WO2008142175A1

WO2008142175A1 - Method for obtaining squalene

Info

Publication number: WO2008142175A1
Application number: PCT/ES2008/000083
Authority: WO
Inventors: Manuel Aguado Ramos; Antonio Martin Martin; Arturo Chica Perez; Maria Angeles Martin Santos; Mónica BERRIOS CABALLERO; Jose Angeles Siles Lopez
Original assignee: Universidad De Cordoba; Pradomudo, S.L.
Priority date: 2007-05-18
Filing date: 2008-02-14
Publication date: 2008-11-27
Also published as: ES2308924A1; ES2308924B1

Abstract

Method for obtaining squalene from distillates from the physical refining of olive oil. The method comprises performing an alkaline saponification of the raw material in the presence of a hydrotrope and a high-vacuum molecular distillation of the unsaponifiable fraction.

Description

PROCEDURE FOR OBTAINING SCALES

The present invention falls within the chemical and agri-food sector, more specifically the invention relates to a process for obtaining squalene from distillates of physical refining of olive oil.

STATE OF PREVIOUS LATÉCNICA 0 Squalane is spinacene (2,6,10,15,19,23-hexamethyl- 2,6,10,14,16,18,20-tetracosahexane), an aliphatic hydrocarbon of formula (I)

5 (I)

It is a natural raw material found in human sebum (5%) and in shark liver oil as the main source of animal origin. It is also found in vegetable oils especially in olive oil.

It is a product of great interest in biochemical and pharmaceutical research since it is the natural precursor of cholesterol biosynthesis in vivo. In addition, it is a product of interest in the cosmetic and medical sector where it is mainly used5 as a vehicle for active ingredients.

Squalene is a precursor of cholesterol and this in turn of the hormone Dehydroepiandrosterone (DHEA), which gives an important role in restoring the normal values of this hormone, which with age or significantly decrease in humans. This hormone is attributed with properties such as the improvement of neurological performance, concentration, memory and the decrease in fat accumulation at the abdominal level. Squalene also has antioxidant properties and the ability to repolarize cell membranes, controlling their repair. 5

In the preparation of squalene, various sources of raw material have been used, such as shark liver oil, rice bran, germ of wheat or olive, olive oil, etc.

Olive oil is made up of a saponifiable fraction, which represents more than 98% of the oil and an unsaponifiable fraction constituted between 1.5% and 0.5%. Among the constituents of the saponifiable fraction are triglycerides, diglycerides, terpenic alcohols and free fatty acids. The constituents of the unsaponifiable fraction are sterols, terpene dialcohols, triterpenic alcohols, aliphatic alcohols, tocopherols, steroidal hydrocarbons, terpenic hydrocarbons (squalene), phenolic and flavonoid compounds, pigments, volatile compounds responsible for the aromas of the oils.

In olive oils, two terpene hydrocarbons have been found in considerable quantities, squalene and β-carotene. Squalene is the main constituent of the unsaponifiable fraction and can reach up to 40% of the total weight.

Patent ES2162742 describes a process for obtaining squalene from the by-product obtained from the physical refining (neutralizing distillation / deodorization) of olive oil, which consists in extracting the physical refining fatty acids, with water, an apolar solvent. and another polar, to obtain two miscelas: one polar and one non-polar. Then the polar miscela is decomposed to produce two miscelas: one of alcohol / water and the other of alcohol / polar solvent / fatty acids. Then, the solvents of the said miscelas are recovered to obtain a squalene concentrate from the non-polar miscellany and a fractional distillation of the squalene concentrate thus obtained is carried out, to obtain three fractions: one light, one squalene and another heavy Finally, the fraction rich in squalene is cooled for purification.

