FIELD OF THE INVENTION

The present invention relates to a method of extraction of carotenoids from a natural source. More particularly, the present invention relates to a method of extraction of lycopene from tomatoes.

BACKGROUND OF THE INVENTION

Lycopene is a carotenoid responsible for the red colour of a large number of fruit and vegetables. This compound has some remarkable properties as a colorant. It is authorized for use as a food colorant. Lycopene is found in tomatoes, guava, apricots, watermelon, papaya, chilli, marigold, pink grapefruit and many others. Tomato and tomato products are considered one of the best sources of lycopene.

Studies have shown that lycopene has a strong antioxidant potential that makes it an excellent deactivator of the oxygen singlet and free radicals. This antioxidant potential confers it an anti-carcinogenic activity and a capacity to help prevent cardiovascular diseases. Lycopene has also been found to play an effective role in treating heart diseases, infertility in men and prevention of smoking- related diseases. Lycopene, because of its special properties, is also used in the nutraceutical and cosmetic industry. Therefore, there is a huge demand for lycopene in the industry of natural extracts.

In the last few years, supercritical fluid extraction of lycopene (usually supercritical C02 in all applications) has become very important in the alimentary and pharmaceutical industry. The costs of the industrial process are quite high, because of the severe operation conditions (temperature and pressure) and the compulsory use of a co- solvent such as acetone, methanol, ethanol, vegetable oils (soybean oil, sunflower oil and hazelnut oil). Another limitation of the supercritical extraction processes is the probable isomerisation of lycopene (from the trans to the cis form).

Extraction of lycopene with organic solvents is known. This technology allows the treatment of by-products as such, by conveniently combining well-established separation or purification processes (evaporation under vacuum, crystallisation, chromatographic or membrane separation) in order to obtain lycopene with the desired purity. A critical aspect of the extraction with a solvent is the selection of the solvent that must belong to the classes of compounds that are allowed in the food and pharmaceutical industry and give high enough extraction yield, in order to reduce their concentration and make them easily removable form the final lycopene product.

However, pure solvents give low extraction rates. International Application WO03/079816 discloses a process for the extraction from tomatoes as such by means of a solution of ethyl acetate saturated with water. Lycopene content of the extract is between 5% and 20%.

In our earlier Indian Patent application 2608/CHENP/2008, we have disclosed a method of extraction of mixed carotenoids rich in lycopene from a natural source using an organic solvent without a pre-treatment step.

Extraction of lycopene using alkali treatment are known in the art. Alkalis are used since they increase the stability of lycopene and also neutralize the acid in tomato tissues.
One of the main reasons for the difficulty in efficient lycopene extraction using alkalis without enzyme pre-treatment is the cellular localization of lycopene which is deeply embedded within the chromoplast of the plant structures where the pigment accumulates (W.M. Harris and A.R. Spurr, "Chromoplasts of tomato fruits II. The red tomato", American Journal of Botany, Vol. 56, pp. 380-389, 1969).

Other problems with the existing technologies are that they do not provide complete extraction of lycopene, do not provide stable lycopene, the yields are low and the methods are time intensive.

Thus the need of the lycopene industry is to develop a convenient, effective and affordable method that substantially increases the lycopene extraction, provides lycopene having suitable characteristics and is also feasible at industrial scale.

SUMMARY

The invention relates to a method of extraction of lycopene from a natural source which comprises treating the natural source with at least one lipolytic enzyme and at least one cellulolytic enzyme; subjecting the enzyme treated natural source to an alkali treatment; separating the alkali; and, extracting lycopene from the alkali treated natural source with an organic solvent.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs, la and lb demonstrate the absorption spectra of lycopene extracted by using different treatment processes: (1) Sigma standard. (2) Na2C03 (3) KOH (4) NaHCOS (5) cellulase (6) lipase+ CaCOS +EDTA (7) cellulase, lipase+ CaC03 +EDTA (8) cellulase, lipase+ CaC03 +EDTA (dried tomato) (9) Control 2 process (10) CaC03.

Figs. 2a and 2b give HPLC chromatograms of lycopene extracted by using different treatment processes (1) Sigma standard. (2) Na2C03 (3) CaC03 (4) KOH (5) lipase+ CaC03 +EDTA (6) cellulase (7) cellulase, lipase+ CaC03 +EDTA (8) cellulase, lipase+ CaC03 +EDTA (sundried tomato) (9) NaHCO3(10) Control 2 process.

