FIELD OF THE INVENTION
The present invention relates to a method of extraction of mixed carotenoids from natural sources. More particularly, the present invention relates to a method of extraction of mixed carotenoids rich in lycopene from tomato.
BACKGROUND OF THE INVENTION
Carotenoids are class of pigments synthesized de novo in photosynthetic plants, algae, bacteria and can also be found in animals. In animals all the carotenoids found are ultimately derived from plants, unicellular or acellular organisms. Carotenoids are fat soluble yellow, orange or red pigments such as beta-carotene, lutein, xanthophylls, zeaxanthin and lycopene. Carotenoid compounds found in fruits and vegetables benefit human health by playing an Important role in cell function. The dietary necessity of the carotenoid beta-carotene, the precursor of vitamin A, has been recognized for many decades. More recently, lycopene has attracted substantial interest among carotenoids and medical researchers.
Lycopene, a potent antioxidant is a member of the carotenoids family and is the natural pigment responsible for the deep red color of several fruits. Lycopene is found in tomatoes, guava, apricots, watermelon, papaya, and pink grapefruit. Tomato and tomato products are considered as one of the best source of lycopene.
There has been a significant research especially in the use of lycopene in treating cancer, heart disease, infertility in men, prevention of smoke related diseases, restorative role of lycopene in the prevention of osteoporosis in postmenopausal women by preventing the formation of free radicals there by limiting oxidative stress. Studies have also shown that lycopene and other carotenoids from tomatoes have synergistic antioxidant activity. Studies carried out have opened up interest in production of carotenoids especially lycopene from tomatoes by researchers and food, nutraceutical and pharmaceutical industries.

Several methods for separation and extraction of carotenoids from fruits and vegetables are known.
US 2003/0180435 discloses a method for separation of carotenoids from fruits and vegetables by a supercritical SFE-CO2 fluid extraction. Such method however, has limitations with respect to operational size to achieve any quantifiable production for industrial scale.
US 7,138,152 discloses a multi-step process for extracting carotenoids from a source of fruit or vegetable processing waste by means of admixing the source, a first organic solvent and a surfactant to form a slurry. The slurry is then treated with a second organic solvent thereby separating the treated slurry into a liquid fraction and a solid fraction. Carotenoids are collected from the first portion of the liquid fraction by concentrating the carotenoids; treating the concentrated mixture to precipitate the carotenoids in crystalline form and separating the crystalline carotenoids from the treated first portion. This process is lengthy, tedious and involves many steps.
EP0818225 discloses a process for extraction of lycopene from partially dehydrated fresh material. The extraction is carried out with aliphatic or aromatic hydrocarbons or water-immiscible solvents in the presence of phospholipids as surfactants and stabilizing agents. The extract is concentrated to an oil or fractioned to the desired lycopene concentration. The process is lengthy, tedious and involves heating at high temperature and hence not suitable for extraction of lycopene along with other heat sensitive carotenoids.
Such prior art methods have limitations of being time-consuming, have multiple steps, involve pre-treatment steps for example pre-cooking and are not economical for large scale separation and extraction of carotenoids containing lycopene. Besides prior art processes have poor extraction efficiency with regard to the total amount of the carotenoids in particular lycopene extracted.

SUMMARY OF THE INVENTION
The object of the invention is to provide a method of extraction of lycopene rich mixed carotenoids having improved extraction efficiency.
It is an object of the present invention to provide a method of extraction of carotenoids rich in lycopene from tomato using a single organic solvent, without pretreatment step.
Accordingly, the present invention provides a method of extraction of carotenoids rich in lycopene from natural source comprising the steps of; subjecting the natural source material to primary crushing; separating serum from pulp and skin; subjecting serum to suitable separating means to separate residue; subjecting the said residue pulp and skin to secondary crushing for reducing the particle size to micron size dimensions and extracting with a single organic solvent for 15 minutes to 20 minutes at an ambient temperature and separating the organic solvent for obtaining the carotenoids extract rich in lycopene.
In one of the embodiment the natural source used for extracting lycopene rich mixed carotenoids is tomato.
In yet another embodiment the organic solvent used for the extraction is hexane.
As per one of the embodiment, the particle size of the combined mixture of the pulp, skin and the residue is reduced to the dimension in the range of 5 to 15 micron, preferably In the range of 6 to 10 microns. In one of the embodiments the secondary crushing and extraction of the said residue, pulp and skin is carried simultaneously. In alternate embodiment the extraction of the said residue, pulp and skin is carried out after secondary crushing.
DETAILED DESCRIPTION
The present invention is directed to a method of extraction of carotenoids rich in lycopene from a natural source without the need of subjecting the natural source material to pre-treatment step such as heating, treatment with enzyme or other agents prior to extraction.

