NO890895L - PROCEDURE FOR THE PREPARATION OF "NATURAL" METHYLANE TRANILATE. - Google Patents

Description

The present invention is aimed at a microbial method for producing methyl anthranilate from naturally occurring methyl N-methylanthranilate.

In recent years there has been a significant consumer demand for better and more "completely natural" flavourings. In order to fulfill this need, it is necessary to develop new methods for providing chemicals with natural aroma.

Concord grape ( Vitis labrusca) flavor represents a type of product that has found wide application in the food industry. Methyl anthranilate is known to be the main contributor to the typical aroma of this fruit, and it is a valuable component in many other flavor systems. Although methyl anthranilate is widely distributed in nature, its isolation is generally not economical due to the extremely low levels present. Consequently, an alternative method for obtaining natural methyl anthranilate is required.

Although methyl anthranilate is not easily obtained from natural sources, methyl N-methylanthranilate (hereafter referred to as dimethylanthranilate) is easily extractable from "petitgrain mandarin leaf oil"

(Citrus reticulata) in high yield. Consequently, it would be desirable to take advantage of this good availability to prepare methyl anthranilate by an N-demethylation process.

Microbially mediated N-demethylation reactions have been reported in species of Pseudomonas (Taylor, B.F., i., Appl. Envoron. Microbiol.46:1286.1983), Streptomyces (Yamano, et al., in Ann. Rep. Takeda Res. Lab... 21:88, 1963 also Beilet and van Thuong U.S. Patent 3,520,778 in 1970), Arthrobacter

(Kaczkowski, J. , in Acta Soc. Botan. Polon.. 28:677, 1959 )

Cunnlnghamella. Xvlaria (Mitscher et al. , in Experientla.24:133.1968), Clavice<p>s (Voigt and Bornschein, in Pharmazle. ,22:258, 1967 ) and Ascer<g>ilus (Funderburk et al in Proe. Southern Weed Conf.. 20:389, 1967). Various types of N-dealkylation reactions have been reported in the degradation of lignin by dry rot fungi, including members of the genus Phanerochaete (Kuwahara et al. in J. Ferment. Technol.. .62:237, 1984 ), Polyporus, Ganoderma. Fomitopsis (Reade and . McQueen, in Can. J. Microbiol.. 29:4.57, 1983), Polvstictus (Shimazono and Nord, in Arch. Biochem. Bio<p>h<y>s.. 87:140, 1960) , Trametes and Fornes (Smolikova and Tichy, in Biologia..5:211, 1976 ).

Despite these reports, however, the microbial demethylation of dimethyl anthanilate has not been indicated, one reason being that both the starting material and the product are highly toxic to most microorganisms. Furthermore, the substrate stereochemistry causes difficulties because the microbiological demethylations are enzymatic and consequently very selective. As a result, no reported microbiological reaction can be used as a general method for the demethylation of substituted amines.

It is therefore an object of the invention to develop a microbiologically mediated demethylation method which will unexpectedly give reasonable yields of methyl anthranilate. Another purpose is the development of a microbiological method which is effective despite the toxicity of the starting material and the product.

These and other purposes are achieved by the present invention, which is aimed at a method for microbiological conversion of the substrate dimethylanthranilate to methylanthranilate. According to this method, the dimethylanthranilate is converted by culturing it with a fungal microorganism. The microorganism is cultivated in a nutrient broth, and the substrate is added to the culture medium after a suitable incubation period. The amount of substrate used can be up to approx. 10 weight #, preferably up to approx. 2.0% by weight, more preferably up to approx. 0.5, relative to the combined weight of the broth, culture and substrate. A substrate analog, such as 2,5-dimethylaniline or N,N-dimethylaniline can be added to the culture medium before the substrate addition to increase the yield of the method. Protein synthesis inhibitors can also be used. A preferred microorganism for use in the present method is a wood rot fungus or a fungus capable of degrading cellulose or lignin.

