Title of Invention	"AN IMPROVED PROCESS FOR THE PREPARATION OF VANILLIN"
Abstract	An improved process for the preparation of vanillin which com¬prises] cultvating Spirulina sp. in a conventional nutrient medium having been supplemented with a compound convertable to vanillin selected form isoeugenol, eugenol, ferulic acid and coniferyl aldehyde for at least 18 hrs. in a known manner then recovering venillin by conventional solvent extraction methods from the cells and medium*

Full Text

This invention relates to an improved process for the preparation of vanillin. Particularly this invention relates to the biotransformation of isoeugenol, eugenol- a clove principles, and ferulic acid and coniferyl aldehyde to vanillin using axenic cultures of Spirulina spp. In this process, vanillin is produced from the micro alga Spirulina cultures when fed with precursors-isoeugenol, eugenol, ferulic acid and coniferyl aldehyde. In the past decade, there has been an increasing and steady shift in consumer preference toward the use of food ingredients derived from natural sources in view of their safety over the synthetic ones (Dziezak, J.D. (1986) Food Technol. 40, 108; Tyrell, M.H. (1990) Food Technol., 44, 68; Stofberg, J.(1986) In: Biogeneration of aromas (ed.) Parliament T.H. and Croteau, R. ACS Symposium series No. 317, 2; Sahai, O.M. (1994) Plant tissue culture. In: Bioprocess production of flavors, fragrances, and color ingredients (ed.) Gabelman, A. 239-275). Among the natural flavors, vanillin, a prime industrial molecule, is used in food and flavor industry. It is a key component of frozen desserts, yogurt, baked goods, candies, liquers, sweets, pudding, cakes, and beverages (Roger and Diane (1994) Trends in Biol. Sci. 19, 521; H&R Contact, No.59, 15-19; Webster, T.M. (1995) Cereal Foods World, 40, 198-200). The very high price of natural vanilla flavor isolated from vanilla pod, and the consumer demand for naturally produced foods have stimulated the search for alternative means of vanillin production (Gatfield, IL,(1995) Perfumery & Flavourist, 20, 5-14). Natural vanilla is a complex mixture of flavor components extracted from the cured beans Vanilla planifolia. The extraction process involves an initial curing process, during which vanilla-precursor glycosides in the bean breakdown to form natural vanillin (4-hydroxy-3-methoxy benzaldehyde) and its related flavor components such as vanillic acid, vanillyl alcohol, p-hydroxybenzoic acid and p-hydroxybenzadehyde, etc., followed by one or more alcohol extractions to remove the relatively hydrophobic flavor components from the bean. Each of these steps may be relatively time-consuming and costly (Riely, K.A and Kleyn, D.H (1989) Food Technol. 43, 64- 67; Webster, T.M. (1995) Cereal Foods World 40, 198-200). The cost of natural vanilla is US $ 4, 000/Kg flavor solids and US $ 70- 90/Kg beans, whereas synthetic vanillin at US $ 15/Kg (Roger, C. and Diane, E.G.(1994), Trends Biol. Sci. 19, 521; Westcott, R.J. et al., (1994) Phytochem. 35,135-138). A very promising way to obtain natural flavors is the use of microorganism since, up to now, the European Economic Community (EEC) legislation incorporates under the term "natural products" those produced from biological sources, by living cells, or their components, including enzymes (Hardinge, J. (1990) Chemistry and Industry (London) Nov. 694- 698, Sahai.O.M (1994) In: Bioprocess production offlavor, fragrance, and color ingredients (ed.) Gabelman, A. 30-39). Current legislation in the Code of Federal Regulations (CFR) governing natural flavors or natural flavorings in the United States defines naturals in 21 CFR 101.22 (a) (3) as isolates from both plant and animal sources, fermentation products and commonly called "Process flavor products" which are flavors derived from thermal reaction and enzymatic treatment (Tyrell, M. (1995) Perfumery & flavourist 40,13-19). The biotransformation of isoeugenol and eugenol to vanillin obtained in an edible alga Spirulina spp. is useful for the production of food grade