Patent Description:
Cholesterol is a sterol (or modified steroid), lipid molecule and is biosynthesized by animal cells as it is an essential structural component of animal cell membranes, which is required to maintain both membrane structural integrity and fluidity. This substance can be found in various sources, such as egg yolk, spinal cord, bones, brain tissue of cows, goat, pig, poultry, fish organs and shrimp exoskeleton and it is even possible to find it in vegetable oils in low concentrations. However, the most important source of cholesterol is lanolin, obtained from sheep's wool. These sources generally contain cholesterol in its free form, as well as in the esterified form (cholesterol molecule bound to a fatty acid).

Cholesterol is an important pharmaceutical intermediate required for the biosynthesis of steroid hormones, bile acids, and vitamin D<NUM>. There is currently an increasing demand for pharmaceutical grade cholesterol having a cholesterol content of <NUM>% or more for the production of vitamins D<NUM>, vitamin D<NUM> derivatives, hormones and emulsions in cosmetics. Among other uses is the synthesis of anti-inflammatory steroid drugs, production of creams for skin lubrication and cosmetics. Other cholesterols with a much lower purity are used for feeding crustaceans and aquaculture.

Cholesterol can be found in egg yolks, organ meats, shrimp, calamari, beef, pork, poultry, fish, wool grease, full-fat dairy products, butter, hard margarines, lard, coconut oil , butter (clarified butter), vegetable shortening and palm oil. These sources generally contain cholesterol in its free form, as well as in the esterified form. The most important source of cholesterol is lanolin, which is obtained from sheep's wool. However, since the fatty acid composition in wool fat is complex in nature due to the presence of free acids and alcohols that can form variable esters, cholesterol extraction is cumbersome and expensive.

Crude fish oils mainly contain triglycerides that make up about <NUM>% of the composition. The other components of the composition comprise partial glycerides (ie, mono- or diglycerides), free fatty acids, phospholipids, and a group of chemicals as the non-saponifiable fraction that includes cholesterol-sterol, glyceryl ethers, fatty alcohols, vitamins, and oxidized pigments. The content of the unsaponifiable fraction varies with seasonal and feeding conditions and varies in the range of <NUM>-<NUM>%.

Fish oil residues from the fish refining industry contain useful products such as cholesterol, proteins, and enzymes, among other fatty acids. Due to the importance of cholesterol as a precursor in the manufacture of vitamin D<NUM>, fish oil and fish oil residues (after removal of polyunsaturated fatty acids) that form the waste stream in the fish oil industry are now considered as an alternative source of cholesterol.

Due to the importance of cholesterol as an intermediate in the production of vitamin D and its derivatives, there is always a need to provide alternative and improved isolation methods for cholesterol production.

Currently, cholesterol on an industrial scale is produced primarily from wool wax alcohols, ie, the unsaponifiable fraction of wool fat, which contains from about <NUM>% to about <NUM>% cholesterol. The most common processes for producing cholesterol involve the formation of an insoluble addition product by reacting cholesterol with a metal salt, followed by decomposition and recovery of the cholesterol. Such processes can meet the purity requirements for cholesterol applications in the pharmaceutical industry. In this sense, the state of the art has presented a variety of disadvantages. In particular, some prior art examples include United States patent <CIT>, which discloses a process for producing a cholesterol concentrate that includes the steps of distilling a fish oil that has no more than <NUM>% free fatty acids in a mixture with an auxiliary fluid in a vacuum distillation column to obtain a first distillate and a first residue; and distilling the first distillate in a vacuum distillation column to obtain a second distillate and a second residue, wherein the second residue includes the cholesterol concentrate. These prior art processes of using high temperatures during molecular distillation generates degradation of the omega-<NUM> fatty acids such as EPA (eicosapentanoic acid) and DHA (docosahexanoic acid), so that the content of trans fatty acids is increased and further promotes the polymerization of unsaturated fatty acids.

The same happens with the teachings of Great Britain patent <CIT> that uses a material that contains a compound that has a cholesteric nucleus, such as sterols, saponins and bile acids, which are subjected to a short path high vacuum distillation to separate the substance thereof, and then the compound is purified. Specified raw materials include animal oils and waxes, such as whale oil, fecal fats and oils, and china wax; vegetable oils, such as soybean oil, cottonseed oil, wheat germ oil, and bean oil; and fungal growths, such as ergot, each of which are sources of zoosterols, phytosterols, and mycosterols, respectively. In the same way, in Japanese patent <CIT>, which discloses a fish oil, preferably subjected to degumming treatment, obtained from a fish such as sardines, mackerel, saury fish, etc., it is subjected to molecular distillation using, for example, in an apparatus such as a falling film type, or centrifugal type.

