Patent Description:
Cutin is a natural cross-linked polyester, mainly formed by condensed polyhydroxy acids. Cutin is located in the cuticle of the plants, of which it is the main component (<NUM>-<NUM>%, w/w). The cuticle is a hydrophobic layer deposited exclusively on the outside of the epidermis cells. The primary role attributed to the plant cuticles is to avoid the water loss from internal tissues; moreover, the cuticle also has a function of gas barrier and thermal regulation, as a defence against pathogens, reduction of nutrients leaching and protection from mechanical injury and UV radiation damage.

The by-products of tomato processing are a rich source of cutin and their recovery is considered important from an economic and environmental point of view. The tomatoes world production is about <NUM> million tons per year, mainly concentrated in the northern hemisphere, of which about <NUM> million tons are processed into different products, such as tomato purée, diced tomato, tomato paste, peeled tomatoes and tomato skins. The waste produced, peels and seeds, every year, at an industrial level, are significant: about <NUM> million tons, of which <NUM>,<NUM> tonnes in Europe alone.

Chemically, cutin is defined as a polymeric network of C16 and C18 polyhydroxylatedfatty acids cross-linked by ester bonds (<NPL>). Many of them are exclusively C16 fatty acids belonging to the family in which <NUM>,<NUM>-dihydroxyhexadecanoic acid, and its positional isomer <NUM>,<NUM>-dihydroxyhexadecanoic acid, are the main components of cutin. Only a small fraction of cutin is made up of fatty acids belonging to the C18 family, the most abundant components of which are <NUM>,<NUM>-epoxy-<NUM>-hydroxyoctadecanoic and <NUM>,<NUM>,<NUM>-trihydroxyoctadecanoic acids, although some derivatives may be present in the form of unsaturated cutin (<NPL>).

Cutin is generally extracted from plant material following isolation of the cuticle. Enzymes such as pectinases and cellulases in acetate buffer are usually used for cuticle isolation. Isolated plants cuticles are dewaxed by immersion in organic solvents, and polysaccharides are removed by acid hydrolysis. However, the degradation efficiency of polyesters by cutinases is too low for industrial applications.

In a recent paper, a structure-based protein engineering strategy was successfully used to improve the hydrolysis capacity of a T. fusca cutinase (<NPL>).

In the enzymatic method, the cuticular membrane is isolated by means of enzymatic treatment to degrade the polysaccharides. The cuticle is then extracted (solid-liquid extraction using a Soxhlet extractor with the solvent CHCl<NUM> and MeOH) to remove soluble material, waxes and other small molecules, obtaining cutin-enriched residues (<NPL>; <NPL>).

Only dewaxed and sugar-free peels can be obtained with this method, but not cutin. This method lacks a procedure to isolate cutin from the dewaxed peels. Furthermore, this method is expensive in terms of solvent use; it should also be considered that Soxhlet extraction uses toxic organic solvents, such as chloroform (carcinogenic) and methanol, and this extraction must be repeated to increase extraction yields. Thus, the procedure is not particularly environmentally friendly.

Cutin can be depolymerized by cleavage of the ester bonds by means of alkaline hydrolysis, using NaOH or KOH in water, transesterification in methanol containing BF<NUM> or NaOCH<NUM>, reductive cleavage by treatment with LiAlH<NUM> in THF, or by TMSI in organic solvents. These methodologies are unsuitable for large-scale cutin extraction due to the many steps involved and the high costs of solvents and reagents.

Another solvent used to extract cutin is acetone, as reported in the <CIT>, in which cutin is isolated by extraction with acetone in a Soxhlet apparatus and then with a reflux system in a basic solution. This method was tested; however, the final extract did not have the usual cutin appearance. As proof of this, the IR spectrum carried out on the product did not show the usual appearance of cutin spectrum. Furthermore, this method is expensive both in terms of solvent used and time.

Finally, another possibility reported in the literature to extract cutin is by acid hydrolysis (<NPL>). In this method, cutin samples were obtained following hydrolysis of the dewaxed cuticles in a <NUM> HCl solution for <NUM> at <NUM> to remove the hydrolyzable polar components and subsequently depolymerized in a <NUM>% (w/v) sodium methoxide solution for <NUM> at <NUM>. After the extraction of the tomato cutin monomers in an organic phase (diethyl ether), the solvent was evaporated in order to quantify and identify the cutin monomers by gas chromatography-mass spectrometry. This method was tested, but the yield was very low, and the extract showed little ability to form a new bio-lacquer.

The international patent application <CIT> describes an extraction process that provides a cutin with a high degree of purity; however, the process, which uses organic solvents and high temperatures and pressures, is particularly complex and expensive.

