The present invention relates to purified (such as by extraction) polyunsaturated fatty acid(PUFA)-containing (microbial) oils, especially oils with a sterol content of less than 1.5%.
There is a growing tendency to include lipid products containing polyunsaturated fatty acids derived from various fermentation processes in foodstuffs. This is of importance in the recently established desirability to incorporate certain polyunsaturated fatty acids in an infant formula.
Various processes have been described for the fermentative production of lipids or oils containing polyunsaturated fatty acids. Examples are EP-A-155,420 for the production of γ-linolenic acid(GLA)-containing lipid from Mortierella, EP-A-223,960, EP-A-276,541 and WO-A-92/13086 for the production of arachidonic acid(ARA)-containing oil from Mortierella and/or Pythium, WO-A-91/07498 and WO-A-91/11918 for the production of docosahexaenoic acid(DHA)-containing oil from Crypthecodinium cohnii or Thraustochytrium, and WO-A-91/14427 for the production of eicosapentaenoic acid(EPA)-containing oil from Nitzschia. Typically, the microbial species producing the lipid containing the desired polyunsaturated fatty acid(s) is cultured in a suitable medium and the biomass is harvested before the desired lipid obtained.
To obtain a lipid concentrate which has a relatively high triglyceride content typically a nonpolar solvent for the lipid (e.g. hexane) or supercritical CO2 is used in the extraction process. For example, EP-A-246,324 describes a fractional extraction process for the isolation of lipids from Mortierella, to obtain different extracts which are enriched in either polar or nonpolar (neutral) lipids. The neutral lipid extract still has, however, a relatively low triglyceride content (89.3%) and a high sterol content (9.4%). US patent no. 4,857,329 describes an extraction process comprising the use of supercritical CO2 to selectively elute neutral lipids from Mortierella biomass. However, the triglyceride content of the lipid extract does not exceed 86%.
Yamada et al, Industrial applications of single cell oils, Eds. Kyle and Ratledge, 118-138 (1992) describe an arachidonic acid-containing oil extracted from Mortierella alpina biomass using hexane. The purified oil has a triglyceride content of 90%.
JP-A-62/179598 describes a process wherein fat (or oil)-containing material is dispersed in aqueous ethanol and crushed. The crushed material is separated from the dispersant. The separated material is extracted with n-hexane to obtain fat (or oil).
JP-A-62/065689 relates to a refined glyceride oil from which a gum such as phospholipid, triglyceride, free fatty acid has been removed by extracting a crude glyceride oil composition from a microorganism, diluting the composition with an organic solvent and contacting with a semi-permeable membrane.
Thus, until now it has not been possible to obtain a microbial triglyceride oil with a high triglyceride content, i.e. 95% or higher, using previous fermentation and extraction technology. It has also not been possible to prepare oils having a particularly low (e.g. less than 1.5%) sterol content.
The present invention generally relates to a process for preparing a (microbial) oil with a high triglyceride content and a low content of "unsaponifiables", where an oil extracted, obtained or derived from a microbial biomass is treated with a polar solvent.
The present invention can thus provide a microbial (or microbially derived) oil having a high triglyceride content, such as ≥ 95%. However the oil may have a triglyceride content of at least 97%, preferably ≥ 98%, and optimally ≥ 99%. The (microbial) oil also has a low (≤ 1.5%) sterol content. Preferably the sterol content is ≤ 1%, such as ≤ 0.6%, optimally ≤ 0.3%.
The oil of the invention can be used in various compositions such as pharmaceutical (or therapeutic), cosmetic, feedstuff or food compositions (for human or animal consumption), especially in an infant formula or nutritional supplement.
A first aspect of the present invention therefore relates to a process of treating a microbially derived oil (an oil derived from a microorganism), the process comprising: (a) contacting the oil with a polar solvent to extract at least one sterol that is soluble in the solvent; and (b) separating at least some of the solvent containing the sterol from the (so treated) oil, so that the resulting oil has a sterol content of less than 1.5%.
The microbially derived oil can be extracted, obtained, or produced by one or more microorganism(s). Often this will be the same species of microorganism, but a mixture of two or more different microorganisms are envisaged by the invention. The process of the invention may therefore be subsequent to the production of the oil itself. The oil may be one that is produced by, or exists inside (e.g. intracellularly) the microorganism(s). Alternatively, it may be obtained from a (usually aqueous) composition obtained or resulting from fermentation (of the microorganisms). This (aqueous) composition may contain the microorganisms themselves: in that case, it is usually a fermentation broth. The microorganisms (or biomass as referred to in the art) can be removed (after fermentation) by a number of methods, for example filtration, centrifugation or decantation. The oil can be extracted or obtained from this biomass.
It is usual that the microbial oil will have been obtained by extraction. This preferably will have involved extraction using a non-polar, or preferably a water-immiscible, solvent, or at least a solvent that is capable of extracting oily components. Such a solvent may be a C6-10 alkane, for example hexane, or (supercritical) carbon dioxide.
Different microorganisms will produce different oils. These can differ in the amount of polyunsaturated fatty acids (PUFAs) as well as in other components, and indeed the PUFAs may be in different forms, for example diglycerides, triglycerides and/or phosolipids. As such, even microbially derived oils can differ significantly from oils containing one or more of these PUFAs that have been obtained from other (e.g. animal or fish or vegetable) sources.
The microorganisms contemplated can vary widely, although preferably they will be able to produce one or more PUFAs, for example on fermentation. Microorganisms can be bacteria, algae, fungi or yeasts. Suitable fermentation processes, microorganisms and PUFA-containing oils are described in co-pending International application no. PCT/EP97/01448 (filed on 21 March 1997 in the name of Gist-brocades B.V.).