Accordingly, the present invention provides a method of extraction of carotenoids rich in lycopene from natural source comprising the steps of; subjecting the natural source material to primary crushing; separating serum from pulp and skin; subjecting serum to suitable separating means to separate residue; subjecting the said residue pulp and skin to secondary crushing for reducing the particle size to micron size dimensions and extracting with a single organic solvent for 15 minutes to 20 minutes at an ambient temperature and separating the organic solvent for obtaining the carotenoids extract rich in lycopene.
A variety of fruits and vegetables may be used as a natural source for extraction of carotenoids rich in lycopene, including a mixture of vegetable and fruits. In the preferred embodiment the natural source is tomato. The tomatoes used in the present invention may be obtained from a variety of sources and cultivars. Unripe, ripe or overripe tomatoes may also be used for the extraction, preferably ripe tomatoes are used.
Tomatoes may be used as a whole in the form of pulp and skin or either pulp or skin may be used for extraction of carotenoids rich in lycopene.
For extraction the tomatoes are sorted to separate foreign matter. The sorted tomatoes may be cleaned by washing with water.
The sorted and cleaned tomatoes are subjected to primary crushing for separating serum from pulp and skin. For primary crushing, any suitable means may be used such as blender, mixer, knife mill or homogenizer. The tomatoes are 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, preferably at A'C.
The physical separation of serum from pulp and skin is carried by using appropriate method such as decantation, filtration or centrifugation. In some of the embodiments

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 is used for further processing or discarded or used for other applications such as food products or a raw material for food products. In the present invention 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 primary crushing is used for extraction. The said 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 the final carotenoids extract rich in 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.
The pulp, skin and residue is then subjected to secondary crushing for reducing the particle size to micron size dimensions and extracted with a single organic solvent. The particle size of the pulp, skin and the residue is reduced to the dimension in the range of 5 to 15 micron, preferably in the range of 6 to 10 microns. The secondary crushing is carried out by using suitable homogenizing means which can convert the pulp, skin and residue to the desired micron size dimensions. The means used for the secondary crushing may be selected from homogenizers, mixers preferably high shear mixers, mills such as bead mills or any suitable means. In the present invention any suitable organic solvent may be used for extraction. In preferred embodiments the solvent used is n-hexane. In some of the embodiments extraction is carried out after secondary crushing. In the preferred embodiments, extraction and secondary crushing are carried out simultaneously. The extraction is carried out for the period of about 15 minutes to 20

minutes. It was observed that reduction in the particle size of the pulp, skin and the residue to the dimension in the range of 5 to 15 micron, preferably in the range of 6 to 10 microns significantly increases the surface area for extraction without destroying the carotenoids and improves the extraction efficiency. The increased surface area and the simultaneous secondary crushing as well as extraction also reduces the overall requirement of the organic solvent required for the extraction as compared to the prior art methods. In the present invention the quantity of the organic solvent used for the extraction is in the ratio of 1:1 to 1:3, preferably it is in the range of 1:2.
Example 1
Extraction of mixed carotenoids rich in Iycopene from tomatoes:
10 kg of tomatoes were sorted and weighed. The tomatoes were then cleaned by washing with water. After washing the tomatoes were subjected to primary crushing using pulper. The crushed tomatoes were centrifuged using centrifuge REMI C24BL for 15 minutes at 10,000 rpm speed to separate serum and pulp. The serum constituted about 80 to 85% and pulp and skin portion was about 1500 g to 2000 g. The serum was further centrifuged by high-speed centrifuge at 20,000 rpm speed for 15 minutes -20 minutes and residue was separated from almost clear liquid. . The separated residue, which was about 100 gm, was mixed with the pulp and skin. The combined mixture was subjected to secondary crushing using homogenizer of 20,000rpm to reduce the particle size to about 6-10 microns. Simultaneously, when the secondary crushing was taking place 3500 ml to 4000 ml of n-hexane was added to carry out the extraction, the extraction was done twice. The simultaneous crushing and extraction was carried out for about 15 minutes to 20 minutes.
The above extracted mixture was centrifuged at 9000-10000 rpm for 10 minutes-15 minutes at room temperature and 7500 ml of extract was separated. The solvent that is n-hexane was separated from the extract under reduced pressure of 300-350 mbar and temperature of 40-50°C. The quantity of the oleoresin extract was 9850 mg.

