PROCESS AND METHOD FOR OPTIMAL RECOVERY OF CAROTENOIDS

FROM PLANTS

DESCRIPTION

TECHNICAL FIELD

The present invention relates to a process for producing carotenoids, particularly lycopene, from plants or plant constituent parts, particularly from fruits and vegetables.

BACKGROUND OF THE INVENTION

Dietary carotenoids are known to have health benefits as supplements in decreasing the risk of diseases, particularly certain cancers and eye diseases. The carotenoids that have been most commonly studied in this regard are β-carotene, lycopene, and lutein. The health benefits of the carotenoids as dietary supplements are thought to be due to their activity as antioxidants. Carotenoids can be industrially obtained by extraction from fruits and vegetables, which have high concentrations of these substances by using organic solvents or supercritical fluids. Other methods utilized for carotenoid extraction are the fermentation of certain species of microorganisms (fungi or bacteria) or chemical synthesis. Carotenoids, which are well credited for important health-promoting functionalities can also be found in the waste products originating from certain processing plants such as tomato peel from tomatoes dice, pure, sauce or paste production. To date, several problems related to the recovery and purification of carotenoids from plant products have been addressed. Generally, the core problem remains to be able to recover such organic molecules from a matrix, which is substantially cellulosic in nature.

Lycopene is a carotenoid responsible for the red color of a large number of fruits and vegetables. Consumption of lycopene from tomatoes has been associated with protection against oxidative DNA damage in addition to their anticancer properties (Agarwal & Rao, 2000) and their functionality in the prevention of cardiovascular diseases. Studies so far have indicated that consumption of tomatoes and tomato sauce is directly associated with a reduced risk of developing different types of cancers of the digestive system and prostate cancer. Apart from potential health benefits, lycopene is also used as an alternative to synthetic food colorants. Here, it should be noted that lycopene covers a wide range of colors ranging from light yellow, passing through orange, to an intense color of red.

Lycopene is insoluble in water, and can be dissolved only in organic solvents and oils and is susceptible to degradation by UV light, which limits its shelf-life after extraction from the raw plants.

The extraction efficiency of carotenoids can be improved by using solvent combinations to facilitate partitioning. At present, the production of most extracts or products rich in lycopene, or in other carotenoids available on the market requires the use of organic solvents or solvent combinations such as hexane, ethanol, acetone, etc.

In its natural form, lycopene is heat resistant and present in a thermodynamically stable state as all-trans isomer crystal within the chloroplasts of plant cells (Harris & Spurr, 1969). Conventional extraction often requires heat to facilitate the migration of solvent to extract pigment compounds. Although increased temperatures correspond to improved solubility and organelle membrane disruption, heat and UV exposure should be limited when possible due to the thermo labile nature of carotenoids once they are in the solvent (Rodriguez- Amaya, 2001). Previous studies have demonstrated that heat treatments, longer than 1 hour, favored the trans-to cis isomer conversion of lycopene, while light irradiation induced czs-isomer degradation over time in tomato products (Chen, Shi, Xue & Ma, 2009; Shi, Dai, Kakuda, Mittal & Xue, 2008).

There are earlier research outcomes indicating that the solvent systems containing hexane and ethyl acetate are the most efficient for carotenoid extraction from tomato seeds and peels (Strati & Oreopoulou, 2011). However, (i) the time consumed for purification of the lycopene from the organic solvents which are carcinogenic (such as hexane) and (ii) the fact that the extract is prone to degradation when exposed to heat or UV light for extended periods of time, tend to make the product more expensive and less reliable as a health supplement. Additionally, the use of organic solvents for extracting carotenoids can be toxic and in any case they must be removed from the final product after extraction. Yet their complete removal is not always plausible to some desired levels, such as those of the U.S. FDA requirements. Particularly hexane is known to be a strong carcinogen compound and its remains in the lycopene extracts are not acceptable at any level.

Extraction efficiency of carotenoids including lycopene can be improved by using solvent combinations. Comparison of efficiency among different solvents for carotenoid extraction from various plant materials is presented in the literature, but most of them deal with optimization of the extraction conditions.