Fig. 3 gives the TLC profile of lycopene extracted using enzymatic pretreatment Lanes (1) Lycopene Sigma standard, (2) P -carotene sigma standard (3) Lycopene from lipase treatment (4) Lycopene from cellulase treatment.

DETAILED DESCRIPTION

The present invention is directed to a method of exfraction of lycopene from a natural source which comprises treating the natural source with at least one lipolytic enzyme and at least one cellulolytic enzyme, subjecting the enzyme treated natural source to an alkali treatment, separating the alkali, and, extracting lycopene from the alkali treated natural source with an organic solvent.

According to an embodiment of the invention, the natural source includes any material which is rich in carotenoids especially lycopene such as vegetables and fruits. In a preferred embodiment the natural source is tomato. The tomatoes used in the present invention may be obtained from a variety of sources and cultivars. Preferably the natural source is selected from ripe tomatoes, over ripe tomatoes, damaged tomatoes, rejected tomatoes, unripe tomatoes, tomato pomace, dried tomatoes or tomato by-products. Preferably, ripe tomatoes are used as natural source. However, any suitable source rich in lycopene such as guava, apricots, watermelon, papaya, chilli, marigold, pink grapefruit, red carrots, red capsicum etc. can be used.

According to yet another embodiment of the invention, the method fiirther comprises prior to treating the natural source with the lipolytic and cellulolytic enzymes, at least one of the steps of homogenizing, grinding or crushing the natural source, separating serum from pulp by mechanical or chemical means, concentrating the pulp to reduce the moisture content to 30-40% and/or drying to remove more than 90% moisture.

According to an embodiment of the invention, the natural source is sorted to separate foreign matter. The sorted natural source may be cleaned by washing with water.

According to another embodiment of the invention, the cleaned natural source is sliced and dried to remove more than 90% moisture. Preferably, the natural source is dried to remove more than 95% moisture. Preferably, the natural source is sun-dried.

According to a fiirther embodiment of the invention, the sorted and cleaned natural source is subjected to crushing for separating serum from pulp and skin. For crushing, any suitable means may be used such as blender, mixer, knife mill or homogenizer. The natural source is crushed to particular form where in about 60 to 90 % of serum is separated, preferably 80-90% of serum is separated. The separated pulp and skin may be used immediately or stored, if stored pulp and skin should be kept in a dark container and stored at a low temperature.

According .to yet another embodiment of the invention, the serum is.separated from skin and pulp by mechanical or chemical means such as decantation, centrifligation, filtration, distillation, or rotary vacuum evaporation. In an embodiment, separation may be carried out by centrifugation, preferably at 9000-10000 rpm speed for 10 minutes -15 minutes at room temperature. The serum thus separated may be discarded or used for other applications such as food products or a raw material for food products. Alternatively, the serum is further processed by subjecting it to a separation step using a suitable means to separate the residue and almost clear liquid. The said residue along with the pulp and skin obtained after crushing is used for extraction. The separation of residue from the liquid is carried out by subjecting the serum to any suitable method of separation such as by filtration, centrifugation and decantation. It was found that combining of the residue with the pulp and skin and extraction of such combined mixture improves the yield of lycopene and thus improves the extraction efficiency. Further, in preferred embodiments the separated liquid is subjected to treatment like reverse osmosis to get almost purified water which may be recycled back and used in the method of the present invention.
Alternately, the purified water may be used for any potable purposes.

According to still another embodiment of the invention, the pulp is separated from the serum by filtration. This pulp is then subjected to concentration in a distillation imit to reduce the moisture content to 30-40%.

According to an embodiment of the invention, the dried natural source or the wet natural source i.e. the pulp of the natural source separated from the serum is subjected to enzymatic treatment with at least one lipolytic enzyme and at least one cellulolytic enzyme. The dried or wet natural source may prior to or during treatment with lipolytic enzyme be contacted with at least one cellulolytic enzyme.

According to an embodiment of the invention, the lipolytic enzyme is selected from the group comprising of lipases, phosphatases and hydrolases. Preferably, the lipolytic enzyme is lipase having an activity between 1200U/ml/kg to 1700 U/ml/kg. More preferably, the lipase has an activity of 1500 U/ml/kg. A variant derived from one or more of the above enzymes by substituting, deleting or inserting one or more amino acids can also be used.