Patent ES2186380 describes a process for obtaining phytosqualane from a residue from the process of obtaining various vegetable oils. The residue is subjected to an esterification step of the fatty acids it presents, to a distillation and is hydrogenated. Finally it is treated with various reagents, washed and deodorized to obtain high purity phytosqualane. Patent ES8602102 refers to a process for obtaining squalene and squalene from by-products of physical refining and / or deodorization of vegetable oils. The described procedure consists in subjecting the by-products of the physical refining and / or deodorization of olive and pomace vegetable oils, previously catalytically hydrogenated, to an alkaline saponification followed by a continuous extraction with solvents of squalene and / or squalane from the mass of soap. 0 In the US patent application US2004015033 the same raw material is used as in the aforementioned patents, differentiating the procedure in which an extraction is carried out under supercritical conditions. In the patent application WO 9426683 a procedure for obtaining squalene from olive oil residues is described, where the process comprises a stage of saponification of the raw material, followed by the esterification of the fatty acids it presents and finally an extraction with super and / or subcritical gases, until obtaining a or concentrate concentrated in squalene

Patent ES2238183 describes a process for obtaining compounds of high added value from the olive leaf. In a first stage, the sheets are subjected to solid-liquid extraction with organic solvents, preferably hexane or ethanol, and the crude extract obtained is concentrated in vacuo. In a second stage a fractionation of the crude extract is performed by countercurrent extraction in column with supercritical carbon dioxide. This procedure allows to extract from the olive leaf natural products such as waxes, squalene, oleuropein, hydroxytyrosol and other phenolic compounds and terpenic alcohols.

In the article by Serra Masiá, A. et al., Fats and Oils, VoI. 32 (5): 313-317 (1981), describes the separation and obtaining of squalene from the oleins from the physical refining of olive oil. This article5 describes three different processes:

(a) Separation by thiourea complexes. Thiourea forms addition complexes with squalene, but according to Angla (B. Angla, Ann. Chim., (12) 4, 667, 1949) structures with double bonds have an unfavorable effect on the addition with thiourea. The relative stability of these complexes together with the change of scale makes it unfeasible as an industrial process;

(b) Separation by fractional distillation. Fractional distillation gives neither the purities nor the yields commented on at the laboratory scale.

In addition, as a technique it is not suitable for such high purities, there are many fractions that are carried along with the squalene. In the article by Serra Masiá in this section, it is said that the laboratory-scale experiences were not encouraging, in terms of operating conditions, they were very difficult to achieve and stabilize. Consequently, good separations were not achieved;

(c) Separation by saponification in the presence of a hydrotrope. In this procedure, squalene is separated in the unsaponifiable fraction in the presence of other compounds, which makes its isolation more complex. In addition, strong mineral acids such as sulfuric acid are used for the fluidization of the gels formed during the saponification process, which is undesirable due to its corrosive effect and highly dangerous in its handling.

According to the foregoing, it would be desirable to have a process for obtaining squalene that minimizes the above inconveniences and allows squalene to be obtained with an adequate degree of purity.

EXPLANATION OF THE INVENTION

The invention provides a process for obtaining squalene, from distillates of physical refining of olive oil.

According to a first aspect of the invention, this provides a process for obtaining squalene from the distillation of physical refining of olive oil as a raw material, characterized in that it comprises the steps of: a) alkaline saponification of the raw material: b) physical separation of the saponifiable and unsaponifiable fraction; and c) molecular distillation of the unsaponifiable fraction.

In the saponification process of the present invention, it is preferred the use of reaction times between 1 and 6 hours, with a time between 1 and 3 hours being more preferred.

In accordance with an embodiment of the process of the invention, an excess of base is used in the alkaline saponification stage with respect to the saponification index of the oil used. Preferably, oils with a saponification index of between 175 and 240 mg base / g fat are used. More preferred are oils with a saponification index of between 180 and 210 mg base / g fat.

According to a preferred embodiment, the raw material used to obtain squalene is the oleins from the physical refining of olive oil. Preferably, said oleins have the analytical characteristics indicated in Table 1.

Table 1. Analytical characteristics of the oleins used as raw material.

According to another preferred embodiment of the invention, a base weight / fat ratio of between 0.2 and 1 is used, 0.5 being more preferred. Regarding the weight / total water / fat ratio, preferably said ratio is between 1 and 12, with 5 being more preferred.

In the process of the present invention, preferably alkaline saponification is carried out using a base selected from the group consisting of NaOH, KOH, Ca (OH) ₂ , Mg (OH) ₂ . The use of KOH being particularly preferred.

According to a particular embodiment of the process of the invention, the saponification process is carried out at a temperature between 40 and 100 ⁰ C. Preferably the temperature is between 70 and 90 ⁰ C. In accordance with a preferred embodiment of the invention, the saponification stage (a) is performed in the presence of a hydrotrope.

