ENZYMATIC PROCESS FOR PREPARING SPICE OIL Field of the invention The present invention relates to an enzymatic process for the preparation of spice oil. The main usage of the invention is in the recovery of volatile oils from spices such as ginger, clove and garlic using enzymatic mixture to aid optimized extraction. Background of the invention Reference is made to traditionally followed process for the extraction of spices where in spices are mechanically crushed and steam or hydro distilled to collect volatile spice oil. In dry processing, fresh spice such as ginger is sliced, dried at 60°C for 10 h and powdered in a blender. Dry powder is mixed with water at lOOg: 2 Uter and distilled for 5 h and oil is collected. The drawbacks are (a) Even when the fresh spice is used for processing, the distillation time is not reduced; (b) There is no enhancement in the yield of spice oil; (c) The extraction of oil by the traditional distillation is slow and inefficient because of the barrier offered by the tough cell walls and cell matrix of the plant material used. Reference may be made to Bartley, J.P. and Foley, P (1994). Supercritical fluid extraction of Australian grown ginger (Zingiber ojficinale). J. of the Sci. of Food and Agri. 66(3), 365-371, wherein ginger oil is extracted by supercritical fluid extraction method. The draw back of this method is that the process is complex, involves high technology and it is not suitable for all plant materials. Reference may also be made to Low cost oil-extraction gadgets: M. Somashekar, RRL, Trivandrum, wherein a novel dewatering step prior to distillation is incorporated to retain desirable fresh flavour and the envisaged draw back is that there is no quantitative enhancement of the oil extracted. Objects of the invention The main object of the present invention is to provide an enzymatic process for the preparation of spice oil, which obviates certain drawbacks as detailed above. Summary of the invention Accordingly the present invention provides an enzymatic process for the preparation of spice oil, which comprises: (a) preparing an enzyme complex having cellulase, pectinase, protease and xylanase using aqueous medium, (b) homogenizing spices selected from ginger, garlic and clove in the enzyme complex solution and incubating, (c) filtering the homogenized spice enzyme solution to obtain clear liquid solution, (d) distilling the liquid solution, and
(e) collecting spice oil. In one embodiment of the invention, the enzymes are taken in an amount of 0.35 units/ml to 500 units/ml and the ratio of enzyme to aqueous medium is 1 : 16. In another embodiment of the invention, the spices have a mesh size of 300 to 700 micron and the ratio of spices to enzyme complex solution is in the range of 1 : 1 to 2: 1. In another embodiment of the invention, the incubation is carried out for a period of 2-3 h and at a temperature ranging from 30°C to 50°C. In yet another embodiment of the invention, the distillation is carried out at a temperature in the range of 95-100° C and for a period ranging from 2 to 3 h. In a further embodiment of the invention, the yield of the spice oil is 0.86-1.9 ml for
100 g of dry ginger weight, 1.69-2.53 ml for 100 g of dry weight of garlic, 10-15 ml for 100 g dry weight of cloves. . In another embodiment of the invention, the spice oil contains up to 73% of zingiberene, curcumene and sesquiterpene alcohol in ginger oil, up to 76% of diallyl trisulphide, allyl methyl trisulphide and diallylyl disulphide in garlic oil, and up to 80% of eugenol in clove oil. In another embodiment of the invention the cellulase is taken in an amount of 0.35-4.6 unit/ml, pectinases 80-500 units/ml, proteases 30-150 units/ml and xylanase 17.5-125 units/ml. Detailed description of the invention The present invention provides an enzymatic process for the preparation of spice oil, which comprises preparing a enzyme complex having cellulase, pectinase, protease and xylanase in a quantity of 0.35 units/ml to 500 units/ml using aqueous medium in a ratio of 1: 16. Ginger, garlic and clove having mesh size of 300 to 700 micron are homogenized in the enzyme complex solution in a ratio of 1:1 to 2: 1 and incubating for a period of 2-3 h at a temperature ranging from 30°C to 50°C. The homogenized spice enzyme solution is filtered to obtain clear liquid solution, which is then distilled at a a temperature of 95-100° C for a period ranging from 2 to 3 h to obtain spice oil having an yield of 0.86-1.9 ml for 100 g of dry ginger weight which is 30% more than that of untreated ginger, 1 ,69-2,53 ml for 100 g of dry weight of garhc which is 50% more than that of untreated garlic, 10-15 ml for 100 g dry- weight of cloves which is 50% more than that of untreated cloves. The total concentration of major constituents in above obtained oil was up to 73% of zingiberene, curcumene and sesquiterpene alcohol in ginger oil, up to 76% of diallyl trisulphide, allyl methyl trisulphide and diallylyl disulphide in garlic oil, and up to 80% of eugenol in clove oil.