The oieoresin extract thus obtained was analyzed by spectrophotometer and HPLC.
By spectrophotometry standard Lycopene (Sigma Chemicals, USA, batch no L 9879) reconstituted in hexane showed three distinct peaks at 446, 471 and 503nm. The extract obtained in the present invention also showed three distinct peaks at 446, 471 and 503 nm similar to standard lycopene, which confirms the presence of lycopene in the oieoresin extract. Lycopene was also quantified using the standard graph. The quantity of lycopene obtained was 2-gms/10 kg of tomato fresh weight or 2000 mg/10 kg of tomato fresh weight.
The oieoresin extract was also subjected to HPLC analysis to quantify lycopene and other carotenoids. For lycopene quantification HPLC method followed was of Lee, M.T. and Chen, B.H. "Separation of Lycopene and Its cis Isomers by Liquid Chromatography" reported in Chromatographia, 54, November (No. 9/10) (2001). For the analysis the mobile phase used was n-Butanol: Acetonitrile: Dichloromethane (30:70:10) the solvents used were HPLC grade solvents with the flow rate of 2ml/minutes. HPLC lycopene content was about 88% ± 2% and beta-carotene content was about 12% ± 2%.
The quantity of lycopene obtained was about 200mg per kg of tomato fresh weight and beta-carotene was 20mg/kg of tomato.
The foregoing description and the example are intended as illustrative and are not to be taken as limiting. Such modifications and alterations may be included insofar as they come within the scope of the invention.

WE CLAIM
1. A method of extraction of carotenoids rich in lycopene from natural source
comprising the steps of:
subjecting the natural source material to primary crushing;
separating serum from skin and pulp;
separating residue from serum using a separating means;
subjecting skin, pulp and residue to secondary crushing for reducing the particle size to micron size dimensions and extracting with a single organic solvent for 15 minutes to 120 minutes;
separating the organic solvent and obtaining lycopene rich mixed carotenoids extract.
2. A method as claimed in claim 1 wherein the natural source material is selected from tomatoes, over ripe tomatoes, damaged tomatoes, rejected tomatoes, unripe tomatoes, or tomato by products.
3. A method as claimed in claim 1 wherein the serum is separated from skin and pulp by mechanical or chemical means preferably decantation, centrifugation, filtration, distillation, or rotary vacuum evaporation.
4. A method as claimed in claim 1 wherein the serum separated from skin and pulp is in the range of 60 to 90%, preferably in the range of 75-90%.
5. A method as claimed in claim 1 wherein the separating means used for separating residue from serum is selected from ultra-filtration, centrifugation preferably at high speed.
6. A method as claimed in claim 1 in which the particle size of the pulp, skin and the residue is reduced to the dimension in the range of 5 to 15 micron, preferably in the range of 6 to 10 microns.

7. A method as claimed in claim 1 in which secondary crushing and extraction of the
said, pulp and residue is carried out simultaneously.
8. A method as claimed in claim 1 in which extraction of skin, pulp and residue with
micron size particle is carried out after secondary crushing of the said skin, pulp and
residue.
9. A method as claimed in claim 1 in which the organic solvent used for the extraction is
hexane.