vanillin. Vanillin has been obtained by biotransformation using microbial agents and also found in plant cell cultures. The following are the patents currently available pertaining to this. The production of vanillin described by Robenhorst and Hopp (1991) involves the incubation of Serratia spp., Enterobacter spp., and Klebsialla spp. cells with 2% eugenol or isoeugenol after 20 hrs of inoculation. The reaction was carried out till 300 hrs. The production of vanillin is more in isoeugenol than in eugenol incubated cultures (EP 405197 A1). In another patent (US 5068184) Knuth and Sahai explains the production of vanillin from callus/suspension cultures of Vanilla planifolia and optimization of media composition, analysis of flavor composition, and in situ recovery of vanilla flavor using suitable adsorbants has been described. The production of vanillin through the bioconversion of vanillin precursor, ferulic acid with a tissue culture of undifferentiated cells derived from Vanilla plant/ and enzymes obtained therefrom in the presence of a water soluble sulphydryl compound and optimally, also in the presence of assimilate carbon source has been described (Labuda I.M. et a/., US 5279950). The process described by Mane and Zucca (*1993) involves the production and effective release of vanilla flavor from pods using hydrolytic enzymes has also been patented (FR 2 691 880 A1). A process described by Westcott, R.J. et a/., involves producing natural vanillin from ferulic acid as a precursor has been developed on the use of Vanilla plant aerial roots as the biocatalyst (Phytochemistry (1994) 35, 135-138). 1. The chemical process is not an ideal alternative to biological process due to the demand for the latter for natural flavor production. The synthetic vanillin/vanilla flavor lacks all the essential attributes of natural (Sahai, O.M. (1994) Plant tissue culture In: Bioprocess production of flavor, fragrance and colour ingredients (ed.) GabelmanA., 239-275). 2. Conventional process involves specific agroclimatic requirements and curing and drying of pods is tedious (Knuth et al., (1991) Flavor composition and method, US patent No.5068184). 3. The biotechnological process involves growth of plant cells or microbial cells as biological agents in vanillin production and these culture systems are heterotrophic needing more costly nutrient inputs for their growth and scale up also is difficult. The main object of the present invention is to provide an improved process for the preparation of vanillin. Another object of the present invention is to provide a process for the preparation of vanillin using an autotrophic organism -Spirulina species. Accordingly the present invention provides an improved process for the preparation of vanillin which comprises: cultvating Spirulina spp. in a conventional nutreint medium having been supplemented with a compound convertable to vanilliniStteh-as isoeugenol, eugenol, ferulic acid and coniferyl aldehyde for at least 18 hrs. in a known manner then recovering vanillin by conventional solvent extraction methods from the cells and medium. In an embodiment of the present invention a compound convertable to vanillin used may be such as eugenol, isoeugenol, ferulic acid and conferyl aldehyde. In an embodiment of the present invention the solvent used to extract vanillin used may be such as ethyl acetate, diethyl ether and hexane. The steps involved in the process is as follows: 1. Developing of axenic cultures of Spirulina spp. by plating techniques on Zarrouk's medium (1966). Composition of the Zarrouk's medium: (Table Removed) 2. Developing of suspension cultures of Spirulina spp. from axenic cultures in zarrouk's liquid medium. 3. Feeding of isoeugenol and eugenol to Spirulina suspension cultures. 4. The separation of Spirulina cells from liquid medium by filtration on whatman filter paper. 5. Extraction of vanillin using suitable organic solvent. 6. Concentration of organic solvent fraction containing vanillin under reduced pressure. 7. Preparation of ethanolic extract of vanillin. 