Supercritical fluid extraction process of lycopene has been studied by many researchers. Machmudah et al. (Machmudah et al., J. Food Engineering, 108 (2012) 290-296) have found that the optimum operating condition to extract lycopene from tomato peel by-product containing tomato seed using supercritical carbon dioxide, under which 56% of lycopene was extracted, is 90 °C, 40 MPa, and a ratio of tomato peel to seed of 37/63. They have also found that the presence of tomato seed oil helped to improve the recovery of lycopene from 18% to 56%.

Baysal et al. (T. Baysal, S. Ersus, D.A.J. Starmans, Supercritical C02 extraction of β-carotene and lycopene from tomato paste waste, J. Agric. Food Chem. 48 (11) (2000) 5507-5511) observed highest yield (54%) of lycopene at 55 °C, 30 MPa and a flow rate of 4 kg/h with 5% ethanol used as a modifier.

Karaj et al. (D. Karaj, A. Mele, Kinetics of lycopene and β-carotene extraction from tomato skin in the presence of oleic acid by near critical liquid C0₂, International J. Engineering Inventions Volume 4, Issue 8 [January 2015] PP: 01-05) investigated the extraction of lycopene and ^-carotene from tomato skin using near critical liquid carbon dioxide in the presence of oleic acid as a modifier. They observed that the oleic acid as co-solvent has a beneficial role in the stability of cis-isomer of lycopene (0.19-0.067 μg/g sample).

In supercritical fluid extraction processes, the mobile phase is subjected to pressures and temperatures near or above the critical point for the purpose of enhancing the mobile phase solvating power. The process begins with C0₂ in vapor phase, which is then compressed into a liquid before becoming supercritical. It is a relatively rapid method because of the low viscosities and high diffusivities associated with the supercritical fluids. The extraction can be selective to some extent by controlling the densities of the medium. The extract is easily recovered by simply depressurizing in the system, allowing the supercritical fluid to return to the gas phase and evaporate, leaving little or no amount of solvent residue. However, this process has some disadvantages which make its applicability limited at the industrial scale for lycopene production including: (i) a high operational pressure is required which increases the processing cost in addition to the related safety concerns and environmental hazards;

(ii) whilst recycling and cost saving can be achieved by recompression of the solvent, equipment installation can be costly and complex; and

(iii) a high capital investment is necessary for the equipment.

Accordingly, there is a need to improve the available industrial processes for production of carotenoids from plant materials with improved cost efficiency as well as extraction ability while protecting the extracts for a longer time. Therefore, it is one of the aims in the present invention to provide an improved process and method for extracting a carotenoid substance from plants or plant constituent parts, particularly from fruits and vegetables, that can be applied at the industrial scale to obtain enhanced purity of the product with self-protection ability against heat and UV light and with improved cost and yield efficiency.

BRIEF DESCRIPTION OF THE INVENTION

It has been found that the technical problems outlined above can be addressed by the process set up in claim 1.

An objective of the present invention is to provide a process for production of carotenoid extract from carotenoid sources.

In one aspect, a process producing carotenoid extract from a carotenoid source, the process comprising: Providing a carotenoid source;

providing an extraction solvent including

at least one acetal compound, and

at least one component selected from medium chain fatty acids, water-soluble primary alcohols and water-soluble secondary alcohols, and isomers thereof;

mixing the carotenoid source with the extraction solvent; and

extracting soluble components from the cartenoid source to produce carotenoid extract. In another aspect, the present invention provides a method for production of carotenoid from a carotenoid source.

In a third aspect, the present invention provides a caretonoid extract product comprising at least one acetal compound, and at least one component selected from medium chain fatty acids, water-soluble primary alcohols and water-soluble secondary alcohols, and isomers thereof.

In yet another aspect, the present invention provides a use of carotenoid extract product as a food colorant or antioxidant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing lycopene concentration in selected solvents immediately after extraction in accordance with an embodiment of the present invention and after 6 months.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides processes for production of carotenoids from plants or plant constituent parts via solvent extraction. From here on a plant or plant constituent part containing carotenoid will be referred to as a carotenoid source. In a first aspect, the present invention provides a process for producing carotenoid extract via the extraction of carotenoid that is contained in a carotenoid source, which from hereon will be referred to as a process of the present invention. The process of the present invention can be applied to any carotenoid source. The carotenoid source can be, for example, tomatoes, watermelons, rosehip and gac fruit as the source of lycopene; orange plants, carrots, pumpkins and sweet potatoes as the source of ^-carotene; tobacco leaves as the source of nicotine; green leafy vegetables such as spinach, kale and yellow carrots as the source of lutein.

