BioMatNet Item: FAIR-CT95-1075 - Ultrahydrophytosqualene: New Processes for the Generation of Squalene by Supercritical Fluid Extraction from Waste of Olive Oil Production and Hydrogenation of Squalene

FAIR-CT95-1075
Ultrahydrophytosqualene: New Processes for the Generation of Squalene by Supercritical Fluid Extraction from Waste of Olive Oil Production and Hydrogenation of Squalene

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Economics : Enviromental Aspects : FAIR Area 2.1 - Chemical and Physical Processes : Pharmaceuticals/Cosmetics : Process Engineering : Separation/Fractionation : Vegetable Oil/Fat

	Contract No:	FAIR-CT95-1075
	Date Prepared:	July 2001, February 1999
	Source:	Final Report Second Annual Progress Report

Final Report

Source: Final Report, 1999

Consortium: The project was co-ordinated by Muller Extract Company GmbH, Coburg (Germany), in partnership with Stazione Sperimentale per le Industrie degli Olie dei Grassi, Milano (Italy), Institudo de Biologia Experimental e. Tecnologica, Oeiras (Portugal), Fabrica Torrejana de Azeites S.A, Torres Novas (Portugal) and Hamburg Technologei GmbH, Technische Universitat Hamburg (Germany).

Abstract

Introduction

Squalene(C₃₀H₅₀) is a triterpene - compound with 6 unconjugated double bounds. This compound is a high value biological raw material which is used in large quantities as an intermediate for the manufacture of pharmaceuticals as well as in cosmetic formulations and health food products. Most of the squalene is hydrogenated to squalane (C₃₀H₆₂) which is the corresponding saturated compound. Squalane is also a high value biological material, that is mainly used in the formulation of cosmetics but is also used as a carrier of lipid soluble drugs.

In general pure (99 %) squalene is obtained from shark liver oil, especially from the livers of rare species of deep sea sharks. Squalane is obtained by traditional hydrogenation of squalene with Raney Nickel as catalyst. Squalene is also found in high concentrations in olive-oil residues (olive oil deodoriser distillates = ODD) after the last production step (deodorisation) and is regarded as a waste product of the refineries. Existing attempts, to obtain squalene from olive-oil residues by distillation methods have not resulted in producing pure squalene in economically viable quantities.

The aim of this project is to replace the traditional shark liver oil based production of squalene and squalane by a new process and a new prototype technology to generate these compounds from the otherwise worthless residues of olive-oil production. However, the process should be such that the quality of the cold pressed olive-oil (virgin oil) as well as the quality of the refined olive-oil should not be affected by the proposed process.

The starting material for squalene enrichment is the waste generated by the olive-oil industry. The objective of this project was to use supercritical fluid extraction with carbon dioxide to obtain a nearly pure squalene and to hydrogenate this squalene to squalane in supercritical carbon dioxide as solvent. The process development has been based on an existing extraction plant that has been extended to include a prototype plant for the economic production of squalene and squalane from olive oil residues.

The benefits of the project includes:

the protection of rare shark species and the production of a clean phytoproduct without solvent residues
no waste waters would arise as a result of the use of carbon dioxide and the avoidance of organic solvents
a waste product which could not be used before will be used in an environmentally friendly process to produce high valuable biological materials.
the phytoproduct should be better for health in comparison to animal products because the shark is the end of the food chain
by replacing the traditional hydrogenation with Raney-Nickel catalyst by an environmentally friendly process with other catalysts the end-product does contain traces of nickel that can lead to allergic reaction on the skin when the hydrogenated products are used in cosmetic formulations.
for those countries of the EC bordering the Mediterranean Sea, a process for the squalene/squalane enrichment from olive-oil residues could be of far reaching economical importance. All olive-oil residues are considered as waste products and have to be disposed of.
the environmental friendly treatment of these residues for the production of squalene and squalane could create a new market and new jobs in these countries.

The project has been carried out by SMEs and research organisations from Germany, Italy, and Portugal. Transfer of this technology is also planned for Greece and Spain.