According to an embodiment of the invention, the cellulolytic enzyme is selected from the group comprising of cellulase, pectinase and hemicellulase. Preferably, the cellulolytic enzyme is cellulase having a cellulolytic activity between 800 U/ml/kg to 1200 U/ml/kg. More preferably, the cellulase has an activity of 1000 U/ml/kg. A variant derived from one of the above enzymes by substituting, deleting or inserting one or more amino acids can also be used.

The extraction efficiency may be increased by use of any of the other hydrolytic enzymes individually or in combination which will be known to a person skilled in the art.

According to still another embodiment of the invention, the enzymatic treatment is followed by treatment with at least one alkali selected from calcium carbonate, magnesium carbonate, potassium hydroxide, sodium hydroxide, sodium bicarbonate and sodium carbonate.

According to an embodiment of the invention, the alkali treatment is followed by separation of the alkali which is carried out by washing with water or treatment with a chelating agent selected from EDTA, citric acid, tetra potassium pyrophosphate, sodium pyrophosphate and trisodium phosphate.

According to a further embodiment of the invention, after separating the alkali, a suitable solvent is used for extracting the lycopene from the natural source.

According to an embodiment, the solvent is selected from hexane, ethyl acetate, acetone, dichloromethane, ethanol ethyl acetate, denatured ethanol, methanol 2-propanol, heptane, tertiary butanol and dichlorodifluoromethane. Preferably, the solvent is hexane. Any other solvent suitable for extraction, known to a person skilled in the art may also be used.

Preferably, the method of extraction using a solvent as disclosed in our earlier patent application 2608/ CHE/2008 is used. However, any suitable method for solvent extraction may be used.

Thus, it was possible to obtain a much higher yield of lycopene using the method of the present invention described above and illustrated in greater detail by means of the following examples. However, it should be understood that the invention is never restricted thereto.

EXAMPLES

Example 1

PROCESSING OF NATURAL SOURCE

20 kgs of tomatoes were sorted and weighed. The tomatoes were then cleaned by washing with water. After washing, the the surface moisture was removed.
Example lA

Wet Natural Source

A first batch of 10 kgs of tomatoes was processed as follows:
Cleaned tomatoes were subjected to crushing using pulper. The crushed tomatoes were filtered to separate the serum from the pulp. One part of this pulp was used for extraction (CONTROL 1) and for different alkali treatments.

The other part of the pulp was distilled to reduce moisture content to 30-40% (CONTROL 2). This was used for enzymatic treatments.

Example IB

Dried Natural Source

10 kgs of cleaned tomatoes were sliced and sun-dried to remove around 96% moisture. For enzymatic treatment, the sun-dried tomatoes in the form of tomato flakes were ground to coarse particles using a grinder.

40 g of sun-dried tomato flakes corresponds to 1 kg of fresh tomato.

COMPARITIVE EXAMPLES OF EXTRACTION OF LYCOPENE USING VARIOUS METHODS

The following examples give the various methods and the yield of lycopene extracted using various methods such as direct extraction with solvent, extraction using alkali treatment, extraction using enzymatic pre-treatment and extraction using a combination of enzymatic and alkali treatment.

Example 2

Extraction of lycopene using Solvent
Extraction of lycopene using solvent was carried out using the method as illustrated in the Indian patent application 2608/CHE/2008.
Wet and dried tomatoes from Examples lA and IB were exfracted using solvent hexane in ULTRA PRIDE table top wet grinder until the solvent was clear. The extract was separated from the pulp by centrifixgation using centrifiige REMI C24BL at 22°C and concentrated in a Rotovapor BUCHIR215 at 50°C bath temperature with 320mbar pressure.

Extraction of lycopene using solvent without enzymatic pretreatment of CONTROL 1 gave a yield of 210mg/kg tomato fresh weight of lycopene.

Extraction of lycopene using solvent without enzymatic pretreatment of CONTROL 2 gave a yield of 428mg/kg fresh tomato and 467.36mg/kg sun-dried tomato.

Example 3

Extraction of Ivcopene using Alkali KOH

The tomato pulpfrom Example 1A was treated witU different concentrations of KOH which were 0.02M, 0.04M, 0.06M, 0.08M and 0.1 M/ kg fresh tomato. After addition of KOH, the mixture was mixed well and the pH was noted, it was then heated at 70°C for 30min. The pulp was then transferred to the distillation unit and water was distilled out.

The pulp was then washed with tap water until pH becomes neutral. Lycopene was then extracted as described in Example 2.