According to the present invention, a method has been discovered by which methyl anthranilate, or more specifically "natural" methyl anthranilate, can be produced by microbial transformation of dimethyl anthranilate. The "natural" dimethyl anthranilate used in this transformation can preferably be recovered from natural sources, such as e.g. from "petitgrain mandarin leaf oil" (Citrus retlculata) by standard techniques such as extraction or distillation. Although dimethyl anthranilate and methyl anthranilate are generally toxic to most microorganisms, certain types of fungi can tolerate useful concentrations of these compounds up to approx. 10$, relative to the total weight of the cultural medium. Very desirable concentrations in this connection are those of up to approx. 2$. The fungus is also capable of general N-demethylation of substituted amines. Microorganisms suitable for these purposes and which provide desired yields of methyl anthranilate include, but are not limited to, various strains of Streptomyces (eg, S. rosechromooenus, S. purpuracens. S. lavendulae. S. griseus. S .Dlatensis). strains of Aspergillus (eg A. oryzae. A. niger).Candia lipolytica Yarowia lipolytica. species of Xylaria (eg X. digitata. X. hvooxylon. X. magnoliae.). species of Arthrobacter (e.g. A. flavencens. A. globiformis. A. mvsorens. A. varlabilis. A. simplex). Cunninghamella echinulata (several strains) and Claviceps purpurea (several strains). Very desirable yields of methyl anthranilate can be obtained with essentially all types of wood rot fungi, preferably with species from the genus Trametes. Pol<y>stictus and Polyporus, and most preferably Trametes cingulata ATCC 26747 and Polyporus sos ATC 10089.

According to one embodiment of the method according to the invention, a culture suspension is prepared by inoculating a suitable nutrient broth with the selected microorganism. A suitable nutrient broth is one that contains carbon sources, nitrogen sources, trace minerals and any required weight factors such as vitamins. Suitable carbon sources are preferably lactose, cellobiose, nitrol, sorbitol, ethanol, glycerol, more preferably monosaccharides, such as maltose, fructose, rhamnose, xylose and most preferably glucose or malt extract. Suitable nitrogen sources include nitrogen-containing organic substances, preferably peptbn, meat extract, corn extract liquor and most preferably yeast extract. Inorganic nitrogenous compounds are also suitable, including nitrates, nitrites and ammonium salts. These nutrients can be supplemented by adding vitamins (especially those from the B group) and/or trace minerals (such as Fe, Cu, Mo, Mn, P, Ca etc.) to the medium, however, the method can be carried out in unsupplemented medium if a small amount yeast extract is added.

The inoculation according to one embodiment of the invention is carried out by adding the selected microorganism culture to a suitable nutrient broth and allowing the organism to incubate at a suitable temperature and pH for 1 to 14 days before adding the substrate (ie dimethyl anthranilate). The length of this incubation period varies with the organism used, but growth of the culture can be monitored by measuring the disappearance of the carbon source, changes in pH, or by visual observations (ie, the appearance of turbidity or fungal mycelia in the culture broth).

The cultivation of the microorganism and the substrate can be carried out as a stationary culture, but often higher yields can be obtained when the microorganism is cultivated in submerged culture (ie shake flask cultures or fermentor culture) under aerobic conditions. A suitable pH range is from approx. 3.0 to approx. 8.0, and preferably approx. 5.0 to approx. 8.0, and especially approx. 6.0 to approx. 7.0. The pH can be regulated by adding inorganic or organic acids or bases. The incubation temperature is appropriate between approx. 8°C and approx. 33°C, a range from approx. 20 to approx. 30°C is preferred, and in particular the range from approx. 22°C to approx. 26°C.

The fermentation can be carried out by using the cells of the microorganism isolated from the culture broth, or by means of enzymes isolated from the cells by usual enzyme extraction techniques. When the isolated enzyme is used, the reaction can conveniently be carried out in aqueous solutions (e.g., physiological saline, buffer solutions, fresh nutrient medium, etc.), or the isolated cells or enzyme extract can be immobilized on a solid support, and the desired products can be formed in the absence of the living organism. The transformation can also be carried out using mutants of the microorganism. These mutants can be readily obtained by methods well known in the art (eg, UV irradiation, exposure to mutagenic compounds, etc.).

