Microbiological process for producing vanillin

Details Microbiological process for producing vanillin Microbiological process for producing vanillin

1998-06-11

2001-05-22

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing oxygen-containing organic compound

C435S123000, C435S155000

Reexamination Certificate

active

06235507

ABSTRACT:

The present invention concerns a microbiological process for the production of vanillin from ferulic acid. According to this process, a culture, preferably a submerged culture, of any bacterium of the order Actinomycetales, preferably of the family Streptomycetaceae is incubated with the substrate ferulic acid to fermentatively produce vanillin. The product vanillin is recovered from the fermentation broth by a designed extraction method allowing also the separation and recovery of valuable fermentation by-products, in order to obtain the analytically and sensorically purified product vanillin, and, in particular the by-product guaiacol.
BACKGROUND
In the use of flavouring compounds, it is more and more important that the flavour compounds can be designated as “natural”. In line with the European and U.S. regulations this means that the compound has to be obtained by physical, enzymatic or microbiological processes and only from materials of plant or animal origin. Various research activities during the last decade were thus focused on the use of renewable, cheap and natural raw material sources for the fermentative production of vanillin. However, commercially attractive volumetric yields have not as yet been reported so far.
Guaiacol is a phenolic, smoky type of molecule which significantly contributes to the characteristic flavour of vanilla extracts. It is thus often used in combination with vanillin for vanilla type flavours. However, the fermentative production of natural guaiacol has not yet been described so far.
In the past 10 years, several methods concerning the microbial or enzymatic production of vanillin have been proposed. In general, a suitable precursor is transformed to vanillin by a microorganism or an enzyme. Suggested precursors are eugenol, isoeugenol, ferulic acid, curcumin or benzoe siam resins. Usually, transformation yields are extremely low. For example, Haarman & Reimer (EP 0 405 197 A1) describe a production of 18 mgL
−1
starting from 0.2 gL
−1
eugenol using the microorganisms Serratia, Klebsiella or Enterobacter. This transformation takes place over a period of 13 days. Pernod-Ricard (EP 453 368 A) describe a process where 46 mgL
−1
vanillin was obtained in a 6 day Pycnoporus fermentation from ferulic acid. Along this line is also Kraft General Foods (U.S. Pat. No. 5,128,253) describing a process where 210 mgL
−1
vanillin is obtained from ferulic acid within 54 days. In order to obtain this titer a reducing agent had to be added as otherwise the formation of vanillin would not occur and only vanillic acid would be formed. Takasago (JP 227980/1993) prepared mutants of Pseudomonas strains that are blocked in the degradation pathway of vanillin. Thus, starting with 1 gL
−1
ferulic acid 0.28 gL
−1
vanillin could be obtained. An application reporting a potentially economical attractive volumetric yields of vanillin in a fermentation process has recently been published by Haarman, & Reimer (EP 0 761 817 A2). They identified two strains of the genus Amycolatopsis which are able to accumulate vanillin up to a concentration of 11.5 gL
−1
in the fermentation broth after feed of ferulic acid.
It can be concluded from the above discussion that high amounts of vanillin are not easily formed in microbial systems. This is mainly due to the cellular toxicity of vanillin, which at concentrations above 1 gL
−1
prevents growth of the vanillin producing microorganisms. In microbial systems, usually the respective alcohol or acid is found and not vanillin. This toxic effect of vanillin was overcome by the use of enzymes (Quest, EP 0 542 348 A2). Treating isoeugenol with lipoxygenase resulted in 10-15 gL
−1
vanillin at a yield of 10-15%. Much lower concentrations were obtained when using eugenol (0.3-0.5 gL
−1
in a yield of 0.3-0.5%) and no turnover is reported for ferulic acid. The method employing lipoxygenase is scarcely attractive from the economic point of view.
Another measure to circumvent the toxicity of this compound is the microbial production of coniferylaldehyde which forms vanillin upon thermal treatment. See, for example, BASF (Offenlegungsschrift, DE 3604874 A1). Similar is also the immobilized cell system recently described in WO 96134971 in which vanillin is accumulated up to a concentration of 1 gL
−1
. A possible economic benefit of using immobilized biomass is given by recycling the biocatalyst.
Many papers deal with the respective metabolic pathways starting from eugenol, isoeugenol or ferulic acid. In general, vanillin is believed to be an intermediate compound in the degradation pathway of these compounds. Two publications have discussed the involvement of vanillin in the degradation of ferulic acid. Toms and Wood, Biochemistry 9 (1970) 337-43, cultivated Pseudomonas sp. on ferulic acid and elucidated the degradation pathway. Though vanillin was not found in the culture supernatant, evidence was given that vanillin is an intermediate compound, since vanillic acid could be detected. Starting from ferulic acid, vanillin was obtained in cultures of
Streptomyces setonii
(Sutherland et al., Can. J. Microbiol. 29 (1983) 1253-57). No indication was given on the amount, but only traces where found when repeating the experiment.
Ferulic acid as a substrate for biotransformations is abundantly available from different natural sources. The acid often occurs in the form of a glucoside in plant materials, such as wood, sugar beet melasse, bran of corn, rice and various types of grasses. It can be isolated from the corresponding glycosides in these products by well-known hydrolysis methods, e.g. using enzymes, and can be used as crude material or purified material. A British source (GB 2301103 A1) describes for instance the enzymatic breakdown of ferulic acid containing plant material by a ferulic acid esterase, in order to obtain the free acid.
SUMMARY OF THE INVENTION
The present new, high-yield microbiological process for the production of vanillin contemplated by the present invention comprises cultivating first in a nutrient broth a microorganism of the order of the Actinomycetales, preferably of the family Streptomycetaceae, most preferably the bacterium
Streptomyces. setonii
ATCC 39116, wherein, preferably, the cultivating period is about 5-40 hours and lasts, until the carbon source glucose is (almost) consumed, then adding the substrate ferulic acid in the range of about 5-40 gL
−1
of fermentation broth, either continuously or batch-wise. After an approximate incubation (biotransformation) period of about 5-50 hours, substrate conversion to vanillin and several by-products is completed. The ferulic acid is consumed and vanillin accumulated up to about 8-16 gL
−1
in the fermentation broth. Typical by-products of the ferulic acid biotransformation are vanillic alcohol, vanillic acid, guaiacol, para-vinylguaiacol and 2-methoxy-4-ethyl-phenol.
Subsequent product recovery consists in the removal of the biomass, conveniently followed by a two-step extraction with an appropriate organic solvent, preferably methyl-tert-butylether. A first extraction is carried out at a pH of greater than about 9, preferably at a pH of from about 10 to about 11 and most preferably from about 10.8 to about 11 in the aqueous phase to selectively extract by-products, such as the sensorically highly active guaiacol. Then, the aqueous raffinate is “acidified” to neutral pH values a pH from about 5 to about 8; preferably from about 6 to about 7.5, most preferably from about 6.9 to about 7.1 to selectively extract the product vanillin. Purification of the raw vanillin extract may finally be done by applying well-known recrystallization methods. Guaiacol may be purified from the raw extract by distillation.


REFERENCES:
patent: 4874701 (1989-10-01), Cooper
patent: 4981795 (1991-01-01), Cooper
patent: 5128253 (1992-07-01), Labuda
patent: 3604874 A1 (1987-08-01), None
patent: 405 197 A1 (1991-01-01), None
patent: 453 368 (1991-10-01), None
patent: 542 348 A2 (1993-05-01), None
patent: 761 817

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