8. Quantification of vanillin flavor. The examples given below which are provided by way of illustration only and should not be construed to limit the scope of the invention. EXAMPLE 1: A 5 day old suspension cultures of Spirulina (40 ml) were fed with filter sterilized isoeugenol at 1.25 mM and cultures were incubated on rotary shaker at 90 rpm under the light intensity of 2000 Lux at 25 ±2°C. The cultures were analysed at 24 hrs interval and experiment has been carried out till 120 hours. The cells were separated from the medium by filtration using whatman No.4. The cells and medium were extracted for isoeugenol and vanillin using ethyl acetate as organic solvent. The solvent fraction was concentrated in vacuuo and fraction was dissolved in 80% (v/v) ethanol, and used for analysis. The analysis and quantification of vanillin was done by TLC, Spectrophotometry, HPLC and FTIR. The Rf values recorded for isoeugenol and vanillin was 0.59 and 0.17 respectively, and quantification was done using calibration curve at 280 nm for vanillin and 260 nm for isoeugenol. HPLC analysis was carried out by eluting fractions of isoeugenol and vanillin from TLC plates, using methanol, acetic acid, and water as solvent. The FTIR was carried out using concentrated fraction of ethyl acetate. The FTIR spectrum showed the strong peaks at 1700 cm'1 (-CHO), 3500 cm"1 (-OH), 1158 cm"1 (-C-O-C) and 1640 cm"1 (-C=C) due to characteristic groups of vanillin and isoeugenol (Fig.4). The maximum production of vanillin was obtained on 48 hrswhich was 1243.57 jig/culture with 20-25% conversion or 31.093 mg/L of medium (Fig.2). The biotransformation of isoeugenol to vanillin was extracellular. EXAMPLE 2: A 5-6 day old suspension cultures of Spirulina (40 ml) were fed with filter sterilised isoeugenol at 2.5 mM and cultures were incubated on rotary shaker at 90 rpm under the light intensity of 2000 Lux at 25 ± 2 °C. The cultures were analysed at 12 hrs interval and experiment has been carried out till 72 hours. The cells were separated from the medium by filtration using whatman No.4. The cells and medium were extracted for isoeugenol and vanillin using ethyl acetate as organic solvent. The solvent fraction was concentrated in vacuuo and fraction was dissolved in 80% (v/v) ethanol, and used for analysis. The analysis and quantification of vanillin was done by TLC, Spectrophotometry, HPLC and FTIR. The Rf values recorded for isoeugenol and vanillin was 0.59 and 0.17, respectively, quantification was done using calibration curve at 280 nm for vanillin and 260 nm for isoeugenol. HPLC analysis was carried out by eluting fractions of isoeugenol and vanillin from TLC plates, using methanol, acetic acid, and water as solvent. The FTIR was carried out using concentrated fraction of ethyl acetate. The FTIR spectrum showed the strong peaks at 1700 cm"1 (-CHO), 3500 cm"1 (-OH), 1158 cm"1 (-C-O-C) and 1640 cm"1 (-C=C) due to characteristic groups of vanillin and isoeugenol. The maximum production of vanillin was obtained on 36 hrs which was 1274.95 ^g/culture with 10-12% conversion or 31.875 mg/L of medium. (Fig.1). The biotransformation of isoeugenol to vanillin was extracellular. shaker at 90 rpm under the light intensity of 2000 Lux at 25 ± 2 °C. The cultures were analysed at 12 hrs interval and experiment has been carried out till 72 hours. The cells were separated from the medium by filtration using whatman No.4. The cells and medium were extracted for isoeugenol and vanillin using ethyl acetate as organic solvent. The solvent fraction was concentrated in vacuuo and fraction was dissolved in 80% (v/v) ethanol, and used for analysis. The analysis and quantification of vanillin was done by TLC, Spectrophotometry, HPLC and FTIR. The Rf values recorded for isoeugenol and vanillin was 0.59 and 0.17 respectively, quantification was done using calibration curve at 280 nm for vanillin and 260 nm for isoeugenol. HPLC analysis was carried out by eluting fractions of isoeugenol and vanillin from TLC plates, using methanol, acetic acid, and water as