The cellulase can be used at a level of 0.35-4.6 unit/ml, pectinases 80-500 units/ml, proteases 30-150 units/ml and xylanase 17.5-125 units/ml. Fungal enzyme having above mentioned enzyme complexes was used for the extraction of spice oils. Fresh spice material such as ginger or garhc were cleaned and mixed with enzyme solution in 1:1 to 2:1 ratio and homogenized in a warring blender to 300-700 micron size and hydrolysed at 30°-50° C up to 3 h. Dry spice such as clove was powdered to 300-700 micron size, and suspended in enzyme solution and hydrolysed. Then the hydrolysate was passed through hydraulic press and the filtrate was collected. The filtrate was set-aside at 10-25° C for 2-3 h to sediment starch material and the supernatant was hydrodistilled to obtain volatile spice oil. The novelty of the present invention is that the process provides an approach to efficient extraction of spice oil from fresh or dry spices by the enzymatic weakening of the cell wall resulting in optimized extraction of oil. An enzyme preparation, which contains cell wall degrading enzymes such as cellulase, hemicellulases, pectinase, protease etc., obtained from fungal or bacterial cultures may be adopted efficiently for this purpose. The .following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. EXAMPLE 1 A comparison was made between the fresh ginger and the dry ginger powder on the efficiency of recovery of volatile oil. Conventional method of extraction was followed for the extraction of dry ginger powder wherein 100 g of material was suspended in 2 1 of water, distilled for 5 h and volatile oil was collected and measured. Cleaned fresh ginger (625 g equivalent to 100 g on dry weight basis) was mixed with water, (2:1) and homogenized in a warring blender to 300-700-mesh size. The homogenized material was extracted in a hydraulic press. Filtrate was kept for sedimentation in cold (10 ° C for 2-3 h). The supernatant was decanted and subjected to hydro distillation and volatile oil was collected and measured. The volume of oil obtained was 1.44 ml for fresh ginger compared to 1.3 ml with dry ginger. The results indicate that the recovery of volatile oil and method of handling of fresh material is efficient and easier compared to preparation and extraction of dry material. EXAMPLE 2 The fungus Aspergillus ustus (accession No. 1134) used for the preparation of the enzyme complex was obtained from the culture collection of the Department of Food Microbiology at C.F.T.R.I., Mysore. The fungus was maintained on potato dextrose agar slants (Hi-Media, Mumbai, India). The culture characteristics of Aspergillus ustus grown on potato dextrose agar are: colonies were light brown in colour, with heavy sporulation,
conidiophores were smooth and heads were irregular in form. Wheat bran used for enzyme production was procured from the local market. Other chemicals used in the media and the reagents were of the analytical grade and were obtained from standard Indian companies. The wheat bran used for growing Aspergillus ustus was mixed with 1% (w/w) of defatted ground nut meal and moistened to 60% with a medium containing MgSO4.7H2O, 0.05; KC1, 0.05; K2HPO4, 0.1; and FeSO4.7H2O, 0.0001, pH of the medium was 4.5. Cultivation was carried out in 500 ml capacity flasks containing 50 g wheat bran (of wet weight) having 60 % moisture content and sterilized for 45 min at 15 lbs. The spores from 7-day-old cultures were inoculated to these flasks, mixed well, and allowed to grow at ambient temperature (26 ° - 30 ° C) for 96h. The enzyme was extruded from the moldy bran by extraction with water (1:4) and various enzyme activities were estimated as follows: Cellulase: 0.5 ml of enzyme solution and 1 ml of citrate buffer (0.1 M, pH 4.8) were taken in 18 mm test tube containing a coiled filter paper strip (Whatman No. 1, 1x6 cm strip, 50 mg). Sample was incubated for 1 h at 50° C and 3 ml of dinitrosalicylic acid reagent was added and the tube placed in boiling water bath for 5 minutes and cooled. A blank was run parallel to this with boiled enzyme solution. Glucose released (mg) was estimated by measuring the colour of the sample at 540 nm in a spectrophotometer. Activity was calculated as micromoles of glucose released /ml of enzyme/minute. Xylanase: Reaction mixture containing larchwood xylan solution (Sigma chemical co. USA, 1% solution) 1 ml, acetate buffer (0.05 M pH 5.5) 0.5 ml, and diluted enzyme solution 0.5 ml was incubated for 30 minutes at 55 ° C. The reducing groups released was estimated by dinitrosalicylic acid reagent as mentioned above and activity was represented as micromoles of xylose released /ml enzyme/minute. Pectinase: The % reduction in viscosity of a 1% pectin solution under specified conditions was considered for this activity. To 20 ml of 1% purified pectin solution (prepared in 0.1 M of citrate buffer, pH 4), 2 ml of enzyme solution was added and incubated at 40° C for 30 minutes. Boiled enzyme solution was used as control. Flow time of the reaction mixture in minutes and that of control was determined by using Ostwald viscometer. Percentage reduction in viscosity was calculated by considering the difference in flow time of control and that of sample.
