A PROCESS TO OBTAIN AQtJEOUS EXTRACTS CONTAINING CAROTENOIDS AND OBTAINED EXTRACTS

DESCRIPTION

The present invention relates to a process to obtain aqueous extracts containing carotenoids and to the obtained extracts. State of the art

The agri-food sector is one of the productive fields that produces the largest amount of special wastes, for the most part non hazardous. At European level farming by-products have a significant potential for the development of bio-energetic industry and are estimated being as much as 250 million ton/year. The management and the disposal of these by-products is a complex issue that has to be faced at social, economical and environmental level, aiming to rationally and sustainably use these resources .

Tomato {Lycopersicum esculentum) , a Solanaceae plant of the dicotyledonous family, is one of the most relevant production of the Italian agri-food sector. Nowadays is cultivated on a total surface of more than 114.000 ha for a total production of more than 59 million of quintal (source: ISTAT; data related to year 2006) . Out of this amount a 30% is for fresh consumption while the other 70% is for food processing industry. The main products obtained by tomato processing are peeled tomatoes, concentrated tomato paste, tomato puree and several variety of tomato sauces. Within the different tomato transformation industrial processes, relevant amount of by-products are produced and the amount can be as much as 0.6 % of the processed products in the production of peeled tomatoes and, for the production of puree, paste and juices, can reach the 2 %. The by-product is essentially constituted by the fibrous parts of the

fruit, seeds and skins. The traditional use of byproducts obtained by tomato industrial transformations, is the animal feeding in the zootechnic sector; in some cases these are used for thermo-valorisation purposes. Lycopene, a terpene of the carotenoid family, is the main antioxidant molecule contained in tomato and its content is estimated in 50-600 μg/g in each tomato skin. Lycopene is the main carotenoid in human body; this is essentially due to the high amount of tomatoes consumed with the diet. Several research groups worldwide focused their attention on the protective effects of this molecule on human health, effects mainly due to the antioxidant properties. Clinical trials shown that lycopene plays a role in preventing some chronic and degenerative disease such as some cancers (prostate, cervical breast and liver) and some cardiovascular diseases (hypertension, stroke, heart attack) .

The antioxidant properties of lycopene allow to use this molecule not only in the pharmaceutical sector, but also as food supplement in new food products (nutraceuticals) or in the cosmetic sector to neutralize free radicals responsible of skin aging. In fact, the oxidative reactions of human metabolism, are responsible of the production of free radicals that have to be balanced with several molecules defined as antioxidants. This increasing interest in using lycopene attracted the attention on new methods of production and extraction of this molecule.

Despite on a national base lycopene is almost completely imported, paradoxically we have to dispose relevant amount of skins derived by industrial tomato processing, that for its content in lycopene is the ideal starting material in order to extract this molecule. Agri-food by-products can therefore be an interesting economical resource. That is the reason why in the last

few years the possibility of performing process of reuse, recycle and re-process of the industrial by-product tomatoes also through lycopene extraction has been evaluated. Today the commercially available lycopene is both produced via chemical synthesis and via extraction with alcoholic and, or organic solvents (EP1103579B1 EP1676888A1) or with supercritical fluids, in particular CO ₂ (EP0818225B1) . In the first process lycopene production has a low cost but the final product is contaminated by residues of toxic-hazardous organic solvents. In the second process, in addition to the issue related to the solvents used for the extraction, there is also the need of long and expensive purification costs in order to separate other molecules (carotenoids, xantophyllins) , extracted in a non selective way that lead to a low purity grade product. Furthermore the vegetal material used for the extraction of lycopene, become a special waste that needs to be properly disposed and will not be reused in any way, with a considerable increase of the overall process costs.

Supercritical CO ₂ replaces traditional solvents on an industrial scale. The process that use this solvent has some advantages i.e. good extraction performances by CO ₂ in the supercritical phase of non-polar compounds, non toxicity of this substance, straightforward availability, non hazardousness, short time of extraction. There are anyway some non negligible drawbacks such as reaching and keeping the supercritical phase of CO ₂ . Considering the high pressure needed there are also relevant problem related to the high industrial costs and safety of the process.