The international patent application <CIT> describes a method for preparing a polymer of the elastomer type based on a monomer selected from polyhydroxylated fatty acids and the esters of a polyhydroxylated fatty acid and a C1-C18 aliphatic chain alcohol. The method comprises preparing a reaction medium by mixing the monomer with a polyol, this reaction medium being catalyst free, then a step of heating the reaction medium in order to implement the copolymerisation of the monomer and of the polyol and the cross-linking of the polymer thus formed, with at least the initial phase of this heating state being implemented at a reduced pressure.

As highlighted above, the methods known to state of the art are mainly based on extraction processes with organic solvents, which are known to make more hazardous and expensive the treatment of the waste.

The enzymatic processes, although suitable for the purpose, have a degradation efficiency that is too low, which makes them not economically advantageous and of low interest for industrial applications.

In cases where the acid hydrolysis was used, followed by depolymerization and extraction in the organic phase, the product obtained was found to have a low yield and a poor ability to form effective bio-lacquers/bio-resins.

The European Patent No. <CIT>) describes a method for extracting cutin from tomato peels that does not use organic solvents. The method, in particular, includes the steps of.

The solid residue obtained is a cutin extract characterised by:.

The method under patent <CIT> uses only tomato peels, which sometimes contain contaminants resulting from farming or tomato processing.

In addition, the use of HCl makes the extraction method known to the state of the art unsustainable due to the chloride salts formed during the acidification phase: for this reason, the liquid residue cannot be discharged into surface waters or biogas plants.

In this context, the technical task at the basis of the present invention is to provide an improved cutin extraction process compared to the one described in the patent <CIT>.

The first object of the present invention is, therefore, a process for the extraction of cutin from tomato waste including the steps of:.

the precipitate obtained being a cutin extract having a <NUM>, <NUM>-dihydroxyhexadecanoic acid purity between <NUM>% and <NUM>% by weight of the total weight of the extract (w/w). Further objects of the present invention are:.

The process of the invention allows the extraction of cutin from tomato processing waste and its reuse in further formulations.

In particular, the process according to the invention provides for the use, in addition to the tomato peels, of generic waste or by-products from the processing of fresh tomatoes from preserved industries. The use of waste or by-products from the processing of tomatoes from organic cultivation also reduces the risk of the presence of contaminants in the peels.

The process of the invention eliminates the use of HCl, replacing it with organic acids. The use of organic acids makes it possible to reduce the risks of toxicity during the processing steps; to reduce the risks of environmental contamination; to reduce the contamination by sodium chloride of the wastewaters used during the process. Sodium chloride is difficult to eliminate, and it is subject to strict environmental legislation.

In this sense, the described cutin extraction process allows not only the valorisation of these tomato processing waste, but also the recycling of the reaction products.

Compared to the process described in <CIT>, the method according to the present invention allows to reduce the processing time, having the Applicants identified suitable temperatures and heating times to speed up the depolymerization phase in alkaline solution without causing degradation of the cutin contained therein.

Still advantageously, the described cutin extraction process allows the production of a bio-resin, starting from tomato processing by-products, with potential application in the preparation of lacquers, coatings, water-based paints, and cosmetic formulations.

Also advantageously, the process for the extraction of cutin allows the use of this bio-resin for the production of multilayer articles preferably for wrapping or packaging, with a consequent reduction in the purchase costs of the raw materials for the production of such articles.

Finally, the process makes it possible to obtain exhausted tomato peel waste free of cutin, which finds application as a raw material for producing food supplements or additives, or as a substrate for extracting carotenoids/lycopene.

As mentioned above, the object of the present invention is a process for the extraction of cutin.

In a step A of the extraction process, waste or by-products obtained from tomato processing are prepared.

Preferably, by tomato waste or by-products is meant the material from fresh tomato processing, selected from the group consisting of peels, seeds, leaf by-products, green tomatoes, peel residues and mixtures of the previous. Preferably, tomato waste or by-products are peels or combinations of peels and seeds.

In accordance with a preferred embodiment, the waste or by-products of tomatoes are tomato peels. It should be noted that different types of peels can be used: peels coming directly from tomato processing, dried peels, frozen peels and peels from which lycopene has been extracted.

According to a preferred embodiment, the tomato waste or by-products preparation step (A) comprises the following sub-steps:.

According to a preferred embodiment, the seeds separated from the peels are intended for the biogas production, animal feed or used for the extraction of oil and proteins.

Preferably, the water used for flotation is recirculated through a vertical centrifuge to remove residual tomato pulp and to clean the water; in this way, the water can be used again for the same purposes, thus minimizing its consumption.

The pulp removed by centrifugation is preferably used for the biogas production.

