• Field of the invention

The present invention relates to an improved method for the extraction of chlorophyll from green leaves. Though any green leaves can be used in the method of the present invention, the preferred leaves are edible grass, bamboo leaves, banana leaves and the like which are fibrous, or their combinations. The chlorophyll extract produced by the method of the present invention is useful for food coloring applications.

• Background of the invention

Synthetic dyestuffs have been extensively used in food coloring. With the growing concern over the carcinogenic properties of synthetic pigments, those derived from natural sources are gaining importance. Green leaves are rich and renewable source for natural green pigments. Green pigments in leaves are primarily chlorophylls. Chlorophyll is not a single molecule but a family of related molecules, designated chlorophyll a, b, c, and d. The most widely distributed form of chlorophyll is chlorophyll a; chlorophyll b is also present in most plants. Chlorophyll a is blue green in colour while chlorophyll b is green. Chlorophyll a is more temperature sensitive than chlorophyll b. Chlorophyll a has absorbance maxima of 430 nm and 662 nm, while chlorophyll b has maxima of 453 nm and 642 nm. Chlorophyll c is found in various algae; Chlorophyll d is found only in marine red algae. c and d chlorophylls are not of much commercial importance. The molecular structure of chlorophylls a and b consists of porphyrin ring and a long phytol tail. In the centre of the porphyrin ring is a magnesium molecule. Chlorophyll c lacks the phytol chain. Chlorophyll d's structure is similar to Chlorophyll b except in the position of-CHO group. Other pigments present in green leaves are carotenes and xanthophylls.

Natural chlorophyll is highly sensitive to heat, light and pH and can easily undergo degradation. As chlorophyll degrades, the initial step is either the loss of the magnesium from the centre of the molecule or the loss of the phytol tail. Loss of magnesium results in the formation of the molecule, phaeophytin, whereas loss of the phytol tail results in molecule termed chlorophyll. Further degradation of either the phaeophytin or the chlorophyllide produces phacophorbide. Phaeophytin is degraded by the loss of the phytol tail and chlorophyllide by the loss of magnesium ion. The degradation route is shown in Figure below:

Efforts have been made to isolate chlorophyll from plant materials such as spinach, alfalfa, mulberry leaves and the like commercially for coloring food products. The acid generated in the fresh leaves immediately after harvesting initiates degradation of chlorophylls present in the leaves Drying of the leaves even if done carefully, can result in the degradation of the chlorophylls.

Extraction of chlorophyll from any fresh leaves is not commercially popular because of many reasons. Water immiscible solvents cannot extract the pigments present in the leaves effectively because water restricts the contact of solvent with the tissues of the leaves. Water miscible solvents are a better choice for chlorophyll extraction from fresh leaves; however, the solvent gets diluted with water requiring frequent rectification, which require additional machinery and results in higher solvent losses and extra processing cost. Moreover, presence of water in the extract accelerates the degradation of the pigments, leading to colour loss. In addition to water, the solvent used, the pH and temperature employed for the extraction are also critical for the stability of the pigments. A process that takes care of these parameters would be required to isolate the pigment from the leaves in the form as present in the green leaves.

Hence extraction of chlorophyll is commercially carried out using dried leaves of the said plant materials. Chlorophyll extracted from dried leaves lacks the original bright green shade present in the fresh leaves. Further the chlorophyll extracted from dried leaves has dull colour and also lacks stability. To reinstate the shade and improve the stability the magnesium ion in the chlorophyll molecule is substituted by copper through Chemical reaction, leading to formation of a copper complex. Copper complex is more stable than the natural chlorophyll, but the resulting chlorophyll being chemically modified, it is not approved in many countries for food colouring.

Acetone is the most commonly used solvent for extraction of chlorophyll, although methanol, ethanol and isopropyl alcohol are also used. These solvents are reported to be more efficient than acetone in extracting chlorophylls from dry leaves. Grinding the leaves is necessary for complete extraction.