The use of enzymatic bio-catalysts in order to perform the hydrolysis of vegetal matrixes derived by agri-food transformations, and the use of surfactants to

-A- pseudo-solubilise apolar molecules in water phase, can be an interesting option with respect to the traditional Chemical processes, both on the economical and the environmental side. Regarding this, the state of the art describes thermal and biochemical pre-treatments based on hydrolytic enzymes (WO2006036125A1) always described as preparatory to extractions with an organic solvent. CN1315953, in addition to the biocatalytic treatment, foresee steps of treatment with surfactants solubilised in organic solvents with the only aim of improving the solvent extraction performances. In the state of the art there is no description of a process that foresee an extraction with water.

The central point of this invention is the use of surfactants that allows to pseudo-solubilise lycopene in water phase and to extract with relevant yields. This direct use on vegetal matrixes is not described in the state of the art.

It is therefore an object of the present invention a process to obtain an aqueous extract containing carotenoids, in particular lycopene, comprising the following steps: a) mixing of plant material and, or fibrous plant byproducts containing carotenoids with an amount of water from 1 to 10 times the weight of said materials and, or by-products until a mixture is obtained; b) adding said mexture of an enzymatic catalytic system characterized by the fact that after or during step b) a surfactant is added in order to obtain an aqueous extract containing carotenoids.

According to the invention examples of carotenoids are lycopene and, or β-carotene and as plant materials and, or fibrous by-products are advantageously used whole

tomatoes and, or carrots and, or fibrous by-products of the agri-food transformation industries. Among fibrous by-products tomato skins, solid cellulosic residues derived from peeled tomatoes and puree productions can be cited. It is possible to use both fresh and cryoconserved skins, generally at temperature of - 2O ⁰ C, defrosted and processed within 24 hours from de-icing.

For special applications to suspend the skins it is also possible to use thermal water, that is to say spring water characterised by chemico-physical properties and peculiar inorganic salts due to the specific geochemical properties of the lands surrounding the spring.

It is important to point out that the invention give the possibility to add the surfactant both after and at the same time of the enzymatic system.

The enzymatic biocatalytic system, used in the step b) of the present invention at T 20 - 50 ⁰ C and pH 3 - 6 comprises enzymes chosen from the class formed by cellulases, pectinases, xylanases, amylases, proteases, cutinases and arylesterases . Advantageously enzymes having the following activities: pectinasic 100-1000 KU/L, xylanasic 160-1600 Kϋ/L, amylasic 100-1000 Kϋ/L and arylesterasic 360-3600KU/L are used.

In order to accelerate the reaction before than step b) it is possible to perform a thermal and, or mechanical treatment of the mixture referred to in step a) . As mechanical treatment homogenisation and whipping can be cited.

The surfactants used, acting in aqueous solution, are .part of the class of food-grade, natural and, or synthetic surfactants. As favourites surfactants can be cited surfactants belonging to the classe formed by polysorbates, lecitins, saponins, alkyl-glucosides, sodium dodecyl sulphate, cetylstearilic alcohols, betaines, imidazolines, sarcosinates, sulphosuccinates,

quaternium and polyquaternium. Advantageously, food grade surfactants are used, among these can be cited Tween

(polysorbates) classes or lecitins in particular soy lecitin. The use of food grade surfactants allow to directly use the extracts in sectors such as human food consumption permitting also not to consider the vegetal matter used for the extraction of carotenoids as hazardous wastes.

The aqueous extract undergoes a filtration process to separate particles larger than 100 μm, as the smaller particles of plant material and, or by-products still contain significant amounts of lycopene; the main uses of lycopene allow to use this extract. For specific industrial needs it can be advantageous that the extract obtained by the process according to the present invention undergoes a pasteurisation process, advantageously at T 90 ⁰ C for 20-200 minutes, and hot- packed and, or added with stabilizers chosen among those generally used in the relevant technological sector. The use of enzymatic catalytic systems gives several advantages. The enzymatic products, as well as the surfactants, are water soluble so the entire process can be performed in water phase. Furthermore, an extraction performed using non-hazardous enzymes and surfactants can be carried out by non specifically trained operators, i.e. in a farm, as no toxic-hazardous substance is used. The enzymes contribute to make the recovery of chemicals more efficient as, hydrolyzing in a selective way plant material, the enzymes promote the release and the pseudo-solubilisation of these chemicals.

According to the process of the invention, moreover, the overall cost of the lycopene extraction process is reduced if compared to similar processes. Furthermore in this way, the solid by-products of this process after contact only with water, enzymes (considered safe as

already used for food applications) and surfactants (food grade or natural) are not considered as special wastes. This allows both a disposal in landfill and their reuse of the production of bio-polymers or dried products used in agriculture as fertilizer or for animal consumption or as bio-fuels, allowing to recover part of the energy used during the productive processes.