In one preferred embodiment of the present invention, the carotenoid source corresponds to the industrial waste generated in the tomato processing industry. Such waste is comprised of tomato peels which are rich in fibers and pulp attached thereto, as well as tomato seeds. The waste material contains high amounts of lycopene (40-50 g/kg) when dried depending on the variety of the tomato.

The use of waste product from industrial tomato processing as a carotenoid source for the present invention allows the recovery of a material which is currently either used as animal food or even discarded. Hence the extraction of lycopene from the waste of industrial tomato processing is an example of bio refinery; i.e., a process which integrates an established industrial process by-product, in this case from industrial tomato processing, to generate substances with a high added value. Such an achievement is a very important progress in improving the sustainability of the modern industry. In another preferred embodiment of the present invention the carotenoid source corresponds to the leaves of green leafy vegetables such as spinach, kale and yellow carrots. These products, when appropriately treated, are excellent sources of lutein.

The carotenoid source, before being extracted, is dried until it reaches a suitable degree of humidity in order to avoid degradation of the carotenoid. In a preferred embodiment, the tomato waste as the lycopene source is dried between 30 - 60°C for 2-5 hours to reach a 3-5 wt% moisture content. The carotenoid source, with the appropriate degree of humidity, is grounded or milled to obtain suitable particle size in order to increase the total surface area of the reaction and to facilitate extraction. In a preferred embodiment, the tomato waste as the carotenoid source is grounded or milled to obtain a predetermined particle size. In a preferred embodiment, the mean particle size is between 50 to 500 micrometer. In general, reduction of the particle size increases the yield of the carotenoid and the extraction rate.

The process of the present invention utilizes an extraction solvent including medium-chain fatty acids (MCFAs) in combination with , water-soluble primary and secondary alcohols including ethanol, propanol, buthanol, and isomers thereof, and acetal compounds as the extraction solvent, from here on referred to as an extraction solvent of the present invention.

In this description "water-soluble primary and secondary alcohols" refers to the primary and secondary alcohols having a minimum of at least 5% by volume of water- solubility at 1 atm25 °C. As the number of Carbon atoms in the chain increase, the less soluble will be the alcohol in water. For ease of solvent recovery and achieving optimum viscosity, solubility of the extraction solvent is required. The viscosity of the extraction product to be obtained is around 1.00 + 0.5 MPa.s within the temperature range of 0-100 °C. The suitable alcohols of the present invention include ethanol, propanol and butanol, and isomers thereof.

In one embodiment the alcohol of the present invention is at least one selected from ethanol, propanol and butanol, and isomers thereof.

In another embodiment, the alcohol of the present invention is at least one selected from propanol and butanol, and isomers thereof.

In another embodiment, the alcohol of the present invention is 1 -propanol.

It has been surprisingly found that when the extraction solvent of the present invention is used, a greater yield of carotenoid extraction is possible from carotenoid sources as compared to the pure MCFAs and pure solvents. The use of MCFAs in combination with water-soluble primary alcohols, water-soluble secondary alcohols, and acetal compounds as the extraction solvent contributed to the yield of carotenoid extract. In this description, the expression "medium-chain fatty acids" refers to the fatty acid composed of a glycerol backbone and three fatty acids in which 2 or 3 of the fatty acid chains attached to glycerol are medium-chain in length. In one preferred embodiment of the present invention the medium-chain fatty acid is selected from the group consisting of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, linolenic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid and cerotic acid.

The chemical formulas of the said medium-chain fatty acids are shown in Table 1 hereunder:

Table 1

In one embodiment, the medium-chain fatty acid of the present invention is at least one selected from the group consisting of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, linolenic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid and cerotic acid.

In another embodiment, the medium chain fatty acid of the present invention is at least one selected from oleic acid and linolenic acid.

In another embodiment, the medium chain fatty acid of the present invention is oleic acid. Suitable acetal compounds of the present invention include dimethoxymethane, diethoxymethane, dipropoxymethane, dibutoxymethane and 2-ethylhexylal.

In one embodiment the acetal compound of the present invention is at least one selected from dimethoxymethane, diethoxymethane, dipropoxymethane, dibutoxymethane and 2- ethylhexylal.