solvent. The FTIR was carried out using concentrated fraction of ethyl acetate. The FTIR spectrum showed the strong peaks at 1700 cm"1 (-CHO), 3500 cm"1 (-OH), 1158 cm"1 (-C-O-C) and 1640 cm"1 (-C=C) due to characteristic groups of vanillin and isoeugenol. The maximum production of vanillin was obtained on 36 hrs which was 1778.92 jag/culture. The biotransformation of isoeugenol to vanillin was extracellular. EXAMPLE 4: A 5-6 day old suspension cultures of Spirulina (40 ml) were fed with filter sterilised eugenol at 1.25 mM and cultures were incubated on rotary shaker at 90 rpm under the light intensity of 2000 Lux at 25 ±2°C. The cultures were analysed at 12 hrs interval and experiment has been carried out till 72 hours. The cells were separated from the medium by filtration using whatman No.4. The cells and medium were extracted for eugenol and vanillin using ethyl acetate as organic solvent. The solvent fraction was concentrated in vacuuo and fraction was dissolved in 80% (v/v) ethanol, and used for analysis. The analysis and quantification of vanillin was done by TLC, Spectrophotometry, HPLC and FTIR. The Rf values recorded for eugenol and vanillin was 0.82 and 0.58 respectively, quantification was done using calibration curve at 280 nm for vanillin and 260 nm for eugenol. HPLC analysis was carried out by eluting fractions of eugenol and vanillin from TLC plates, using methanol, acetic acid, and water as solvent. The FTIR was carried out using concentrated fraction of ethyl acetate. The FTIR spectrum showed the strong peaks at 1700 cm'1 (-CHO), 3500 cm"1 (-OH), 1158 cm'1 (-C-O-C) and 1640 cm"1 (-C=C) due to characteristic groups of vanillin and eugenol. The maximum production of vanillin was obtained on 36 hrs which was 153.58 ng/culture. The biotransformation of eugenol to vanillin was extracellular. The principle involved in the method is as follows: The vanillin is produced from Spirulina spp. cultures when fed with isoeugenol and eugenol, a clove principles, (which are not naturally present in the Spirulina system). Hence, this biotransformation is a novel method wherein foreign compounds, isoeugenol, and eugenol has been converted into value added product, vanillin. The formation of vanillin from isoeugenol involves two steps. (Fig.3). In the first step, formation of ferulic acid from isoeugenol reaction wherein methyl group of aromatic side chain is converted to carboxylic acid group. In second step, formation of vanillin from ferulic acid might be via feruloy CoA through a reaction to similar to p - oxidation of fatty acids (Funk and Brodelius 1990). Eugenol can bioconverted to either coniferyl aldehyde or ferulic acid; both of which are useful intermediates in the bioconversion to vanillin. EXAMPLE 5: A 5-6 day old suspension cultures of Spirulina (40 ml) were fed with filter sterilized ferulic acid at 1.25 mM concentration and cultures were incubated on rotary shaker at 90 rpm under the light intensity of 2000 Lux at 25±2°C. The cultures were analysed periodically at 1 day interval and experiment was carried out for 5 days. The cells were separated from the medium by filtration using Whatman No. 4. The medium was extracted with ethyl acetate after its pH adjusted to 2-3 using concentrated HCI. The cells were homogenized in mortar and pestle with ethyl acetate. The solvent fraction was concentrated under reduced pressure and the concentrated residue was dissolved in known volume of 80% (v/v) ethanol and is used for analysis. The analysis was carried out by TLC, HPLC and GC. The Rf values recorded for vanillin (0.74), vanillic acid (0.69), ferulic acid (0.66) respectively in Benzene, dioxane and acetic acid solvent system. HPLC was carried out using Shimadzu LC-6A liquid chromatograph with RP-C18 column (150 mm x 4.6 mm i.d, Shim-pack) connected to C18 guard column using methanol, acetic acid and water as solvent system. The GC analysis was carried out using Shimadzu GC-15A SE-30 column with temperature programme using N2 as carrier gas. The analysis of medium of ferulic acid fed Spirulina