The production of an aqueous extract rich in pseudo- solubilised lycopene, or kept in suspension in water phase thanks to surfactants and plant macromolecules of polysaccharidic and, or proteic origin, in water phase gives several advantages:

• The use of agri-food by-products as low cost raw materials for the production of functional drinks and, or "functionalising" additives does give the real opportunity to get, through appropriate processes, profits from wastes. The economic convenience of this technology and the large range of possible applications and products derived from this makes it possible that the presented process could be interesting and suitable also considering the easiness of scale up.

• The use of by-products as raw materials from one side allow to the waste produces to clear the cost, some time quite relevant, for detoxification and disposal, and to transform in a new opportunity to generate profit. From the side of the manufacturer there is the clear advantage of using a low or no cost raw material, so to reduce the cost of the new obtainable products and propose this to the market at a competitive price also considering its novelty.

• The agri-food by-products are generally used in fertiliser production, for animal consumption, to

produce biogas, in the biological depuration and thermo-valorisation processes. Just a small fraction of these by-products nowadays is valorised by the recovery of phyto-molecules through extraction processes.

• This new technology allows the potential zeroing of by-products solving the industrial problem related to disposal costs and, at the same time, generating profits. • The availability of aqueous extracts rich in lycopene, the end product formulators of several sectors (food, nutraceuticals and cosmetic) can insert an "active principle" fat-soluble, that usually needs to be disperse with complicated processes and using chemical dispersing molecules having strong limitations, substituting in an easy and effective way part of the formulation water with the product.

The extracts obtainable through the process of the invention can be used in nutraceutical, pharmaceutical and cosmetic sector.

In the nutraceuticals sector can be produced:

1. "Eat & drink" functional juices, reach in antioxidant molecules, soluble fibres. 2. Additives for functional fruit juices enriched with antioxidant molecules, soluble fibres.

3. Functionalised additivated yogurts, mousses, jellies .

4. Candies, desserts. 5. "Meal substitute" bars, functionalised anti-hunger products .

6. Functional biscuits, oven products and snacks. In the cosmetic sector the extracts can be used as additives in face body, anti-age, eye contour creams,

moisturizing, sun, after-sun cream, make-up products. Another use is in personal care products: soap, shampoos, shower gels, bath foams, conditioners etc.

In the pharmaceutical sector the extracts of the invention can be used to produce creams, ointments, lotions, emulsions, gels, operculum, medical devices, drops, eventually added to other pharmaceutical principles . Example 1 Production of an extract containing lycopene for food applications.

In order to produce an aqueous extract containing pseudo-solubilised lycopene from tomato skins for food applications the transformation of the raw material _. within 12 hours from the production is preferred. The skins are re-suspended in tap water or deionised water in a proportion equal to 2 folds the weight of skins to be treated. The water re-suspended skins are treated at a temperature of 75°C for 90 minutes. The pre-treated skins are cooled down to 3O ⁰ C and undergo a biocatalytic process for 4 hours at a constant temperature of 30 ⁰ C with food grade enzymatic products having cellulasic and pectinasic activities and at least one of the following other activities: xylanasic, amylasic, proteasic, cutinasic and aryl-esterasic.

After the biocatalytic treatment the hydrolysed skins are added with a food grade surfactant (i.e. Tween 20/60/80) in concentration of 5% of the skin weight at a temperature of 30 ⁰ C for 4 hour. The raw extract undergoes to several filtration treatments (using bag, cardboard and cartridge technologies) . The filtrate extract is added with suitable preservatives (potassium sorbate 0.3- 2 % and/or citric acid 0.1-5% w/v and/or lactic acid 0.1- 6%) and pasteurised at 9O ⁰ C for 30 minutes and hot-packed

in suitable containers (bag-in-boxes, tanks and cisterns among others) .

Typical data of a tomato aqueous extract for food applications : Dry weight: 30 g/kg pH: ^' 2.8 - 3.5

Total phenolics : > 100 mg/Kg Total sugars: > 6 g/Kg Lycopene : > 15 mg/Kg Example 2

Production of an extract containing lycopene for cosmetic applications.