• Prior art

Conventionally commercial extraction of chlorophyll is done from dried leaves using organic solvents. There is no process known to the best of our knowledge for the extraction of chlorophyll from fresh leaves on a commercial scale.

In the conventional extraction of chlorophyll on a commercial scale the raw material namely, the harvested green leaves such as spinach or alfalfa, are dried using a rotary drum or fluidized bed dryer. Acetone and methanol are the commonly used solvents for extraction. 90% isopropyl alcohol has also been reported to be efficient for chlorophyll extraction from spinach leaves. The extract is pH controlled and solvent removed by distillation. However, substantial degradation of chlorophyll takes place during drying. It has been observed that once initiated, the degradation gets carried on to subsequent process stages, resulting in a dull colour of the resulting product. The colour shade of the product is evaluated in terms of absorbance ratio, which is basically the ratio of absorbance of a dilute chloroform solution at two wavelengths. (AOAC Method)

Chlorophyll extraction from un-dried fresh leaves, therefore, faces two challenges:
- extracting fresh leaves requires the use water miscible solvents. The water tends to dilute the solvent thus reducing extraction efficiency during repeated extraction.

- chlorophyll is highly susceptible to degradation in the presence of water.
- Effective loading quantity of the vegetable matter is substantially reduced due to the presence of water.
For these reasons, the raw material used namely, the green leaves, are dehydrated prior to the commercial extraction. But,

- drying (dehydrating) results in partial degradation of chlorophylls, usually
25% to 50%, even if done carefully.

- the partial degradation of chlorophylls in the dried leaves leads to the production of a dull green extract
- extraction of chlorophylls from dried leaves using common extraction solvents is comparatively slower than from fresh leaves and hence require longer process time.

With the increasing awareness of the carcinogenic properties of synthetic pigments, those derived from natural sources are gaining importance. Green colour finds application in all kinds of food products and synthetic green dyes still dominate the field, primarily due to the non-availability of a 100% natural replacement with the true colour shade.

Under the circumstances explained above, currently there is a need to develop a process for extraction of chlorophyll commercially which is simpler, faster and cost-effective and can yield a product with bright green shade.

• Objectives of the invention

Therefore the main objective of the present invention is to provide an improved method for the extraction of chlorophyll from fresh green leaves, avoiding the drawbacks of the hitherto known processes.

Another objective of the present invention is to provide an improved method for the extraction of chlorophyll from fresh green leaves which is simple, cost effective, and commercially applicable.

Yet another objective of the present invention is to provide an improved method for the extraction of chlorophyll from fresh green leaves employing a solvent that has high extraction efficiency for chlorophyll pigments resulting in a product with minimum degradation.

Yet another objective of the present invention there is provided a process for the extraction of chlorophyll, the resulting product being greener and brighter than the product obtained by conventional prior art processes from dried leaves.

• Summary of the invention

Accordingly the present invention provides an improved method for the extraction of chlorophyll from fresh green leaves which comprises
(a) squeezing the green leaves by conventional method to remove the water.
(b) mixing the resulting squeezed leaves obtained in step (a) with a solvent selected from ethyl acetate, methanol or a mixture of ethyl acetate and hexane, preferably ethyl acetate
(c) heating the mixture obtained in step (b) to a temperature in the range of 45-50 deg C to obtain an extract
(d) Filtering the extract obtained in step (c)
(e) Rejecting the steps (b), (c) and (d) until the extraction is complete and combining the resulting extracts
(f) Adjusting the pH of the combined extract obtained in step (e) using methanol ie alkali solution selected from methanol potassium hydroxide or methanolic sodium hydroxide, preferably methanolic potassium hydroxide
(g) evaporating the solvent from the combined extract obtained in step (f) at a temperature not exceeding 80 deg C
(h) dissolving the concentrate obtained in step (g) in a hydrocarbon solvent selected
from hexane, heptane, preferably hexane
(i) washing the solution obtained in step (h) with aqueous solution of a mild alkali
selected from sodium carbonate, potassium carbonate, preferably sodium carbonate,
and removing the aqueous layer by gravity separation and
(j) distilling the solution obtained in step (j) at a temperature not exceeding 70 deg C
and under vacuum to get the chlorophyll.
In an embodiment of the present invention, the green leaves used in the process is selected from edible grass, bamboo leaves, banana leaves and the like, which are fibrous and rich in chlorophyll.
In another preferred embodiment of the invention the ratio of the concentrated extract to the hydrocarbon solvent used in step in (h) may be around 1:10