KOH treatment at O.IM concentration gave the highest yield i.e. 491.25mg/kg fresh tomato of lycopene.

Example 4

Extraction of lycopene from using Alkali Na2C03

The tomato pulp from Example lA was treated separately with O.OIM, 0.02M, 0.03M, 0.04M and 0.05M/ kg tomato of Na2C03 and mixed well. The pH was noted and the fiirther treatment was carried out as described in Example 3 (but in the wash step unlike with KOH, in Na2C03 only one wash was given). Lycopene was extracted as described in Example 2.

Na2C03 0.05M gave the highest lycopene yield i.e. 769.25mg/kg fresh tomato of lycopene.

Example 5

Extraction of lycopene from using Alkali NaHC03

The tomato pulp from Example lA was treated separately with O.OIM, 0.02M, 0.03M, 0.04M and 0.05M/ kg tomato of NaHC03. The pH was noted and the further process was carried out as described in Example 3. Lycopene was extracted as described in Example 2.

NaHC03 at 0.05M gave highest yield i.e. 421.15mg/kg fresh tomato of lycopene

Example 6

Extraction of lycopene from using Alkali CaC03

The tomato pulp from Example lA was treated separately with 0.025M, 0.05M, 0.075M and 0.125M/ kg tomato of CaC03. The mixture was then heated at 60°C for 30min and the mixture washed to remove CaCOS. Lycopene was extracted as described in Example 2.

With CaC03 treatment four different concentrations were used 0.025M, 0.05M, 0.075M and 0.125M/ kg tomato among which 0.05M, 0.075M and 0.125M were found to give similar extract values and hence 0.05M/kg concentration was used for fiirther processing.
CaC03 at 0.05M gave the highest yield i.e. 630 mg/kg fresh tomato of lycopene.

Example 7

Exfraction of lycopene using Alkali CaC03 and chelating agent EDTA

The tomato pulp from Example lA was treated with an optimized CaC03 concenfration of 0.05M/kg tomato as described in Example 6, where after treatment with CaC03, CaC03 was removed with the chelating agent EDTA at 35mg/kg tomato and lycopene was extracted as described in Example 2.

Example 8

Pulping of natural source for enzymatic pretreatment

The tomato pulp of Example lA (CONTROL 2) and sun-dried tomatoes of Example IB were pulped using a grinder and distilled to remove moisture before carrying out the enzymatic pretreatments as mentioned in the examples below:

Example 9

Preparation of cellulase solution

Food grade c,ellulase was obtained from Tex Biosciences Chermai, India, cellulase solution was prepared by adding 10,000U/ml cellulase to 10ml of 0.2M acetate buffer of pH 6.0.

Example 10

Extraction of lycopene using enzymatic pretreatment with cellulase

The pulp from Example 8 was treated with the cellulase solution of Example 9 at a concentration of 10,000U/ml/kg tomato at 50°C for 20min.

cellulase pretreatment gave the highest yields of 956mg/kg of fresh tomato and 983mg/kg of sun-dried tomato of lycopene.

Example 11

Extraction of lycopene from wet natural source using enzymatic pretreatment with lipase+ CaC03+ EDTA treatment

1500U/ml/kg tomato of lipase (obtained from Tex Biosciences Chennai, India) was added to the pulp obtained from Example 8. After mixing well, the pulp was incubated at 40°C for 20min. The mixture was then treated with 0.05M/kg concentration of CaC03 and 35mg/kg of EDTA as described in Example 7. Lycopene extraction and quantification was done as in Example 2.

Lipase pretreatment gaye the highest yields of 730 mg/kg fresh tomato and 791 mg/kg sun-dried tomato of lycopene.

Example 12

Extraction of Ivcopene according to the present invention using enzymatic pretreatment with cellulase and lipase followed by CaC03 and EDTA

The pulp after treatment with cellulase as described in Example 10 was treated with lipase followed by CaC03and EDTA treatment as described in Example II. Lycopene extraction and quantification was done as in Example 2.

The highest lycopene extract yalue of 1390mg/kg with fresh tomato and 1546mg/kg with sun-dried tomato was obtained using this treatment.

Example 13

Analysis of Lycopene by UV-Spectrophotometer

Sigma standard (Sigma Aldrich- Mumbai) was prepared by dissolving Img lycopene in hexane and the mixture was vortexed until completely dissolved. From this stock solution (Img/ml) working standard with 10ug-50ug concentration was pipetted out and made up to 3ml with hexane and the working standards were read at 472nm and also scanned at 300-1000 nm.