In general, the substrate can be added directly to the fermentation broth after the microorganism has matured. An emulsion of dimethylanthranilate and "Tween-80" (mono-oleate) can be prepared so that the final concentration of dimethylanthranilate in the broth is up to approx. 10$, preferably up to approx. 2%, and more preferably up to approx. 0.5$, while the concentration of "Tween-80" is up to approx. 1%. This reaction mixture is allowed to incubate for a further 1 to 14 days, preferably 3 to 10 days and especially 4 to 8 days (depending on the organism used), or until no further production of methyl anthranilate is observed.

Under the usual conditions used for the method according to the invention, a smaller amount of N-formyl methyl anthranilate is also produced. These two products are typically present as a mixture in the broth when fermentation is complete. This mixture of N-formyl methylanthranilate and methylanthranilate in combination with unreacted dimethylanthranilate can be isolated and the three individual components can be separated by conventional techniques (e.g. solvent extraction, distillation and chromatographic separation techniques). N-formyl methylanthranilate is readily converted to methylanthranilate by base- or heat-catalyzed decarbonylation and, if desired, the yield of methylanthranilate can be increased by using this reaction. In all cases, there was a surface yield of 95 to 98 wt-# of the starting materials and products (i.e. none of the microorganisms degraded the starting material to unwanted by-products) and GLC purities of

> 95$ was usually obtained after simple solvent extraction.

The following examples serve to illustrate embodiments of the invention. These examples are in no way intended to limit the scope of the following claims.

EXAMPLE I

Trametes versicolor fermentation using yeast-malt culture

Five flasks containing 100 ml each of yeast-malt (YM) broth (Difco Laboratories) were autoclaved at 121°C for 20 min. After cooling, each flask was inoculated with 5 ml of a 3 day old YM broth culture of Trametes versicolor ATCC 42394. Each culture was incubated at 30°C and 200 rpm on a rotary incubator/shaker for 3 days. Different portions of an emulsion of "Tween-80" and dimethylanthranilate (DMA) were added to each culture to give final dimethylanthranilate concentrations of 0.2, 0.5, 1.0, 5.0 and 10.0% in the fermentation broth. Each culture was incubated for a further number of days and the results, such as the percentage concentration of methyl anthranilate (MA) are reproduced in Table 1.

EXAMPLE II

Trametes fermentation using mineral salt culture By using methods and materials similar to those described in example I, but by using a mineral salt solution containing NH4NO3 (3 g/l), K2PO4 (1.3 g/l), MgS047H20 (0.5 g/l), KC1 (0.5 g/l), FeSO4 (0.01 g/l), yeast extract (5 g/l) and glucose (15 g/l) instead of YM broth , to cultivate the microorganism, results were obtained which were similar in MA concentration and incubation period to the results indicated in the preceding Table I.

EXAMPLE III

Other Trametes fermentation

Using the methods and materials described in Example I, but other members of the genus Trametes. including T. versicolor strains ATCC 34578, 42394, 32085, 11235, 32745, 12679, 34584, 38068, 38070, 42462, 44677, T\ carbonaria 26746, T. clngulata 26747, T. cubensis 14590, T\ extenuatus 2674 gibbosa 34670, or T. hispida 36206, or substituting members of the genera Streptomyces, Candida. Xylaria. Arthrobacter. Cunninghamella and Claviceps. results similar to those described in Example I are obtained.

EXAMPLE IV

Polyporus sps fermentation

Using methods and materials similar to those described above for Example I, but using Polyporus sps ATCC 10089 as the microorganism, results similar to those from Example I were obtained. Using a 7-day-old culture of this organism (cultivated in double strength YM broth) to which 2% dimethyl anthranilate and 1% "Tween-80" were added, and allowing the reaction to continue for an additional 7 days, 19.7% conversion to methyl anthranilate and 9.8% conversion to methyl anthranilate were measured to N-formyl methyl anthranilate.