The tomato waste obtained from the preparation step (A) are immersed in an alkaline solution to obtain an alkaline mixture with a pH > <NUM>. Thealkaline mixture comprises a liquid phase and a dispersed solid residue.

Preferably the alkaline mixture has a pH ≥ <NUM> and ≤ <NUM>, preferably ≥ <NUM> and ≤ <NUM>, preferably equal to about <NUM>.

According to a preferred embodiment, the alkaline solution is a solution comprising one or more basic substance chosen from the group consisting of: NaOH, KOH.

Preferably, the basic substance is NaOH.

According to a preferred embodiment, the alkaline solution has a concentration between <NUM> and <NUM>, preferably between <NUM> and <NUM>, preferably between <NUM> and <NUM>, preferably between <NUM> and <NUM>, preferably equal to about <NUM>.

According to a preferred embodiment, the combination ratio (weight/volume) of the tomato waste or by-products (weight) with the alkaline solution is between <NUM> and <NUM>.

The alkaline mixture obtained from the immersion step (B) is heat treated at a temperature preferably between <NUM> and <NUM>, preferably between <NUM> and <NUM>, preferably between <NUM> and <NUM>, preferably equal to about <NUM>.

According to a preferred embodiment, the heat treatment is carried out for a period of time preferably between <NUM> minutes and <NUM> hours, preferably between <NUM> minutes and <NUM> hours, preferably between <NUM> and <NUM>, preferably between <NUM> minutes and <NUM>, preferably equal to about <NUM> minutes.

The term "isolation" refers to the separation of one or more phases from a suspension. Suitable techniques, for the purposes of the invention, to carry out the isolation step are those known to the person skilled in the art, preferably chosen between filtration and centrifugation.

Preferably, centrifugation is performed by means of a horizontal centrifuge, decanter.

The liquid phase is an alkaline solution of cutin. Note that the liquid phase has a brown or yellow colour and does not contain fine solid particles.

The solid residue is exhausted tomato peel waste, free of cutin.

Preferably, the exhausted tomato peel waste is characterised by a solid content of between <NUM>% and <NUM>% (w/w).

For the purposes of the present invention, the term exhausted tomato peel waste is understood to mean a tomato peel waste which is no longer suitable for re-entry in the preparation of tomato peel waste step (A) according to the process of the present invention. Cutin-free peel waste means a peel waste having, when dried, a cutin content < <NUM>%, preferably between <NUM>% and <NUM>% by weight on the total weight of the exhausted peel waste.

It should be noted that, preferably, the peel waste, when wet, is characterized by a cutin content between <NUM> and <NUM>% (w/w).

The liquid phase obtained from the isolation step (D) consists of an alkaline solution of cutin.

Acidification is preferably carried out until a pH preferably comprised between <NUM> and <NUM>, preferably between <NUM> and <NUM>, preferably between <NUM> and <NUM>.

The acidified cutin suspension comprises a liquid phase and a precipitate.

Preferably, the organic acid is chosen from the group consisting of: acetic acid, citric acid, formic acid, oxalic acid, succinic acid, glutaric acid, maleic acid.

Preferably, the organic acid is citric acid or acetic acid.

More preferably, the organic acid is added in the form of an aqueous solution, said aqueous solution preferably having a concentration between <NUM> to <NUM>, preferably equal to about <NUM>.

The organic acid is preferably added to the alkaline solution at a concentration between <NUM>% and <NUM>% by volume on the total volume of the solution (v/v), preferably <NUM>%.

The organic acid is preferably added to the alkaline solution until a colour change of the solution from brown to ochre is observed.

All the acids considered give an IR spectrum of cutin in which the main peak is around <NUM>-<NUM>-<NUM> and there is an area of the spectrum for values below <NUM>-<NUM> sufficiently clean.

The isolation step (F) yields a cutin extract characterised by <NUM>,<NUM>-dihydroxyhexaenoic acid purity between <NUM>% and <NUM>%, preferably about <NUM>% by weight on the total weight of the extract (w/w).

Preferably, the cutin extract obtained from the isolation step (F) has a water content between <NUM>% and <NUM>% by volume on the total volume of the extract (v/v).

Preferably, the cutin extract obtained from the isolation step (F) has a solid content between <NUM>% and <NUM>% by weight, on the total weight of the extract (w/w).

More preferably, the cutin extract obtained from the isolation step (F) has a sugar content between about <NUM>% and <NUM>% by weight on the total weight of the extract, preferably between about <NUM>% and <NUM>% (w/w), preferably between about <NUM>% and <NUM>% (w/w).

According to a preferred embodiment, the cutin extract obtained from the isolation step (F) has a fat content between about <NUM>% and <NUM>%, preferably between about <NUM>% and <NUM>% by weight on the total weight of the extract (w/w).