In one embodiment the acetal compound of the present invention is dimethoxymethane. One preferred embodiment of the extraction solvent of the present invention comprises one or more component selected from the group consisting of ethanol, propanol, butanol and isomers thereof, and diethoxymethane.

In one embodiment, the extraction solvent of the present invention comprises at least one medium chain fatty acid and an acetal compound. Preferably, at least one medium chain fatty acid is oleic acid or linolenic acid, and at least one acetal compound is selected from dimethoxymethane, diethoxymethane, dipropoxymethane, dibutoxymethane and 2- ethylhexylal. More preferably, at least one medium chain fatty acid is oleic acid and at least one acetal compound is diethoxymethane.

Alternatively, in one embodiment, the extraction solvent of the present invention comprises at least one alcohol selected from the group consisting of water-soluble primary alcohols, water- soluble secondary alcohols and isomers thereof, and at least one acetal compound. Preferably, at least one alcohol is selected from ethanol, propanol and butanol, and isomers thereof, and at least one acetal compound is selected from dimethoxymethane, diethoxymethane, dipropoxymethane, dibutoxymethane and 2-ethylhexylal. More preferably, at least one alcohol is 1 -propanol and at least one acetal compound is diethoxymethane.

The use of acetal compounds in combination with water-soluble primary alcohols, water- soluble secondary alcohols, and isomers thereof, and in combination with medium chain fatty acids, as well as the use of medium chain fatty acids in combination with water-soluble primary alcohols, water-soluble secondary alcohols, and acetal compounds has additional advantages, apart from producing a carotenoid extract with a higher carotenoid content compared to the use of pure MCFAs and pure solvents, as described below. i) The component of the extraction solvent of the present invention, i.e. medium chain fatty acids, are abundantly present in plants and plant constituent parts making the process of extraction in the invention easily applicable industrially. For example, many of the vegetable oils such as hazelnut oil, sunflower oil and olive oil contain oleic acid. The fact that lycopene in fruits and vegetables is stable as long as the plant is fresh is also an indicator that the extract is better stored in the fatty acid based solvents. Hence, the proposed extraction process is believed to improve the shelf life of the extract. Furthermore, the fact that the tomato peels (as a waste product from the tomato paste production) are dried prior to the extraction process is another factor to enable the lycopene extraction independent of the seasonal growth of the tomato. ii) The extraction of carotenoids with the use of the extraction solvent mixture of the present invention has a beneficial role in the stability of carotenoids and avoids the degradation of the carotenoid itself during the extraction period. For instance, the use of oleic acid in combination with ethanol has a benefit of preventing the degradation of lycopene. Thus lycopene produced by using the process of the present invention provides an endurance of lycopene in its cis- isomer till 70 °C in confectionary as a food colorant, and till 140 °C for 1 minute for the use of lycopene in dairy products without degradation.

To carry out the process of the present invention, a carotenoid source that is adequately treated is introduced into an extractor and the extraction solvent mixture of the present invention is passed through the starting material of the carotenoid source, under ambient pressure and pre-set temperature conditions which permit the solubilisation of the carotenoid in the extraction solvent. As the extraction solvent is exposed to the starting material, the extraction solvent extracts the soluble components from the starting material of the carotenoid source. Then the soluble components are moved to the separators where the desired product is obtained.

In general, in the extraction stage, the pressure and temperature conditions are chosen such that they permit an adequate solubility of the carotenoid. In one embodiment of the present invention, the extraction of lycopene by the use of oleic acid in combination with ethanol is carried out under pressure of 1 atm (ambient) and at a temperature of 25-100 °C. In another embodiment of the present invention, the extraction of lycopene by the use of oleic acid in combination with diethoxymethane is carried out under ambient pressure and at a temperature range of 25-100 °C.

The extraction product obtained in the extraction stage is purified and the remaining solvent mixture is removed from the medium. Furthermore, it is possible to recycle and reuse the fatty acids that are used for the extraction process which may further contain the fatty acids from the product itself.

Although lycopene is known to decompose at around 70 °C and above using the process disclosed in the present invention lycopene is protected up to a minimum of 100 °C.