Objectives and tasks

The objectives of the project were to:

obtain extracts enriched, as far as possible, in terms of the squalene content from olive oil residues
to develop an economical production process.

Three possible ways of achieving this using carbon dioxide as the extraction medium have been, tested intensively as part of the research activities. These are:

the direct enrichment of squalene from the olive oil waste by using high pressure counter-current extraction columns for carbon dioxide extraction.
the saponification of olive oil residues and the extraction of the solid soaps in a second step using supercritical carbon dioxide as solvent. This approach reflects the fact that the separation of squalene from methyl- and ethyl -esters and free fatty acids is very difficult. Together with squalene these compounds appear in olive oil residues in high quantities. By transferring the methyl- and ethylesters as well as the free fatty acids into soaps the separation from squalene is easier, since in contrast to squalene the soaps should be not soluble in carbon dioxide.
to transfer methyl- and ethylesters as well as free fatty acids into the corresponding trimethylol-propane-esters, because the separation of squalene from these compounds is much easier.

In addition to the use of carbon dioxide as the main option for squalene enrichment, other options were investigated using traditional solvents.

The project was based on a series of tasks.

The first task was to improve the olive oil waste which is obtained during the olive oil refining process. The starting material should have a squalene content as high as possible and a low level of isomerisation. However, the alternative refining process developed should not alter the quality of the olive oil which is obtained.
The second task was to develop methods to saponify a part of the improved starting materials.
The third tasks concerned the enrichment of extracts in squalene using the improved starting material. The improved olive oil residues have been used for squalene enrichment tests using various counter-current extraction columns with C0₂ as solvent. The corresponding soaps have been used for experiments concerning the extraction of the solid soaps in pilot scale and production scale by using supercritical carbon dioxide as solvent. The optimum extraction conditions, in terms of temperature, The third tasks concerned the enrichment of extracts in squalene using the improved starting material. The improved olive oil residues have been used for squalene enrichment tests using various counter-current extraction columns with CO₂ as solvent. The corresponding soa
Another objective of this project was the hydrogenation of squalene to squalane and the comparison of the hydrogenation in carbon dioxide with hydrogenation in traditional solvents. The corresponding task consisted of a series of basic experiments for the evaluation of a hydrogenation process in supercritical carbon dioxide in pilot scale. The experiments were carried out in pressure vessels of various height equipped with a sampling system to enable reaction velocities to be measured.
A further task was to test a possible improvement of the extraction and hydrogenation through the use of ultrasonics. It might be anticipated that the use of an ultrasonic generator inside the extraction vessel would increase the extraction yields. In the same way the use of ultrasonics inside the autoclave during hydrogenation should result in a more intensive mixing of the catalyst with the substrate, as compared to the use of a traditional magnetic stirrer.

All stages of the project were dependent on the availability of analytical results. Hence, other tasks covered the development of various analytical methods in order to characterise various starting materials, intermediate products, extracts and hydrogenated products which occurred during the tests and experiments.

Results

This project brings together several partners that are specialists in their own areas of knowledge, all of which are necessary and important for the development of phytosqualene and phytosqualane. By working together the partners obtained the following results representative of a successful project.

Optimised olive oil residues with a high squalene content of 35 % to 45 % and low isomerisation have been obtained. The quality of the refined olive oil is very high and not affected by the production of residues with a high squalene content. Soaps with a high (optimised) squalene content of 30%-35% and low isomerisation have been produced from the olive oil residues.

Phase equilibrium measurements and tests with the extraction columns show that the enrichment of squalene directly from olive oil residues with carbon dioxide as solvent in a high pressure counter-current extraction column is difficult. Therefore the olive oil residues have to be modified before squalene enrichment takes place. Two kind of modified olive oil residues have been produced. Saponified olive oil residues have been produced for a solid extraction process with carbon dioxide as solvent. With trimethylol propane esterified olive oil residues have been produced for an extraction process in a high pressure counter-current extraction column with carbon dioxide as solvent. The extraction of saponified olive oil residues has been optimised in terms of various extraction parameters.