Hydrotropy is the property that various compounds (alkaline and alkaline earth salts of organic acids) possess in whose concentrated aqueous solutions certain organic compounds that are otherwise insoluble or hardly soluble (for example proteins, carbohydrates, aniline, nitrobenzene) are well solubilized. ). The hydrotropic compounds reduce the surface tension of the water, thus facilitating the dispersion of the substances to be dissolved. The dissolved substance can be separated by hydrotropic dilution. Hydrotropic compounds find a wide scope of application in the chemical industry as solvents. They are fireproof, harmless and easily regenerable.

Preferably, the hydrotrope is selected from the group consisting of sodium xylenesulfonate, ammonium xylenesulfonate, metaxylenesulfonate, ethylbenzene sulfonate, urea, sodium eumene sulfonate, ammonium sulfonate, paratoluen sulfonic acid and sodium toluene sulfonate. Particularly preferred is sodium xylenesulfonate, metaxylenesulfonate and ethylbenzene sulfonate. Particularly preferred is the use of a 45% solution of sodium xylenesulfonate (w / w) in water.

Any suitable method for the physical separation of the saponifiable and unsaponifiable fractions can be employed. Preferably, said separation can be carried out by centrifugation.

In accordance with a preferred embodiment of the process of the present invention, the percentage of squalene wealth in the unsaponifiable fraction after stage b) is at least 75%; while the percentage of squalene richness after the molecular distillation process is at least 90%, preferably more than 95%, and particularly preferred more than 98%.

In accordance with the preferred embodiment, successive liquid-liquid extractions of the fat-soluble impurities present in the unsaponifiable are performed, which are preferably carried out with water in a 1/1 ratio (VA /) regarding the unsaponifiable.

According to a preferred embodiment, after the physical separation of the saponifiable and unsaponifiable fraction, a stage of purification of the unsaponifiable fraction is carried out by the use of adsorbent materials for the elimination of undesirable materials. Preferably, said adsorbent material is an adsorbent clay or non-activated earth (<10% by weight). Preferably said purification is carried out under vacuum at a temperature between 40 and 100 ⁰ C.

The process of the invention can be carried out in accordance with the following general description. The numbering indicated refers to the one included in Figure 1.

The oleins (2) together with the base (4), the hydrotrope (3) and the water (5), are added to the reactor (1) and once the saponification process is finished, the saponifiable fraction (7A) and unsaponifiable fraction are separated (6A). The saponifiable fraction is centrifuged to recover the unsaponifiable 6B occluded therein, the majority of the squalene present in the starting sample remaining in the unsaponifiable fraction (6A + 6B). The unsaponifiable fraction is centrifuged (8) to remove the remains of soap (9) it contains and then it is subjected to successive liquid-liquid extractions, in a discontinuous full-mix extractor (10), with water (11) to remove water-soluble impurities (12). Between consecutive washes the sample is centrifuged again (13) to remove the aqueous remains it contains (14). In this way the fraction of unsaponifiable free of water-soluble substances is obtained. The unsaponifiable fraction enriched in squalene is subjected to an adsorption purification process, preferably on an adsorbent clay (15) to remove undesirable substances (16). Next, the molecular distillation process (17) is carried out.

With the molecular distillation process, fractions with a squalene richness between 95 and 99% by weight (18 and 19) are obtained. The determination of the percentage by weight of the squalene present in the fractions obtained has been determined by chromatographic techniques (20). A method of determining the percentage of squalene applicable is by means of the gas chromatography, using the technique proposed by the European Union for the joint determination of squalene and waxes, separating the apolar fraction in the column and using a squalane solution as an internal standard (J European Union Commission, Regulation EEC / 183/93, Off. J. Eur. Common. L248 1993).

Another applicable analytical method is the analysis by high performance liquid exclusion liquid chromatography (HPSEC) with refractive index detector, which allows quantifying the remaining triglycerides in the sample, as well as the main groups of intermediate reaction compounds, monoglycerides and diglycerides , due to their differences in molecular weights. Free fatty acids are also included in the hydrocarbon peak when the proportion found in the sample is very low (Standard IUPAC Method 2,508). The quantification of the different groups of compounds is carried out based on their percentage over the total area obtained, since each of the chromatographic peaks corresponds to a complex group of compounds and the equality of the response factors is assumed.

