillin by bioconversion of benzene precursors. According to this method, a Basidiomycete fungus is used which is first cultivated in a liquid medium and, after three days of culture, the bioconversion substrate is added to the culture medium. The bioconversion substrate is a benzene derivative such as ferulic acid or isoeugenol. The microorganism is free in the culture medium.

 This process has a number of drawbacks. First, the use of fungi is not very convenient, then it would be preferable to be able to bring the culture medium and the bioconversion substrate into contact more quickly and, finally, it would be desirable to improve the yield of the process which is, again, about 20 eo.

 In this regard, it can be noted that when the substrate used is particularly expensive, it is important that the process has a maximum yield.

US Pat. No. 5,128,253 describes a process for the production of vanillin by microbial conversion of a substrate chosen from the group comprising in particular ferulic acid, eugenol or coniferyl derivatives, by means of a free microorganism belonging to the species Pse udom on as, Corynebacterium, Rhodotorula glutinis and
Aspergillus niger. The conversion is carried out in the presence of a sulfhydryl type derivative and, if necessary, in the presence of a carbon source.

 Here again, the bioconversion medium is complex and the yield of the process is approximately 20%.

The object of the invention is to provide a process for the production of vanillin having in particular the following advantages - the vanillin prepared is obtained by bioconversion, this
bioconversion can be carried out from natural substrates without
use synthetic additives or reagents - the process must be simple to industrialize, the bioconversion medium
must be a defined environment (without complex biological products
like yeast extracts) and simple and the vanillin obtained should
be easy to extract from the bioconversion medium - the process must be profitable: the production yield compared
to the precursor must be improved compared to that obtained according to the
prior art, and the process must be able to work
long enough with the same bioconversion system.

 To do this, the subject of the present invention is a process for the production of vanillin by bioconversion of benzene precursors of vanillin, in particular ferulic acid or sodium ferulate, characterized in that an immobilized culture of microorganisms is used Actinomycetes, in particular of the genus Streptomyces, in a defined medium in which the benzene precursor of vanillin has been added, this medium comprising, in addition, an organic phosphate, and the vanillin produced by bioconversion of said precursor is recovered, the Streptomyces strain being selected for its property of accumulating vanillin in the medium from the medium containing the benzene precursor of vanillin.

 The method according to the invention uses an immobilized microorganism. This immobilization gives rise to a much higher, more reproducible vanillin production, which can be carried out over longer periods of time than when the microorganism is free in the bioconversion medium. In other words, it gives the system better stability.

 The bacteria according to the present invention are used in immobilized form, that is to say encapsulated in an appropriate matrix as is known to those skilled in the art.

 However, it will be preferred to use microparticles containing the microorganisms and, although these particles may be made of synthetic polymer, it will be preferred to use polymers such as alginates, gelatin or other products of the same type, alginates being very widely preferred. The technology for preparing alginate beads or particles containing microorganisms by crosslinking with Ca2 + is known.

 The term “bioconversion process” will denote a process in which there is practically no growth of the bacterial mass and in which the elements of the medium are essentially intended to ensure the enzymatic conversion of the substrate into vanillin.

 The so-called "defined" medium is a medium which does not contain mixtures of complex proteins such as yeast extracts or peptones, which play a role in bacterial growth, this medium preferably not allowing the growth of microorganisms.

 Among the precursors of vanillin, mention should be made in particular of ferulic acid and its salts, in particular of sodium, but other benzene precursors are possible, in particular eugenol, isoeugenol and conyferilic alcohol, even if their properties are not always compatible with good bioconversion.

 It is also possible to provide for the in situ production of the precursors from natural sources, which may be degraded to derivatives of ferulic type by enzymatic systems.

 The natural sources of these products are quite abundant, especially in many by-products of the sugar industry, such as beetroot chips but also wheat or rice bran.

 The bioconversion medium will also contain elements ensuring the activity of the enzymatic system and the maintenance of this activity, so it is necessary to provide for the presence of certain elements such as magnesium, which sometimes intervenes in the synthesis activity. enzymatic, or other salts such as Nazi, or, possibly elements allowing to modify the pH or the osmotic pressure.

 The key element of the bioconversion medium is organic phosphate.

 As organic phosphate, advantageously one of those chosen from the group comprising the nucleotides, natural or not, or the phosphate polyols such as glycerophosphate, particularly calcium glycerophosphate, will be used.

 Preferably, the organic phosphate is glycerophosphate.

 As the nucleotide, inosine monophosphate, guanosine monophosphate, uridine monophosphate or cytidine monophosphate can be used.

 The other phosphate polyols generally have a prohibitive price.

 Tests carried out with mineral phosphates have shown that these have, in particular on the immobilization of microorganisms, in particular on the encapsulation matrix, harmful effects.