Still preferably, the cutin extract obtained from the isolation step (F) has a lycopene content between about <NUM>% and <NUM>%, preferably equal to about <NUM>% by weight on the total weight of the extract (w/w).

It should be noted that the extraction process, according to the invention, allows the cutin extract to be obtained with extraction yields, relative to the initial by-products, comprised between <NUM>% and <NUM>%, preferably between <NUM>% and <NUM>%, preferably between <NUM>% and <NUM>%, preferably equal to about <NUM>%.

Preferably, the isolation step (F) is performed by horizontal decanter centrifugation, preferably at a rotational speed between <NUM>-<NUM> rpm, preferably at <NUM> rpm.

The use of the horizontal decanter centrifuge advantageously allows to conduct the isolation phase (F) continuously, preferably obtaining an extract quantity of about <NUM>/hour, corresponding to about <NUM>/hour, corresponding to about <NUM>/min.

In accordance with a preferred embodiment, the liquid phase obtained from the isolation step (F) is re-entered into the immersion step (B) as an alkaline suspension of cutin, after alkalinization to a pH > <NUM>, preferably pH ≥ <NUM> and ≤ <NUM>, preferably ≥ <NUM> and ≤ <NUM>, preferably equal to about <NUM>.

Alternatively, the liquid phase obtained from the isolation step (F) is discarded.

According to a preferred embodiment, the step of washing with water provides that the precipitate (volume) and water (volume) are combined in a volume/volume ratio between <NUM> and <NUM>. The washing step is useful to remove any salts deriving from the acidification step.

According to a preferred embodiment, the process according to the present invention includes, downstream of the isolation step (F), a dehydration step (G), which is carried out in a way chosen from: thermal evaporation, lyophilisation, or application of infrared radiation.

The cutin extract obtained from the dehydration step (G) preferably has a water content < <NUM>%.

A further object of the invention is the exhausted tomato peel waste according to the present invention, free of cutin, having a composition comprising or consisting of:.

According to a preferred embodiment, the exhausted waste can be used as.

Note that the cutin-free exhausted waste is that obtained from the isolation step (D) according to the process of the present invention.

A further object of the present invention is a bio-resin comprising at least the cutin extract obtained by means of the process of the present invention and an organic solvent.

Preferably, the bio-resin comprises or consists of:.

The organic solvent is preferably chosen from the group consisting of butyl glycol, <NUM>,<NUM>- propanediol, ethanolamine, diethylamine and ethanol. Still preferably, the organic solvent is butyl glycol or <NUM>,<NUM>-propanediol.

A further object of the present invention is a multilayer article comprising:.

The first layer of the multilayer article, according to the present invention, is preferably an outer layer of the article.

The second layer of the multilayer article, in accordance with the present invention, is preferably an inner layer of the article.

In particular, the inner layer is the one intended for contact with the products or foodstuffs contained therein.

The advantage of multilayer articles thus produced is that the bio-resin, comprising a cutin extract that has been obtained without the use of organic solvents or hydrochloric acid, does not cause toxicity or contamination phenomena of the food with which the bio-resin comes into contact.

The articles, in fact, are preferably food packages or wrappings.

Preferably, the multilayer article according to the present invention is chosen from the group consisting of: can, tray, bag, pouche, jar, tube, pot, cassette, bottle and drum.

A further object of the present invention is the use of the bio-resin according to the present invention as:.

Claim 1:
Process for the extraction of cutin from tomato waste comprising the following steps:
A. Preparing waste or by-products obtained from tomato processing;
B. Immersing the tomato waste in an alkaline solution to obtain an alkaline mixture having a pH > <NUM>, the mixture comprising a liquid phase and a dispersed solid residue;
C. Subjecting the alkaline mixture to a heat treatment, at a temperature > <NUM> and ≤ <NUM>, for a time period ≥ <NUM> minutes and < <NUM> hours;
D. Isolating the liquid phase of the alkaline mixture subjected to heat treatment from the solid residue, the liquid phase being a cutin alkaline solution, the solid residue being an exhausted waste of tomato peels free of cutin;
E. Acidifying the alkaline solution of cutin by adding an organic acid, so as to obtain an acidified suspension of cutin having a pH < <NUM>, the acidified suspension of cutin comprising a liquid phase and a precipitate;
F. Isolating the precipitate of the acidified suspension of cutin from the liquid phase by decanter horizontal centrifuge and washing the precipitate with water,
the precipitate obtained being a cutin extract having a <NUM>, <NUM>-dihydroxyhexadecanoic acid purity between <NUM>% and <NUM>% by weight on the total weight of the extract (w/w).