Furthermore, the above prior art reveals temperatures that generate large concentrations of pollutants such as polychlorinated biphenyls (PCB's), polyaromatic hydrocarbons (PAH), heavy metals, pesticides and degradation products of the aforementioned compounds, which are persistent and bioaccumulative.

However, starting from a raw material other than fish oil, such as wool fat/lanolin, does not allow the recovery of fatty acids in the form of saponified soap during cholesterol esterification. The obtained soap is split to obtain free fatty acids and then esterified by means of an acid catalyst to obtain fatty acids in the form of ethyl ester. Starting from the resulting ethyl ester, it is possible to concentrate omega-<NUM> by means of molecular distillation.

<CIT> describes a process for the extraction of cholesterol from animal tissues such as the brain, spinal cord, etc. by saponification and extraction with a water immiscible solvent. These known processes generate large amounts of liquid industrial waste, the management and/or disposal of which significantly increases production costs.

<CIT> concerns polyunsaturated fatty acid and fatty acid ester mixtures free of sterols and phosphorous compounds and a process for removing sterols and phosphorous compounds from naturally occurring lipid mixtures.

<CIT> concerns a <NUM>-step process for producing cholesterol from fish oil. Given the problems of the prior art, the inventors have discovered that cholesterol can be extracted with high yields and good quality from fish oil by ultrasonic transesterification, molecular distillation, saponification, solvent extraction and cholesterol purification methods through crystallization.

Therefore, a first object of the present invention is to avoid the drawbacks of the prior art. Particularly, the main object of the present invention is to create a process for separating the cholesterol present from fish oil, which process does not generate any amounts of contaminants.

According to the present invention, the main object of the invention is to provide beneficial processes wherein cholesterol purity of at least <NUM>% is obtained, wherein such processes can be conducted on an industrial scale capable of satisfying the market demand for pharmaceutical grade cholesterol, as the raw material. It is also commonly easy to obtain and provide an alternative for use of a co-product used to feed crustaceans, which production cost is low and the solvents used are recovered and reused in the production process.

Another no less important object is the separation of cholesterol present in the heavy fraction from fish oil by methods of saponification and extraction and purification of cholesterol through crystallization.

Finally, another object of the invention also resides in the relationship with molecular distillation or short-path distillation. This is an operation technique for liquid-liquid separation done continuously under vacuum pressure. Considering a high vacuum pressure, the distance between the heating surface and the surface of the internal condenser of the evaporator is less than or equal to the mean free path of the molecules during the separation. In this process, a thin film is formed on the hot surface and the most volatile compounds evaporate, condensing when they come into contact with the internal condenser of the evaporator. The present invention meets these needs and provides other related advantages, including obtaining pharmaceutical grade cholesterol from fish oil with a purity of at least <NUM>%.

The novel features which are considered to be the basis of the invention are set forth in particular in the appended claims and the additional advantages thereof, will be better understood from the following detailed description of the preferred embodiments.

The present invention relates to a process for separating cholesterol present in fish oil by methods of transesterification, molecular distillation, saponification, extraction and purification of cholesterol through crystallization. The present invention relates to a process as defined in independent claim <NUM>.

In one embodiment, the invention then refers to the process based on the separation of cholesterol present in fish oil by the method of transesterification, molecular distillation, saponification, solvent extraction and purification through crystallization and obtaining cholesterol in crystalline form.

In one aspect, the present invention refers to a process for producing cholesterol from fish oil, which generally comprises the steps of esterifying the refined oil with an acid value of less than <NUM> KOH/g using alcohol under the action of a basic catalyst with the aim of converting the glycerides present in the oil to the ethyl ester form or other esters.

The alcohol used for the present invention may be selected from the group consisting of methanol, propanol, butanol and ethanol, and the catalyst may be selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium
ethoxide or sodium methoxide.

Next, the process of the invention comprises concentrating the cholesterol present in the ethyl ester obtained by using a series of short-path evaporators, and then obtaining in turn another stream concentrated in EPA and DHA.

Subsequently, dissolve the concentrated cholesterol in a base in a determined amount in water with the addition of an alcohol. The base used in the present invention may comprise, without being limited to, potassium hydroxide, calcium hydroxide, sodium hydroxide, or calcium chloride.