In another preferred embodiment of the invention the mild alkali solution used for washing in step (i) may be 1% aqueous solution of sodium carbonate or potassium carbonate, preferably sodium carbonate.
In another preferred embodiment of the invention, the ratio of the solution of the extract in hydrocarbon solvent to the alkali solution used in step in (i) is 3:1 and washing may be repeated three times.

• Detailed description of the process of the present invention
The green leaves are separated from sterns and spoiled leaves. The selected leaves are chopped to 30-40 mm pieces in a suitable cutting machine. The chopped material is fed into a screw press and water squeezed out. The de-watered material may be pressed again to reduce the moisture further. The machine used for squeezing may be cooled by circulating cold water in the jacket to prevent heat build up. The pressed material is charged in an extractor and the solvent is pumped in to cover the charge. The charge is heated using steam in the jacket to a temperature in the range of 45-50 deg C and the extract circulated for better extraction efficiency. The extract is drained off and the process repeated till the charge is fully exhausted.

The pH of the combined extracts is adjusted to 8-8.5 using 0.1% methanolic alkali solution and the solvent distilled off in a hot water or steam jacketed stirred vessel or a rising film evaporator. Solvent may be evaporated till the residual solvent level is below 10%. The concentrate is taken up in the hydrocarbon solvent and the solvent layer is washed with the 1% aqueous carbonate solution . The washing is repeated preferably two times or more. Clear solvent fraction is concentrated to get the product.
The present invention is described in detail in the Examples given below which are given to illustrate the invention only and therefore should not be construed to limit the scope of the invention.

Example 1
80 kg of Napier grass was harvested from the farm. Sterns were cut off (25 kg) and the leafy material is chopped in a rotary cutting machine to approx 1 inch size. The chopped material is squeezed through an expeller type juicer machine. 15 kg pressed cake with 25% moisture was obtained. The pressed cake was subjected to extraction at 45-50 deg C using ethyl acetate. Four extractions were carried out and the combined extracts (80 litres) was pH adjusted to 8-8.5 using 0.1% methanolic potassium hydroxide, filtered and concentrated to approx. 3.75 litres at a temperature not exceeding 80 deg C, under vacuum towards flam stages. The concentrate was dissolved in 37.5 litres of hexane. The solution was washed with 12.5 litres of 1% aqueous sodium carbonate solution. The aqueous portion was discarded and the washing repeated twice. The hexane layer was concentrated to yield 220 g product, equivalent to 1.40% of the pressed leaf, with a chlorophyll content of 10% (AOAC Method) and absorbance ratio 1.03.

Example 2
50 kg of Napier grass was harvested from the farm and prepared as in example 1. Sterns (15 kg) were cut off and the leafy potion chopped in a cutting machine to approx 1 inch size. The chopped material was squeezed through the juicer machine to yield 10 kg pressed material, which was extracted with ethyl acetate, at 40-45 deg C. Four extractions were carried out and the combined extracts (50 litres) was pH adjusted to 8-8.5 using 0.1% methanolic potassium hydroxide, filtered and concentrated to approx. 2.5 litres at a temperature not exceeding 80 deg C. The concentrate was dissolved in 25 litres hexane and the solution was washed with 8.3 litres of 1% aqueous sodium carbonate solution. The aqueous portion was discarded and the washing repeated twice. The hexane layer was concentrated to yield 130 g product was obtained, equivalent to 1.30% of the pressed leaf, with a chlorophyll content of 10.2% (AOAC Method) and absorbance ratio 1.00.