cultures showed the formation of vanilla flavor components such as vanillin, vanillic acid, vanillyl alcohol and protocatechuic acid. The cells did not show formation of these compounds. The maximum production of vanillin, vanillic acid recorded on 5th day at 964.10, 3,387.53 fig/culture, whereas vanillyl alcohol and protocatechuic acid showed maximum on 1st day at 90.29, 239.76 jig/culture respectively (Fig. 5). The biotransformation of ferulic acid to vanilla flavor components was found to be extracellular. EXAMPLE 6: A 5-6 day old suspension cultures of Spirulina (40 ml) were fed with filter sterilized coniferyl aldehyde at 1.0 mM concentration and cultures were incubated on rotary shaker at 90 rpm under the light intensity of 1500 Lux at 25±2°C. The cultures were analysed periodically at 1 day interval and experiment has been carried out 4 days. The samples were analysed periodically by separating cells from the medium by filtration using Whatman No.4. The medium was extracted with ethyl acetate after its pH adjusted to acidc side (2-3) with concentrated HCI. The solvent fraction was concentrated under reduced pressure and the concentrated residue was dissolved in known volume of 80% (v/v) ethanol and is used for analysis. The analysis was carried out using HPLC and GC. HPLC analysis was carried out using Shimadzu LC-6A liquid chromatograph with RP-C18 column connected to C18 guard column using methanol, acetic acid and water as solvent system. The GC analysis was carried out with Shimadzu GC 15A using SE-30 column with temperature programme using Na as carrier gas. The analysis of medium showed conversion of coniferyl aldehyde to vanilla flavor components such as vanillin, vanillic acid, protocatechuic acid and ferulic acid. The cells did not show the formation of either precursor or products. Hence, this reaction was found to be extracellular. The maximum accumulation of vanillin was on 2 day at 685.89 ng/culture whereas vanillic acid, protocatechuic acid and ferulic acid recorded maximum on 4th day at 159.89, 129.56, 1508.25 ng/culture,respectively. The formation of ferulic acid was more than other components because ferulic acid is nearer to coniferyl aldehyde in the biosynthetic pathway (Fig.6). 1. The biological agent used for biotransformation is Spirulina which is an edible alga. 2. The feeding of precursor is simple. The isoeugenol and eugenol are easy to obtain from clove oil hence is also from natural source. Similarly ferulic acid and coniferyl aldehyde can be obtained from natural sources such as com hulls and bark of fir tress, respectively. 3. The reaction is extracellular hence downstream processing is easier. 4. The process is ecofreindly in large scale production since it involves a photosynthetic organism as biocatalyst. The novelty of the process involves the use of autotrophic system especially of an approved food grade organism - Spirulina spp. for the production of vanillin by biotransformation. CLAIM 1. An improved process for the preparation of vanillin which comprises: cultvating Spirulinu spjp. in a conventional nutrient medium having been supplemented with a compound convertable to vanillin setected form isoeugenol, eugenol, ferulic acid and coniferyl aldehyde for at least 18 hrs. in a known manner then recovering vanillin by conventional solvent extraction methods from the cells and medium. 2. An improved process as claimed in claim 1 wherein medium containing compound convertable to vanillin in Spirulina suspension cultures is ranging frohi 0.5 mM to 4.0 mM concentration. 3. An improved process as claimed in claim 1 & 2 wherein separation of Spirulina cells from the medium is carried out by filtration usinf whatman No. 1 to 4. 4. An improved process as claimed in claims 1 to 3 wherein the organic solvent used for extraction of vanillin from medium and cells is selected from ethyl acetate, diethyl ether, and hexane. 5. An improved process for the preparation of vanillin as herein described with reference to examples.

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