Similarly, the extracts obtained fi-om the above examples were analyzed for lycopene.
The visible spectra of lycopene extracted from various treatments are shown in Figs la and lb. All extracts showed maximum absorption at 441, 472 and 503nm which are similar to standard lycopene in hexane.

Example 14

Analysis of Lycopene extracts by HPLC

From the stock solution (Img/ml), lOOug lycopene was pipetted out (0.1ml) using sterile syringe and it was made up to 2ml using the mobile phase Acetonitrile: n-butanol: Dichoromethane (70: 30: 10) HPLC grade solvents. The mixture was then filtered through 0.2u button filter (Rankem PTFE). 20ul (lOOng) of this mixture was injected.
The column used was Water's Sunfire C18 column (of 150x 4.6mm dia, Sum particle size) with a flow rate of Iml/min. The sample was also prepared in the same way as standard was prepared and 20ul was injected. The absorbance was monitored at 470nm and 530nm using dual X absorbance detector.

On HPLC analysis, the samples were found to show the presence of only lycopene and no any other carotenoids. A single peak was observed at 3.6min in the extracts and the lycopene standard (Figs 2a and 2b).

Example 15

Analysis of Lycopene by TLC

The lycopene extracts were checked for the presence of any other carotenoid using pre-coated silica gel TLC plates (Merck, Germany). lOul of sample along with lycopene and P-carotene (95% pure) sigma standard were spotted on the activated plates. Lycopene extracts obtained using cellulase and lipase treatments were also run on the activated plates as shown in Fig. 3. The plates were then placed in the pre-saturated TLC chamber containing Petroleum ether: Methanol: Hexane (40:20:20:20).

Thus, it can be seen that enzymatic pre-treatment of the natural source using lipolytic and cellulolytic enzymes gives 6.5 fold increase in lycopene when compared to control 1 process which involves lycopene extraction using hexane without any treatment process. Time taken for the entire process is less than 2 hrs. Further, the dosage of the enzjmies and the cost of the enzymes used in this invention is also less. Also, the method is easily scalable in the industry.

Various modifications may be made to the various embodiments of the present invention in part or whole without departing from the spirit and scope of the appended claims.

We Claim:

1. A method of extraction of lycopene from a natural source which, comprises:

treating the natural source with at least one lipolytic enzyme and at least one cellulolytic enzyme;

subjecting the enzyme treated natural source to an alkali treatment;

separating the alkali; and,

extracting lycopene from the alkali treated natural source with an organic solvent.

2. The method as claimed in claim 1, wherein the natural source is selected from ripe tomatoes, over ripe tomatoes, damaged tomatoes, rejected tomatoes, unripe tomatoes tomato pomace, dried tomatoes or tomato by-products.

3. The method as claimed in any one of claims 1 or 2, which further comprises prior to treating the natural source with enzyme, at least one of the steps of homogenizing, grinding or crushing the natural source, separating serum from pulp by mechanical or chemical means, concentrating the pulp to reduce the moisture content to 30-40% and/or drying to remove more than 90% moisture.

4. The method as claimed in claim 1, wherein the lipolytic enzyme is selected from the group comprising of lipases, phosphatases and hydrolases.

5. The method as claimed in any one of claims 1 or 4, wherein the lipolytic enzyme has lipolytic activity between 1200U/ml/kg to 1700 U/ml/kg.

6. The method as claimed in claim 1, wherein the cellulolytic enzyme is selected from the group comprising of cellulase, pectinases, hemicellulases.

7. The method as claimed in any one of claims 1 or 6, wherein the cellulolytic enzyme has cellulolytic activity between 800 U/ml/kg to 1200 U/ml/kg.

8. The method as claimed in claim 1, wherein the alkali is selected from calcium carbonate, magnesium carbonate, potassium hydroxide, sodium hydroxide, sodium bicarbonate and sodium carbonate.

9. The method as claimed in claim 1, wherein the separation of the alkali is carried out by washing with water or treatment with a chelating agent selected from EDTA, citric acid, tetra potassium pyrophosphate, sodium pyrophosphate and trisodiimi phosphate.

10. The method as claimed in claim 1, wherein the solvent is selected from hexane, ethyl acetate, acetone, dichloromethane, hexane, ethanol ethyl acetate, denatured ethanol, methanol 2-propanol, heptanes, tertiary butanol and dichlorodifluoromethane.