Subsequently the mixture is saponified for a period of time and temperature with constant stirring and reflux. Subsequently, a solvent is added with stirring and alcohol is added to promote correct separation. The above step is followed by a centrifugation process to separate the soap from the extracted phase.

In other aspects of the invention, it is important that a new extraction with a second solvent must be carried out with stirring and addition of alcohol to increase the separation of the unsaponifiable matter contained in the solvent. The solvent is then evaporated using a thin film evaporator to obtain dry unsaponifiable matter. Subsequently, a wash is carried out with a solution of sodium hydroxide or calcium chloride and it is then centrifuged to eliminate the washing water and then a solvent such as acetone, benzene or toluene is added and a first crystallization is carried out. A second solvent selected from the group consisting of hexane, diethyl ether, petroleum ether, acetone and benzene may also be used. Centrifugation and filtration are then carried out and a second crystallization of the previously obtained crystals is carried out with an organic solvent such as diethyl ether or petroleum ether or hexane. Finally, a new centrifugation and filtration is carried out to obtain cholesterol crystals with a purity greater than <NUM>%.

On the other hand, the soap obtained from the last centrifugation is broken down with an acid in the presence of water at a given temperature and reaction time with agitation. Then, it is cooled and the water is separated from the free fatty acids using a centrifuge. Acids suitable for this step include sulfuric, hydrochloric, citric, acetic or phosphoric acid.

The free fatty acids are then pumped to an ultrasonic reactor, where they are esterified with an alcohol and an acid catalyst at a given temperature and reaction time with constant stirring and reflux to obtain fatty acids in the form of the ethyl ester. Said acid catalyst can be selected without being limited from the group consisting of citric acid, acetic acid, phosphoric acid, hydrochloric acid, p-toluenesulfonic acid or sulfuric acid.

Therefore, the invention initially provides a transesterification and molecular distillation process that includes a first stage for obtaining fatty acids in the form of the ethyl esters and which comprises:.

Regarding the fourth step, it must be understood that according to the process of the invention, two streams are obtained: (a) one concentrated in cholesterol in the form of ester and (b) another concentrated in EPA and DHA.

The invention continues with a second stage of saponification and extraction that includes:.

After obtaining cholesterol, it is necessary to purity it. For this purpose, the process additionally includes a third stage that includes:.

For the present invention, the set of evaporators makes it possible to recover and purify the solvent used to reuse it in the extraction process. The product of this stage are crystals of unsaponifiable matter.

Based on the cholesterol separation process of the invention, a concentration of contaminants which are persistent and bioaccumulate is not generated.

Similarly, the invention can be reflected from the examples described below.

Claim 1:
Cholesterol separation process from the heavy fraction of fish oil and characterized in that it comprises a first stage of obtaining fatty acids in the form of their esters and comprising:
(i) a first step of transesterifying refined fish oil with an acid value of less than <NUM> KOH/g from the reaction with methanol, propanol, butanol or ethanol in a ratio between <NUM>:<NUM> and <NUM>:<NUM> w/w with respect to the oil refined, preferably <NUM>:<NUM> w/w with the addition of a basic catalyst selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium ethoxide or sodium methoxide in a concentration between <NUM> and <NUM>% w/w with respect to the oil, where it is allowed react in four ultrasonic reactors arranged in series between <NUM> and <NUM> minutes, at a pressure between <NUM> and <NUM> bar, at a temperature between <NUM> and <NUM> and constant reflux of alcohol;
(ii) a second step to recover the alcohol not consumed in the reaction by flash evaporation in an evaporator that operates between <NUM> and <NUM> bar of pressure and temperature between <NUM> and <NUM>, where the alcohol recovered in this step is reusable in the first step;
(iii) a third step to separate the mixture of the ester and glycerin obtained from the transesterification by means of a disc centrifuge that operates between <NUM> and <NUM> RPM, wherein the reaction reaches a conversion of glycerides to esters is at least <NUM>%;
(iv) a fourth step of concentrating the cholesterol present in the oil in the form of ester through molecular distillation obtaining a heavy fraction, using short-path distillers arranged in series, where the residue from one evaporator is fed to the next distiller and the distillate is returned to the previous still, where the operating conditions of the stills comprise vacuum pressures from <NUM> to <NUM> Pa, temperatures between <NUM> and <NUM> and heat transfer areas between <NUM> and <NUM><NUM> and wherein in this step a stream concentrated in cholesterol content in the form of ester and another stream of esters concentrated in EPA and DHA fatty acids is obtained.