 Indeed, in certain cases, especially when the matrix is an alginate, a partial or total disintegration of the matrix or a relaxation of this medium is observed, thus limiting the lifetime of the system. In addition, in certain cases, cell growth in the medium with the formation of micropellets can be observed with certain nucleotides, which can reduce the performance of the system. It follows that it will be preferred to use, as organic phosphate, glycerophosphate.

According to a preferred embodiment of the method according to the invention, the microorganisms are of the species Streptomyces setonii, in particular the strains Streptomyces setonii CNCM n 1-1555 as well as its mutants having the capacity to convert the precursors of vanillin in vanillin, in particular those obtainable by mutation / selection from said strain, this strain is in particular described by Sutherland,
Crawford and Pometto; Can. J. Microbiol. flight. 29, 1983.

 These microorganisms are preferably immobilized in alginate beads, the latter being produced by dropping, drop by drop, the bacteria incorporated in the liquid sodium alginate in calcium chloride (0.1 M) with stirring. , which ensures the crosslinking of the matrix incorporating the bacteria. It is also possible to provide for the use of a liquid core matrix containing the bacteria or other modes of implementation.

 We can also make beads comprising one or two layers to avoid the escape of bacteria.

 According to another preferred embodiment of the method according to the invention, the medium is maintained at a pH of between 5 and 7.5 and, preferably, around 6.2 to 6.4; the temperature is advantageously between 30 and 50 "C. and preferably close to 45" C.

 The medium preferably contains sodium chloride and magnesium sulfate and the organic phosphate is present in the medium at a concentration between 4 and 10 g / l and preferably between 7 and 8 g / l.

 The process can be implemented by "batch", continuous or semi-continuous. Advantageously, the precursor is added and the vanillin is recovered sequentially or continuously.

 In the case of a "batch" bioconversion, the entire medium is extracted at the end of the conversion.

 Continuous or semi-continuous, the vanillin is extracted as it is produced or at determined times, then the medium is recycled after optional addition of additional elements.

 In this regard, an advantage of this process lies in the fact that the vanillin formed remains in the medium until the practically total consumption of the precursor. It is only then that it degrades into vanillic acid.

 In other words, this process practically gives rise to two distinct stages in the time of formation and degradation of vanillin. This has the effect of facilitating the extraction operation and ensuring an industrially attractive yield.

 It will be possible to recover, preferably, the vanillin when the precursor is no longer detectable in the medium and before the bioconversion of the vanillin into vanillic acid.

 Vanillin is recovered, for example, by liquid / liquid extraction. The solvent can be ether, ethyl acetate or toluene.

 Preferably, the medium also comprises a magnesium salt.

 Advantageously, strains of Streptomyces setonii CNCM n "I-1555 are used in a medium comprising glycerophosphate at a concentration of 7.4 g / l, the pH of the medium being 6.4 and the temperature of 45" C.

 Other characteristics and advantages of the present invention will appear in the light of the detailed description which follows.

 The description refers to Figures 1 to 3.

 FIG. 1 represents the results of bioconversion in a vial of sodium ferulate into vanillin in a medium comprising UMP.

 FIG. 2 represents the results of bioconversion in a vial of ferulic acid into vanillin in a medium comprising glycerophosphate.

 FIG. 3 represents the results of bioconversion of ferulic acid into vanillin in a medium comprising glycerophosphate, the bioconversion however being carried out in successive "batches".

The symbols and abbreviations used are as follows:
X: precursor (ferulic acid or sodium ferulate) 0: vanillin
O: vanillic acid af: ferulic acid
Fna: sodium ferulate van: vanillin av: vanillic acid
I - BIOCONVERSION PROTOCOL 1 - lirincine
Two types of tests are carried out: in a flask or in a "bath".

 For the vial tests, the bioconversion is carried out from sodium ferulate in medium comprising uridine monophosphate (UMP), and from ferulic acid in medium comprising glycerophosphate.

 For the batch tests, the bioconversion is carried out using ferulic acid in a medium comprising glycerophosphate.

 The performance of the process is measured by monitoring the precursor and vanillin concentrations over time.

2 - Strain used
We use a microorganism of the species Streptomyces setonii deposited in the National Collection of Cultures of Microorganisms of the Institute
Pasteur (CNCM), 25 rue du Docteur-Roux - 75015 Paris, under the number "I-1555.

3 - Tests
The following media are prepared
Medium 1 comprises UMP at a concentration of 12.95 g / l, NaCl 0.5 g / l, MgSO4, 7H20 0.1 g / l, the pH is 7.68.

 Medium 2 comprises glycerophosphate at a concentration of 7.4 g / l, NaCl 0.5 g / l, MgSO4, 7H20 0.1 g / l, the pH is 6.4.