The carbon dioxide extraction of saponified olive oil residues in industrial scale leads to an extract with 70%-78% squalene with a high recovery. The squalene can be extracted directly from the soaps. Mixing the soaps with an inert material as matrix like for other processes is not necessary. Two advantages result from this process. Money for inert material is saved and the soaps can be sold after the extraction, as a by- product.

Using a coupled supercritical fluid extraction (SFE)- supercritical fluid chromatography (SFC)- process it is possible to enrich squalene to a purity greater than 90 %, directly from saponified olive oil residues. Phytosqualene of approximately 80% purity has been produced by extraction of solid saponified olive oil residues with carbon dioxide and a further extraction process with carbon dioxide with a high pressure counter-current extraction column. It is also possible to enrich squalene to a purity in excess of 90 %, from esterified olive oil residues, with a high pressure counter-current extraction column in two steps.

The experiments investigating hydrogenation in carbon dioxide as solvent have been successfully. Reaction velocities have been measured. The hydrogenation in supercritical carbon dioxide as solvent is a little faster than hydrogenation with hexane as solvent. Various catalysts and catalyst concentrations have been tested.

The results form the basis for the hydrogenation of phytosqualene to phytosqualane in supercritical carbon dioxide as solvent in a larger scale and the construction of a prototype plant. In a coupled supercritical fluid extraction (SFE)- supercritical fluid chromatography (SFC)- supercritical fluid hydrogenation (SFH)- process it is possible to produce squalane with 80% -90 %, directly from saponified olive oil residues.

A prototype plant which connects the supercritical fluid extraction (SFE) - supercritical fluid chromatography (SFC) and supercritical fluid hydrogenation (SFH) has been constructed and the first samples of squalane at 80%-90% purity have been produced. Phase equilibrium measurements show that the solubility of squalene in the gas phase is strongly depending on the hydrogen concentration. This knowledge is important for processes control of the new prototype plant.

Experiments showed that the use of ultrasonic did not improve the extraction and hydrogenation process.

Various analytical methods for determination of the saponification value, acid value, moisture and volatile substances, free alkali and the determination of the squalene content with HPLC and GC have been developed and used for characterising those products (mainly oils) as required by the project.

Pre-enrichment tests with traditional solvents, such as the saponification of olive oil residues in a double solvent, hexane extraction of saponified olive oil residues and purification of extract in alcoholic alkali solutions have been carried out. These tests provided a lot of information that was used to optimise squalene enrichment from olive oil residues and offered the possibility of evaluating other methods.

The hexane extract, double solvent extract and the carbon dioxide extract have been characterised and the results show why a further squalene enrichment is so difficult. The extracts in general have more or less the same composition. Squalene is surrounded by slightly more volatile hydrocarbons and slightly less lower volatile more polar compounds such as fatty alcohols which have nearly the same solubility in carbon dioxide. Hence, these originally minor compounds became main compounds.

Exploitation

A patent application concerning squalene enrichment and squalane production has been filed. Some articles concerning basic studies have been published. One PhD-thesis which results from the project has been published in the middle of 2000. Phytosqualene-products and soaps have been produced. Samples will be offered to customers in order to evaluate the market.

Future development

The process for producing phytosqualene should be improved with regard to the profitability. The prototype plant for the production of phytosqualene and phytosqualane in supercritical carbon dioxide as solvent should be improved concerning the process control.

Second Annual Progress Report

Source: Abstract from the Progress Report for the period from 01/09/1997 to 31/08/1998

Introduction
Squalene (C₃₀H₅₀) is a triterpene – a compound with 6 unconjugated double bonds. This compound is a highly valuable biological raw material that is used in large quantities as an intermediate product for the manufacture of pharmaceuticals as well as in cosmetic formulations and health food products. Most of the squalene is hydrogenated to squalane (C₃₀H₆₂), which is the corresponding saturated compound. Squalane is a high value biological material mainly used in cosmetic formulations, but also as a carrier of lipid soluble drugs.