Example 3
20 kg of green banana leaves was chopped to approx 1 inch size in a cutting machine. The chopped material was squeezed through a juicer machine to remove water as in example 1. 4.5 kg pressed cake with 20% moisture was obtained, which was extracted with methanol at 40-45 deg C. Four extractions were carried out and the combined extracts (17 litres) was filtered, pH adjusted to 8-8.5 using 0.1% methanolic potassium hydroxide and the concentrated at a temperature not exceeding 80 deg C to approx 1.1 litres. The concentrate was dissolved in 11 litres of hexane. The solution is washed with 3.75 litres of 1% aqueous sodium carbonate solution. The aqueous portion was discarded and the washing repeated twice with 3.75 litres alkali solution. The hexane fraction was concentrated to yield 58 g product, equivalent to 1.29% of the pressed leaf, with a chlorophyll content of 12% (AOAC Method) and absorbance ratio 1.10.

• Advantages of the invention:

1. Provides a method for extracting chlorophyll from fresh green leaves.

2. The chlorophyll obtained has bright green shade and high absorbance ratio.
3. The process eliminates pigment loss associated with dehydration prior to normal extraction.

4. The method is simple, cost effective and commercially applicable

5. The resulting product obtained has minimum degradation.

WE CLAIM

1. An improved method for the extraction of chlorophyll from fresh green leaves
which comprises

(a) squeezing the green leaves by conventional method to remove the water.

(b) mixing the resulting squeezed leaves obtained in step (a) with a solvent selected from ethyl acetate, methanol or a mixture of ethyl acetate and hexane, preferably ethyl acetate

(c) heating the mixture obtained in step (b) to a temperature in the range of 45-50 deg C to obtain an extract

(d) Filtering the extract obtained in step (c)

(e) Repeating the steps (b), (c) and (d) until the extraction is complete and combining the resulting extracts

(f) Adjusting the pH of the combined extract obtained in step (e) using methanolic alkali solution selected from methanolic potassium hydroxide or methanolic sodium hydroxide, preferably methanolic potassium hydroxide .

(g) evaporating the solvent from the combined extract obtained in step (f) at a temperature not exceeding 80 deg C

(h) dissolving the concentrate obtained in step (g) in a hydrocarbon solvent selected
from hexane, heptane, preferably hexane

(i) washing the solution obtained in step (h) with aqueous solution of a mild alkali
sodium carbonate, potassium carbonate, preferably sodium carbonate, and removing
the aqueous layer by simple gravity separation, and

(j) distilling the solution obtained in step (i) at a temperature not exceeding 70 deg C
and under vacuum to get the chlorophyll.

2. A process as claimed in claim 1 wherein the green leaves used is selected from
fibrous fresh green leaves such as grass, bamboo leaves, banana leaves and the like.

3. A process as claimed in claims 1 & 2, wherein the squeezing of the leaves is done in an expeller-squeezer.

4. A process as claimed in claim 1-3, wherein the extraction in steps (b) to (d) is repeated 2-5 times depending on the solvent used, till the chlorophyll is completely extracted.

5. A process as claimed in claim 1-4, wherein the extract obtained in step (f) is concentrated to a solvent content below 10% at a temperature below 70 deg C.

6. A process as claimed in claims 1-5, wherein the solution of the extract obtained in step (h) is washed with an equal volume of 1% aqueous solution of a mild alkali in three instalments.

7. A process as claimed in claims 1-6, wherein the aqueous solution of mild alkali used for washing in step (i) is sodium carbonate or potassium carbonate.

8. A process as claimed in claims 1-7, wherein the distillation in step (j) is done at appropriate vacuum to maintain the temperature below 70 deg C to get the chlorophyll.

9. An improved method for the extraction of chlorophyll from fresh green leaves substantially as herein described with reference to the Examples.