 10 ml of cells in stationary phase (OD = 1.20) immobilized in 40 ml of alginate are introduced into 100 ml of media 1 and 2. The concentration of alginate beads is 140 beads per 10 ml of mixture.

 In medium 1, sodium ferulate (2.22 g / l) is introduced as a precursor of vanillin, in medium 2, ferulic acid (1.62 g / l).

 Medium 3 consists of 1.25 1 of medium comprising glycerophosphate at a concentration of 7.4 g / l, NaCi 0.5 g / l and MgSO4, 7H20 0.1 g / l. 7,000 beads of immobilized microorganism are used and the precursor used is sodium ferulate.

 Bioconversion as a function of time is monitored by means of an HPLC analysis. For this, 0.5 ml of the culture medium is taken, this medium is filtered, it is diluted ten times and analyzed by HPLC.

 FIGS. 1 to 3 represent the results of measurement of the concentrations by HPLC of precursor and of vanillin and of vanillic acid formed. These figures correspond respectively to environments 1 to 3.

a) Svnthèse in vial - mediums 1 and 2
At 45 "C, rapid consumption of the precursor is observed in the two figures. This consumption is almost total for the medium based on UMP and with sodium ferulate as precursor (FIG. 1). based on glycerophosphate with ferulic acid as precursor (Figure 2) In both cases, the synthesis of vanillin is detected from the second day and the amount of accumulated vanillin increases almost until total consumption of the precursor.

 In both cases, the concentration of vanillin reaches approximately 1 g / l, which is a high value, and the maximum molar yield reaches approximately 66% in 90 hours, which is also a high value. The initial concentration of sodium ferulate is 2.2 g / l.

 Finally, it appears that the results obtained in a medium comprising glycerophosphate and with ferulic acid as a precursor are particularly good: the bioconversion lasts only 3 days, the concentration obtained reaches 0.88 g / l and the maximum molar yield is 69% . The initial concentration of ferulic acid is 1.62 g / l.

b) Batch tests - medium 3
It is observed that the system used can operate for approximately 3 weeks, which corresponds to an advantageous operating time from an industrial point of view.

 For all the "batches", a rapid decrease in the precursor concentration is observed at 45 "C. The total disappearance of the precursor corresponds to a maximum concentration of vanillin.

 For all the "batches" (except the first for which the initiation of the reaction occurs) the maximum concentration of vanillin is high (between 0.65 and 0.9 g / l) and the molar yield of bioconversion is also high since it varies between 53 and 69%.

 It therefore appears that the implementation of the method according to the invention in successive "batches" is particularly advantageous since the system works for a long time and gives rise to a production of vanillin with a high yield and a high concentration.

Claims (11)

 1) Process for the production of vanillin by bioconversion of benzene precursors of vanillin, characterized in that - an immobilized culture of Actinomycete microorganisms is used,  in particular of the genus Streptomyces in a defined medium in which the  benzene precursor of vanillin was added, this medium  comprising, in addition, an organic phosphate, and - after the bioconversion, the vanillin produced is recovered, - the strain of Streptomyces being selected for its property  to accumulate vanillin in the medium from the medium containing  the benzene precursor of vanillin.  2) Method according to claim 1, characterized in that the organic phosphate is chosen from the group comprising phosphate polyols and natural or unnatural nucleotides.  3) Method according to any one of claims 1 or 2, characterized in that the organic phosphate is glycerophosphate.  4) Method according to any one of the preceding claims, characterized in that the microorganism is of the species Strep tomyces setonii.  5) Method according to any one of the preceding claims, characterized in that the microorganisms are immobilized in alginate, preferably in alginate beads.  6) Method according to any one of the preceding claims, characterized in that the medium is maintained at a pH between 5 and 7.5, preferably close to 6.2 to 6.4, and at a temperature between 30 and 50 "C and preferably close to 45" C.  7) Process according to any one of the preceding claims, characterized in that the organic phosphate is present in the medium at a concentration of between 4 and 10 g / l, and preferably between 7 and 8 g / l.  8) Process according to any one of the preceding claims, characterized in that the precursor is supplied and the vanillin is recovered sequentially or continuously.  9) Method according to any one of the preceding claims, characterized in that the vanillin is recovered when the precursor is no longer detectable in the medium and before the bioconversion of the vanillin into vanillic acid.  10) Method according to any one of the preceding claims, characterized in that strains of Streptomyces setonii CNCM n "I-1555 in a medium comprising glycerophosphate at a concentration of 7.4 g / l, the pH of the medium being 6.4 and the temperature of 45" C.  11) Method according to any one of the preceding claims, characterized in that the medium further comprises a magnesium salt.