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps.

For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Scheme of the process of obtaining squalene from distillates of physical refining of the olive oil of the invention. DETAILED EXHIBITION OF AN EMBODIMENT

Example 1. Procedure for obtaining squalene. Laboratory scale and pilot plant.

The experimental equipment used on a laboratory scale for the realization of the saponification, was composed of a discontinuous stirred tank reactor that operated under isothermal conditions. The reactor used was made of glass, cylindrical, of 2 L of useful volume and provided with a lid with a hermetic closing system. The cover had several holes; one of them allowed the extraction of samples and another was connected to a condenser that minimized the loss of compounds by volatilization. The condensation system was connected to a Frigiterm Selecta ® thermostatic bath through which water circulated at low temperature (5-8 ⁰ C). The thermostatization of the reactor was carried out through a jacket through which glycerin circulated from a thermostatic bath identical to the previous one, which allowed the heating of the internal contents of the reactor. The process temperature was measured with a Testo 946 temperature probe.

Since the agitation is a key aspect in the reactions that take place in the heterogeneous phase, as is the case of the saponification reaction that was studied, in order to achieve adequate contact between reagents, an Agimatic-HS Selecta ® magnetic stirrer was used and a teflon coated magnet.

At the pilot plant scale, the experimental equipment used consisted of a discontinuous full-mix reactor that was thermostated. The reactor was metallic, 15 L capacity and had a hole in the upper part, where the reagents were added, and a lower opening, whereby the sample extraction was carried out. The reactor had a jacket connected to a closed circuit through which glycerin circulated, which allowed the heating of the reaction medium. Said heating system consisted of a 5 L capacity tank where the heating fluid was heated, a pump that allowed the circulation of the glycerin and several temperature probes.

The homogenization of the internal content of the reactor was achieved by a blade agitator that rotated by the action of an engine. In order to favor mixing, the reactor also presented several baffle partitions inside.

The set was equipped with a closet where the measuring devices and temperature control and degree of agitation were located.

On a laboratory scale, a percentage by weight of squalene was reached in the unsaponifiable fraction of the oleins of 86%. While at the pilot plant scale, a percentage by weight of squalene was obtained in the unsaponifiable fraction of the oleins of approximately 82%.

The unsaponifiable fraction prewashed to remove soluble impurities enriched squalene, he was subjected to a purification process by adsorption with not activated earth (<10 wt%), under vacuum and at a temperature between 40 and 100 ⁰ C to remove substances undesirable

Next, the molecular distillation process was carried out. The distillation system consisted of a KDL 5 Short Route / Molecular Distillation distillation unit of UIC GMBH, whose characteristics are:

- Max flow: 2 kg / h.

- Evaporator temperature max .: 300 ⁰ C.

- Evaporator surface: 4.8 dm ² . - Condenser surface: 6.5 dm ² .

- Working pressure: <0.5 mbar.

- Power supply: 230 V ₁ 50-60 Hz. Total potential: 12 kw.

With the molecular distillation process fractions with a richness in squalene were obtained between 95 and 99% by weight.

The determination of the percentage of squalene was carried out by gas chromatography, using the technique proposed by the European Union for the "Joint determination of squalene and waxes", separating the apolar fraction in a column and using a squalane solution as an internal standard [ J. European Union Commission, Regυlation EEC / 183/93, Off, J. Eur. Commun, L248 (1993)]. The determination was made in duplicate, with a coefficient of variation of less than 5% in all cases.

The chromatographic conditions used are the following:

- Stationary phase: SPB5 column (5% diphenyl-95% dimethylpolysiloxane) (25m ^• 0.25mm LD., 0.25mm film thickness).

- Carrier gas: Hydrogen. Flow: 1 mL / min.

- 12O ⁰ C oven temperature program for 4 minutes and 4 ° C / min ramp up to 310 ⁰ C.

- "On column" injection and flame ionization detector at 325 ⁰ C.

In addition, the samples were also analyzed by high efficiency liquid exclusion liquid chromatography (HPSEC) with refractive index detector, which allows quantifying the remaining triglycerides in the sample, as well as the main groups of intermediate reaction compounds, monoglycerides and diglycerides , due to their differences in molecular weights. Free fatty acids are also included in the hydrocarbon peak when the proportion found in the sample is very low (Standard IUPAC Method 2,508).