At present, 99% pure squalene is obtained from shark liver oil, especially from the livers of rare species of deep-sea sharks. Squalane is obtained by traditional hydrogenation of squalene with Raney-Nickel as the catalyst. Squalene is also found in high concentrations in olive oil residues after the last production step (deodorisation) and is regarded as a waste product of the refineries. Existing attempts to obtain squalene from olive oil residues by distillation methods have not resulted in producing pure squalene in economically viable quantities.

The aim of this project is to replace the traditional shark liver oil based production of squalene and squalane by a new process and a new prototype technology to produce them from the worthless residues of olive oil production.

The quality of the cold pressed olive oil (virgin oil) and also the quality of the refined olive oil should not be affected by the proposed process.

Starting material for squalene enrichment would be the waste left from the olive oil industry. The objective of this proposal is the implementation of the supercritical fluid extraction to obtain a nearly pure squalene and to hydrogenate squalene to squalane in supercritical carbon dioxide as solvent. With regard to the apparatus, an already existing extraction plant shall be extended to include a prototype plant for the economic production of squalene and squalane from olive oil residues.

Benefits of the project
The advantage of the new process is the protection of rare shark species and the production of a clean phytoproduct without solvent residues. By using carbon dioxide and avoiding organic solvents, wastewater will not be produced. A waste product that could not be used before is used in an environmentally friendly process to produce high valuable biological materials. The phytoproduct should be better for health in comparison to animal products because the shark is the end of the food chain. Replacing the traditional hydrogenation with Raney-Nickel catalyst by an environmentally friendly process, means that the end product does not contain traces of nickel that can lead to allergic reactions on the skin when squalane is used in cosmetics.

Applications
For those EU countries bordering the Mediterranean Sea, a process for the squalene resp. squalane enrichment from olive oil residues could be of far reaching economic importance. All olive oil residues are considered as waste products and have to be disposed of. The environmentally friendly treatment of these residues for the production of squalene and squalane could create a new economy and new jobs in these countries. The project is being carried out by SMEs and research organisations from Germany, Italy and Portugal. Transfer of this technology is also planned for Greece and Spain.

Objectives and the corresponding tasks
One objective of the project is to enrich squalene from olive oil residues at as high a level as possible with regard to the squalene content in the extracts, as well as the recovery of squalene to develop an economic production. Two main methods of extraction using carbon dioxide will be tested intensively during this project. The first is the direct enrichment of squalene from the olive oil waste by using high-pressure countercurrent extraction columns for carbon dioxide extraction. The second is squalene enrichment involving the saponification of olive oil residues and the extraction of the solid soaps in a second step by using supercritical carbon dioxide as solvent. Separation of squalene from methyl- and ethyl-esters and free fatty acids is very difficult. Together with squalene, olive oil residues contain these compounds in high quantities. By transferring the methyl- and ethyl-esters, as well as free fatty acids into soaps, it is assumed that a separation from squalene should be possible because, unlike squalene, the soaps should not be soluble in carbon dioxide. In addition to the main possibilities for squalene enrichment with carbon dioxide, other methods will be evaluated in fundamental experiments using of traditional solvents.

The first task is to improve the olive oil waste that is obtained during olive oil refining. The starting material should have a squalene content as high as possible and a low isomerisation. The improved process should not influence the quality of the olive oil obtained by the refining process. The second task is to saponify some of the improved starting materials. Further tasks concern the enrichment of squalene using the improved starting materials produced. These improved olive oil residues should be used for squalene enrichment tests with different countercurrent extraction columns with CO₂ as solvent and the corresponding soaps should be used for experiments concerning the extraction of the solid soaps in pilot scale and production scale using supercritical carbon dioxide as solvent. The optimum extraction parameters in temperature, pressure and flow velocity for the squalene enrichment will be identified in order to improve the process. A subtask concerning squalene enrichment using a high-pressure countercurrent extraction column involves phase equilibrium measurements in special high-pressure phase equilibrium apparatus.