Protease: To 1 ml of 1% casein solution of pH 7, 1 ml of diluted enzyme was added and incubated at 37° C for 20 minutes. To this 3 ml 5% trichloroacetic acid solution was added and kept at room temperature for 30 minutes. The sample was filtered through Whatman No. 1 filter paper circle. To 1 ml of filtrate 2 ml of 0.2 N NaOH and 0.6 ml of Folin reagent was
added and colour that developed was measured after 20 minutes at 620 nm. Boiled enzyme solution treated similarly as above was used as reagent blank. Tyrosine was used as standard. Activity was calculated as mg of product released by 1 ml of enzyme solution. Various enzyme activities produced by the fungus are shown in Table 1. Table 1: Enzyme activities estimated in A. ustus enzyme complex
A batch of 625 g of cleaned and fresh ginger (100 g dry weight) was suspended in 312 ml enzyme solution and homogenized to a mesh size of 300-700 microns. The enzyme suspension contained 142 ml of above enzyme solution and 170 ml of water. Only water was used for control. Homogenized ginger was then incubated at 40° C for 2.5 h and extracted in a hydraulic press. The filtrate was handled and hydrodistilled as under example 1. The yield of oil was 1.9 ml for enzyme treated sample and 1.4 ml for control. EXAMPLE 3 Commercial enzyme complex (Ex. Trizyme 50) was also used for hydrolysis of ginger to release volatile oil. The enzyme contained cellulase 4.6 units/ml, xylanase 125 units/ml, protease 150 units/ml and pectinase 500 units/ml. In this example, 625 g portions of fresh ginger (100 g dry weight) was hydrolysed by various volumes of enzyme solution by considering 500 to 3500 xylanase units activity/100 g dry weight of the material, keeping the substrate to enzyme solution ratio at 2:1. Enzyme solution was prepared by mixing 4 - 28 ml of enzyme in 308-284 ml of water, in which ginger was homogenized and incubated at 40°C for 2.5 h. The extract was obtained and oil recovered by hydrodistillation as mentioned under example 1. The recovery of oil increased with increase in enzyme concentration up to 2500 xylanase units/1 OOg ginger (dry wt.) and thereafter there was no further increase in yield (Table 2). Table 2: Effect of enzyme concentration on the recovery of oil Xylanase units/100 g dry wt. ml of oil/ 100 g dry weight Control 1.40 500 1.50 1000 1.65 1500 1.75 2000 1.80 2500 1.90 3000 1.80
EXAMPLE 4 A batch of 625 g of fresh and cleaned ginger (100 g on dry weight basis) was handled as described under example 3 with enzyme solution containing 2500 units xylanase under different incubation temperatures ranging from 30°C to 50°C for 2.5 h. The oil was recovered as detailed under example 1. Recovery of oil increased with increase in temperature up to 40°C, and decreased thereafter (Table 3). This could be due to the evaporation of the volatile oil with increase in incubation temperature and may also be due to partial denaturation of enzyme at higher temperatures, thus becoming unavailable for cell wall degradation. The oil being volatile, processes, which can be worked out at low temperatures, would be desirable. Table 3: Effect of temperature on the recovery of oil
Temperature (° C) ml of oil/ 100 g dry weight of ginger
30 1.65 40 1.90 50 1.50
EXAMPLE 5 A 5 kg batch of fresh and cleaned ginger (800 g dry weight) was mixed with enzyme solution to contain 2500 units of xylanase/100 g dry weight of ginger. Enzyme solution was prepared by mixing 160 ml of commercial enzyme mentioned under example 3 with 2.34 1 of water. The mixture thereafter was homogenized and incubated at for 2.5 h at 40°C. The extract was obtained and distilled as explained under example 1. Control samples (without enzyme treatment) yielded 11.5 ml of volatile oil compared to 15 ml obtained in enzyme treated ginger. Gas chromatography was carried out using the following conditions: SE 30 column (10%> dia solid L, 60-80 mesh); injection temperature 200° C; detector temperature 235° C; nitrogen flow rate, 40 ml/min. Gas liquid chromatography profile of the major components of the oil given in Table 4 show that the major peak area belonging to zingiberene, curcumene and sesquiterpene alcohols, amounted to about 65% in control ginger (dry) extraction. The recovery of these increased from 8.72 ml in control (out of 11.5 ml oil) to 11.0 ml in enzyme treatment (out of 15 ml). Table 4: Concentration of volatile oil of ginger estimated by gas chromatography
1, 2 and 3=Zingiberene, Curcumene and Sesquiterpene alcohols, 4=others Conventional process a = % of peak area in GC b = ml of component for 100 g ginger (dry weight basis) EXAMPLE 6 A batch of 260 g of fresh garlic (100 g dry weight) was also enzymatically hydrolysed and oil recovered and analysed as per the method given under example 5. The yield of the oil obtained was more in enzyme treated sample (2.53 ml for 100 g dry weight) compared to control (1.69 ml). Major constituents of oil hence obtained contained diallyl trisulphide, allyl methyl trisulphide and diallyl disulphide up to 76%>. EXAMPLE 7 Steps as mentioned under example 5 was repeated to obtain oil from cloves. The spice was powdered to 300-700 micron mesh size, treated with enzyme complex as under example 5, to obtain oil. Yield of oil obtained was 10 ml (for 100 g dry weight) for control and 15 ml for enzyme treated sample. Major constituent obtained was eugenol, which amounted to up to
80%. The main advantages of the present invention are:
2. There is enhancement in the yield of volatile spice oil by about 30-50% over the traditional method.
3. The extraction is more efficient compared to traditional process because the enzyme complex hydrolyses the barrier offered by the cell walls of fresh or dry spice materials to release more oil.