EXAMPLE V

Expanded Polyporus fermentation is seen

Using methods and materials similar to those described above for example I, but by extending the incubation period for Polyporus ses 10089 to 13 days before the addition of 2% dimethyl anthranilate, followed by a further 11 days of incubation, 34.8% of the starting material was converted to methyl anthranilate (representing 7 g of methyl anthranilate/liter of broth) and 1.2% is converted to N-formyl methyl anthranilate (or 2.8 g/l), the remainder being recovered as dimethyl anthranilate.

EXAMPLE VI

A typical natural concord grape flavor in a propylene glycol carrier, suitable for use in a non-carbonated or carbonated beverage at an application level of approx. 0.5-1.0$ was produced as follows:

The ingredients, except for the water and juice concentrate, were mixed together, the water and concentrate were mixed and the mixture was stirred until a clear solution was obtained. Carbonation under pressure produced the desired grape drink.

EXAMPLE VII

A system with typical natural flavor (concord grape type) in a carrier in the form of vegetable oil, suitable for use in chewing gum at an application level of approx. 0.5-1.0$ was produced as follows:

The flavoring may be combined with sugar and a gum arabic base and extruded or molded to provide chewing gum.

EXAMPLE VIII

A typical concord grape WONF flavoring in a grain alcohol carrier suitable for use in a non-carbonated or carbonated beverage at an application level of approx. 0.5-1.0$ was produced as follows:

The ingredients other than the alcohol, water and julce concentrate were mixed, the remaining ingredients were mixed in to form a "single strength" liquid, and the liquid may optionally be carbonated to form the desired beverage.

Claims (16)

1. Process for converting methyl N-methylanthranilate to methylanthranilate, characterized in that it . involves incubation in an aerobic nutrient broth of a culture of a fungal microorganism and methyl N-methylanthranilate at up to about 10 weight-#, relative to the total weight of the broth, the culture and the methyl N-methylanthranilate, for the production of the methylanthranilate. 2. Method according to claim 1, characterized in that the fungal microorganism comprises Streptomyces. Aspergillus. Candida, Xylarla. Arthrobacter. Cunninghamella. Clavicles. Trametes. Polystlctus or Polyporus. 3. Method according to claim 1, characterized in that the microorganism is a wood rot fungus or a fungus that is capable of breaking down cellulose or lignin. 4. Method according to claim 1, characterized in that the microorganism is the genus Trametes. Polystlctus or Polyporus. 5 . Method according to claim 1, characterized in that the microorganism is Trametes cingulata ATCC 26747 or Polyporus sps ATCC 10089. 6. Method according to claim 1, characterized in that it further includes separation of the methyl anthranilate from the nutrient broth and the culture. 7. Method according to claim 1, characterized in that the methyl anthranilate is prepared in a mixture with methyl N-formylan anthranilate. 8. Method According to claim 7, characterized in that it further includes separating the mixture from the nutrient broth and the culture. 9. Process according to claim 8, characterized in that it further includes conversion of methyl N-formyl anthranilate to methyl anthranilate by a base-catalyzed or thermal decarbonylation reaction. 10. Method according to claim 1, characterized in that the pH is in the range from approx. 3 to approx. 9, the temperature is in the range from approx. 18°C to approx. 33"C and the incubation period is from 1 to 14 days. 11. Method according to claim 10, characterized in that the pH is approx. 5 to approx. 8, the temperature is approx. 20°C to approx. 30°C and the incubation period is approx. 3 to 10 days. 12. Method according to claim 10, characterized in that the pH is approx. 6 to approx. 7, the temperature is approx. 22°C to approx. 26"C and the incubation period is about 4 to about 8 days. 13. Method according to claim 1, characterized in that the incubation is carried out as a continuous fermentation. 14 . Method according to claim 1, characterized in that the nutrient broth contains sources of organic nitrogen, carbohydrates, minerals and vitamins. 15 . Method according to claim 1, characterized in that the methyl N-methylanthranilate concentration is not higher than approx. 2.0%. 16. Method according to claim 1, characterized in that the methyl N-methylanthranilate concentration is not higher than approx. 0.5%.