Another objective of this project is the hydrogenation of squalene to squalane and the comparison of the hydrogenation in carbon dioxide with hydrogenation in traditional solvents. Several basic experiments for the evaluation of a hydrogenation process in supercritical carbon dioxide in pilot scale. The experiments have to be carried out in different high-pressure vessels equipped with a sample taking system in order to measure reaction velocities. Different parameters in regard to pressure, temperature, concentrations and catalysts have to be tested.

One further task is to test a possible improvement of the extraction and hydrogenation by the use of ultrasound. By using ultrasound inside the extraction vessel the extraction yields should be enforced. The used of ultrasound inside the autoclave during hydrogenation should result in a more intensive mixing of the catalyst when compared with use of a traditional magnetic stirrer.

Analytical studies are crucial to this scientific research. Analytical methods for characterisation of different starting materials, intermediate product, extracts and hydrogenated product that are produced during the tests and experiments will be developed.

Results at the end of Year 2
This project combines expertise of partners who are all specialists in their own field, each of which is necessary and plays an important role in the development of phytosqualene and phytosqualane. Good results have been obtained so far due to all partners working together.

Optimised olive oil residues with a high squalene content of 35% to 45% and low isomerisation have been obtained. The quality of the refined olive oil is very high and is not influenced by producing a high squalene content in the residues. Optimised soaps with a high squalene content of 30%-35% and low isomerisation have been produced from the olive oil residues.

Physical extraction parameters have been optimised for the extraction of saponified olive oil residues. The carbon dioxide extraction of saponified olive oil residues at industrial scale has resulted in high recovery of an extract containing 70%-75% squalene. The squalene can be extracted directly from the soaps, unlike many other processes, where it is necessary to mix the soaps with an inert material as a matrix. Thus, the additional cost of inert material is avoided and the soaps can be sold as a by-product after the extraction.

Phase equilibrium measurements and tests with the extraction columns show that the enrichment of squalene directly from olive oil residues with carbon dioxide as solvent in a high-pressure countercurrent extraction column is difficult. However, phytosqualene of approximately 80% is available by extraction of solid saponified olive oil residues with carbon dioxide and a further extraction process with carbon dioxide with the high pressure countercurrent extraction column.

The hydrogenation experiments using carbon dioxide as solvent have been successful and reaction velocities have been measured. Hydrogenation using supercritical carbon dioxide as solvent is a little faster than hydrogenation with hexane as solvent. Different catalysts and catalyst concentrations have been tested. The results are a promising base for the hydrogenation of phytosqualene to phytosqualane using supercritical carbon dioxide as solvent at a larger scale. Phase equilibrium measurements show that the solubility of squalene in the gas phase is strongly dependent on the hydrogen concentration. This knowledge is important in carrying out hydrogenation processes using supercritical carbon dioxide as solvent at a larger scale.

Different analytical methods relating to the saponification value, acid value, moisture and volatile substances, free alkali and determination of the squalene content with HPLC and GC have been developed for characterising products (mainly oils) of this project.

Pre-enrichment tests with traditional solvents, such as saponification in a double solvent, pre-esterification and hexane extraction of olive oil residues, have been carried out. Information from these tests was used to optimise the squalene enrichment from olive oil residues and could be used to evaluate other methods. The hexane extract, double solvent extract and carbon dioxide extract with 75% squalene have been characterised and the results show why a further squalene enrichment is so difficult. The extracts have nearly the same compositions. Squalene is surrounded by slightly more and slightly less volatile compounds that have similar solubility in carbon dioxide. These, originally minor, compounds become main compounds.

Publication and exploitation of the results
A patent application is planned towards the end of the project. Some publications are planned after the patent application has been filed. Phytosqualene products and soaps have been produced. Samples should be offered to customers in order to check the market.

Visions
Samples with higher than 80% squalene content should be developed and offered to customers. The process for producing phytosqualene should be improved in regard to the profitability. The hydrogenation of phytosqualene to phytosqualane in supercritical carbon dioxide as solvent should be possible at a larger scale.