In order to produce an aqueous extract containing pseudo-solubilised lycopene from tomato skins for cosmetic applications, fresh tomato skins or stored at - 20 ⁰ C and treated within 24 hours from the de-icing can be used. The skins are re-suspended in tap water or deionised water in a proportion equal to 5 folds the weight of skins to be treated. The water re-suspended skins undergo whipping processes and are treated at a temperature of 90 ⁰ C for 10 minutes. The pre-treated skins are cooled down to room temperature and undergo a biocatalytic process for 12 hours at room temperature with enzymatic products having cellulasic and pectinasic activities and at least one of the following other activities: xylanasic, amylasic, proteasic, cutinasic and aryl-esterasic.

After the biocatalytic treatment the hydrolysed skins are added with a cosmetic grade surfactant, i.e. sodium dodecyl sulfate in concentration of 1% of the skin weight at room temperature for 8 hour. The raw extract undergoes several filtration treatments (using bag, cardboard and cartridge technologies) . The filtrate extract is added with suitable stabilizers (m- parabenzoate 0.01-1 % w/v and/or citric acid 0.1-5% w/v

and/or EDTA 0.1-3% w/v and/or phenoxyethanol 2-5 % w/v) and packed in suitable containers (bag-in-boxes, tanks and cisterns among others) .

Typical data of an aqueous extract of tomatoes for cosmetic applications:

Dry residue: > 10 g/kg pH: 2.8 - 3.5

Total phenolics : > 50 mg/Kg Total sugars: > 3 g/Kg Lycopene: > 25 mg/Kg Example 3

Production of an extract containing lycopene for natural cosmetic.

In order to produce an aqueous extract containing pseudo-solubilised lycopene from tomato skins for cosmetic applications fresh tomato skins or stored at -20 ⁰ C and treated within 24 hours from the de-icing can be used. The skins are re-suspended in tap water or deionised water in a proportion equal to 3 times the weight of skins to be treated. The water re-suspended skins are treated at a temperature of 90 ⁰ C for 20 minutes. After the thermal treatment, the skins are added with a natural surfactant, such as natural saponins, natural alkyl glucosides or grounded Sapindus mukorossi nut in concentration of 3% of the skin weight at a temperature of 60 ⁰ C for 6 hour. The raw extract undergoes several filtration treatments (using bag, cardboard, cartridge and microfiltration technologies) . The filtrate extract is added with suitable preservatives (bitter orange extract 0.01-1 % w/v and/or citric acid 0.1-5% w/v lactic acid 0.1-6%w/v) and packed in suitable containers (bag-in-boxes, tanks and cisterns among others) .

Typical data of an aqueous extract of tomatoes for cosmetic applications: Dry residue: > 8 g/kg

pH : 2 . 8 - 3 . 5

Total phenolics : > 20 mg/Kg Total sugars: > 1 g/Kg Lycopene : > 10 mg/Kg Example 4

Production of an extract containing β-carotene for food applications.

In order to produce an aqueous extract containing pseudo-solubilised β-carotene from carrots for food applications the transformation of fresh raw material is preferred. The skins are re-suspended in tap water or deionised water in a proportion equal to 3 folds the weight of skins to be treated. The water re-suspended carrots are treated at a temperature of 85°C for 90 minutes. The pre-treated skins are cooled down to 30 ⁰ C and undergo a biocatalytic process for 4 hours at a constant temperature of 30 °C with food grade enzymatic products having cellulasic and amylasic activities and at least one of the following other activities: xylanasic, pectinasic, proteasic, cutinasic and aryl-esterasic.

After the biocatalytic treatment the hydrolysed carrots are added with a food grade surfactant (i.e. tween 20/60/80) in concentration of 3% of the skin weight at a temperature of 30 °C for 4 hour. The raw extract undergoes several filtration treatment (using bag, cardboard and cartridge technologies) . The filtrate extract is added with suitable preservatives (potassium sorbate 0.3-2 % and/or citric acid 0.1-5% w/v and/or lactic acid 0.1-6%) and pasteurised at 90 ⁰ C for 30 minutes and hot-packed in suitable containers (bag-in- boxes, tanks and cisterns among others) .

Typical data of an aqueous extract of carrots for food applications: Dry residue: > 25 g/kg pH: 2.8 - 3.5

Total phenolics : > 40 mg/Kg Total sugars: > 10 g/Kg β-carotene: > 10 mg/Kg