In a second aspect, the present invention provides a carotenoid extract obtained by the process of the present invention. In this description, "carotenoid extract" refers to the extract containing carotenoid in a concentration higher than that is present in the natural product from which it has been obtained (carotenoid source). As an example, the concentration of carotenoid in the extract obtained from the industrial waste of the tomato processing, e.g., tomato skins reaches a concentration of 10000-120000 ppm following the process of the invention.

The carotenoid extract product of the present invention comprises at least one acetal compound, and at least one component selected from at least one medium chain fatty acids, water-soluble primary alcohols and water-soluble secondary alcohols, and isomers thereof.

In one embodiment the caretonoid extract product of the present invention comprises at least one acetal compound, at least one medium chain fatty acid and at least one alcohol selected from the group consisting of water-soluble primary alcohols and water-soluble secondary alcohols, and isomers thereof.

The carotenoid extract obtained by the present invention has usage as a colorant and/or antioxidant properties and can be used to elaborate products that contain these extracts. In a third aspect, the present invention provides a composition, from here on referred to as the composition of the present invention that contains this carotenoid extract in combination with an appropriate diluent. Additionally and optionally, the composition of the present invention can contain one or more of the selected additives, for example antioxidants, emulsifying agents or mixtures thereof. The composition of the invention can be a food, cosmetic, pharmaceutical or nutraceutical product.

The composition of the present invention can be obtained by diluting the concentrate of the invention with a diluent to achieve the appropriate carotenoid extract and, alternatively, by adding one or two more appropriate additives, for example, antioxidants, emulsifiers and mixtures thereof. The composition of the present invention can contain a variable amount of carotenoid, depending on the application for which it is put in use.

As a diluent, any substance can be used in which the carotenoid is soluble, for example, fats, oils and mixtures thereof. In one specific application, the diluents are comprised of one or more vegetable oils, for example, olive oil, walnut oil, sunflower oil.

As an antioxidant, any antioxidant can be used, for example ascorbic acid (vitamin C), and/or tocopherols (vitamin E etc.)

As an emulsifier, any emulsifier can be used, for example, lecithin, monoglycerides etc.

The composition of the present invention can be presented in any presentation phase, liquid or solid, for example, encapsulated in soft gelatin capsules.

This invention is of interest to any industry that generates waste products containing carotenoid, for example, in the tomato processing industry where the residual products are an excellent source of lycopene. Lycopene extracts provided by this invention, and the compositions they contain, are mainly destined for the food industry.

The following examples are provided to illustrate the present invention and are not intended as a limitation on the scope thereof.

Examples Tests on a number of samples according to the invention have given the results set out below. Carotenoids were extracted using ethanol, diethoxymethane, hexane and oleic acid. The first series of experiments were conducted with single pure solvents at a solvent to dried tomato peel ratio of 10: 1 (v/w) while the particle size of the dry tomato waste was 1.0 mm.

The second series of experiments were conducted with mixtures of ethanol and oleic acid; diethoxymethane and oleic acid as well as hexane with oleic acid at varying amounts of 10:90 and 80:20 (v/v), solvent to waste ratio varying between 3: 1 and 10: 1 (v/w), and particle size in the range of 0.5 and 1.0 mm.

Lycopene concentrations in the extracting solvent were determined spectrophotometrically at room temperature using 1-cm path length quartz cuvettes and a double-beam UV-VIS spectrophotometer (SHIMADZU UV-3600-UV-VIS-NIR Spectrophotometer). Absorption spectra were identical to that of the lycopene standard and displayed the three characteristic peaks of lycopene at around 445, 472 and 503 nm. To minimize the interference from other carotenoids, the concentration of lycopene was calculated at 503 nm using the molar extinction coefficient 17.2xl0⁴ M^_1 cm^"1. The results of the experiments are presented in FIG.1.

FIG. 1 shows lycopene concentration in the selected solvents immediately after the extraction and after 6 months. FIG. 1 shows the total carotenoid yield after extraction step (repeated twice) and the percentage of individual carotenoids identified by UV VIS measurements. It is shown that the combination of oleic acid with ethanol and diethoxymethane synergistically improved the total yield compared with that obtained by any of the individual solvents as well as that obtained by the combination of oleic acid with hexane. Although hexane is the best solvent to extract the lycopene, it results in the most amount of decay as a function of time. Oleic acid itself or in combination with ethanol or diethoxymethane has been shown to prevent a large percent decay in the lycopene content for six months as compared to pure hexane.