The conditions used in liquid chromatography were the following:

- Two columns 100 and 500 Á of the PL-gel, 30 ^• 0.75 cm LD. connected in series.

- Refractive index detector (HP-1037A).

- Sample concentration: 5-10 mg / mL.

- Injected sample: 10 μL.

- Mobile phase: Tetrahydrofuran. Flow: 1 mL / min.

The quantification of the different groups of compounds was carried out based on their percentage over the total area obtained, since each of the chromatographic peaks corresponds to a complex group of compounds and the equality of the response factors is assumed. The determination was made in duplicate.

By submitting a sample of 1750 g of oleins, with an acid number of 115 mg KOH / Fat (g), a saponification index of 185 mg KOH / Fat (g), a squalene richness of 36.1% and oleic acid of 57.7%, at a saponification treatment under conditions described, 360 g of unsaponifiable fraction are obtained, whose richness in squalene is 83.8% by weight. This degree of purity increases after the process of refining and molecular distillation to 97.7%.

Claims

1. A process for obtaining squalene from distillates of physical refining of olive oil, characterized in that it comprises steps 5 of: a) alkaline saponification of the raw material: b) physical separation of the saponifiable and unsaponifiable fraction; and c) molecular distillation of the unsaponifiable fraction obtained. or

2. The method according to claim 1, characterized in that after step b) a process of purification of the unsaponifiable fraction is carried out.

3. The process according to claim 2, characterized porque5 ground clay adsorbent or not activated (<10 wt%), for the purification of Ia unsaponifiable fraction, under vacuum at a temperature between 40 and 100 ⁰ C is used

4. The method according to claim 1, characterized in that 0 the saponification stage (a) is performed in the presence of a hydrotrope.

5. The method according to the preceding claim 4, characterized in that the hydrotrope is selected from the group consisting of sodium xylenesulfonate, ammonium xylenesulfonate, metaxylenesulfonate, ethylbenzene sulfonate, 5 urea, sodium eumene sulfonate, ammonium sulfonate, paratoluen sulfonic acid and toluene sodium sulfonate

6. The process according to claim 5, characterized in that the hydrotrope is selected from the group consisting of xylenesulfonate or sodium, metaxylenesulfonate and ethylbenzene sulfonate

7. The method according to any of the preceding claims 1-6, characterized in that the percentage of squalene richness in the unsaponifiable fraction after stage b) is at least 75%. 5

8. The method according to any of the preceding claims 1-7, characterized in that the percentage of squalene richness After the molecular distillation process it is at least 95%.

9. The method according to any of the preceding claims 1-8, characterized in that reaction times between 1 and 6 hours are employed.

10. The method according to any of the preceding claims 1 to 9, characterized in that in alkaline saponification an excess of base is used with respect to the saponification index of the oleins used.

11. The method according to any of the preceding claims 1 to 10, characterized in that oils with a saponification index of between 175 and 240 mg base / g fat are used.

12. The process according to claim 11, characterized in that oils with a saponification index of between 180 and 210 mg base / g fat are used.

13. The method according to any of the preceding claims 1 to 12, characterized in that a base / fat weight ratio of between 0.2 and 1 is used.

14. The method according to any of the preceding claims 1 to 13, characterized in that a total weight ratio of fat / water of between 1 and 12 is used.

15. The method according to any of the preceding claims 1 to 14, characterized in that the alkaline saponification is carried out using a base selected from the group formed by NaOH,

KOH ₁ Ca (OH) ₂ , Mg (OH) ₂ .

16. The method according to claim 15, characterized in that the base used is KOH.

17. The method according to any of the preceding claims 1 to 16, characterized in that the saponification process is carried out carried out at a temperature between 40 and 100 ⁰ C.

18. The process according to the previous claim 17, wherein the temperature is between 70 and 90 ⁰ C.

19. The method according to any of the preceding claims 1 to 18, wherein the molecular distillation process is carried out at a temperature of between 200 and 300 ⁰ C.

20. The process according to any of the preceding claims 1 to 19, characterized in that the molecular distillation process is carried out at a pressure of less than 0.1 mbar.