Preferred algae are of the genus Crypthecodinium, Porphyridium or Nitzschia. Preferred fungi are of the genus Thraustochytrium, Mortierella, Pythium, Mucorales or Entomophthora, in particular of the species Mortierella alpina.
The extracted sterol can be an alicyclic alcohol having a four conjugated ring backbone, three aromatic C6 rings and one cyclopentane ring (e.g. desmosterol, cholesterol), an aliphatic or terpenic alcohol, tocopherol). A wax or antifoaming agent, such as polypropylene glycol may be present in the fermentation medium.
Preferred sterols include desmosterol, such as 5-desmosterol. If more than one sterol is present, then suitably 70 to 90%, e.g. 80 to 85%, of the sterols is desmosterol (e.g. for oil produced by Mortierella).
The oil will preferably contain at least one PUFA. This PUFA will usually have been produced by the microbe or microorganism.
PUFAs contemplated by the invention are C20 and C22 ω-3 and C18, C20 and C22 ω-6 polyunsaturated fatty acids. In particular they can include γ-linolenic acid (GLA), dihomo-γ-linolenic acid (DLA), arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). DHA is produced by algae or fungi, such as a dinoflagellate algae, for example of the genus Crypthecodinium, or a fungus, for example of the genus Thraustochytrium. GLA, DLA or ARA can be produced by fungi, such as of the genus Mortierella, Pythium or Entomophthora. EPA can be produced by an algae, such as of the genus Porphyridium or Nitzschia. Typically the oil will dominantly or only contain one PUFA, although oils can contain one or more PUFAs, for example in a lesser amount.
In the processes of the invention after the solvent has been added to the oil, the two phases (oil and solvent) will usually separate. This can easily then allow removal of one phase from the other. That can then give an oil with a low sterol content of no more than 1.5%.
A second aspect of the invention therefore relates to an oil treated or prepared by a process according to the first aspect.
A third aspect relates to a microbial oil comprising at least one polyunsaturated fatty acid (PuFA) having a sterol content of no more than 1.5%. The (total) sterol content may in fact be no more than 1%, for example less than 0.6%. By using the processes of the invention, a sterol content of no more than 0.3% can be achieved.
It will be realised that the oil of the third aspect can be prepared by using the process of the first aspect.
The different oils of the invention can be prepared, for example, by using different solvents, at different temperatures, as will be described later.
The present invention therefore provides a process for preparing an (e.g. microbial) oil, where the oil is treated with one or more polar solvents. These solvent(s) can therefore remove one or more sterols that are soluble in the solvent. This may result in concentrating or enriching of the oil. Therefore, if the oil contains triglycerides, one can concentrate or increase the triglyceride content of the oil. This may be to at least 97%, for example at least 98%, and ultimately at least 99%.
Simultaneously with increasing the triglyceride content, the solvent treatment can advantageously result in the removal of one or more impurities from the oil. In particular, this treatment can result in the lowering of the amount of "unsaponifiables". These unsaponifiables that can be removed by the solvent treatment can include the sterols, aliphatic and terpenic alcohols, waxes and antifoaming agents described earlier. Usually, the treatment of the solvent will not alter the PUFA profile or the oil so treated.
The polar solvent preferably comprises a C1-6 alkanol, for example ethanol. The solvent, however, may be an aqueous one. Preferred solvents therefore comprise an alcohol (e.g. ethanol) and water. However, the solvent may comprise other liquids, and these can be acetone and/or isopropanol.
If the solvent comprises ethanol, this may have a water content of from 0 to 20%, such as from 1 to 7%, and optionally from 2 to 4%. If the solvent comprises methanol, acetone and/or isopropanol (IPA), then the water content is preferably 0 to 2%, 5 to 50% and 5 to 15%, respectively. The solvent may therefore comprise a mixture of two or more liquids. It has been found that ethanol containing a small amount of water (e.g. 97% ethanol, 3% water) can significantly improve the yield of triglyceride after solvent treatment. This is because triglycerides are relatively insoluble in this particular solvent. Having the solvent at a temperature of from 15 to 30°C, e.g. 20 to 25°C, also reduces the amount of triglycerides that dissolved in the solvent.
By using different solvents one can vary the amount of sterol (or indeed PUFA) that is extracted. As has been discussed above, a mix of ethanol and water can provide a high yield of triglycerides since although this solvent will dissolve sterols, triglycerides are nevertheless relatively insoluble in it.
The PUFA will generally exist in several forms, such as triglycerides and diglycerides. These compounds are effectively a glycerol molecule with one or more (although usually only one) of the PUFAs attached to this backbone. Preferably the triglyceride form will be dominant. In the oil of the third aspect (e.g. from the process of the first aspect), the amount of diglycerides present is preferably no more than 2.2%, and preferably less than 1%. The solvent used here is preferably at a temperature of from 10 to 40°C, e.g. 20 to 30°C.
The amount of sterol to be extracted, or the triglyceride content, can be adjusted by varying several process parameters. For example, one can adjust the ratio of solvent to oil, the temperature during extraction and/or by repeating the extraction process. If more than one extraction is to be performed, a counter-current extraction process is preferred, which can minimise triglyceride losses.
Usually the oil will be a crude oil obtained after extraction from a (e.g. dried) microbial biomass with a suitable solvent, followed by evaporation of that (water immiscible) solvent. The oil may be subjected to-one or more refining steps prior to the process of the invention.
The oil of the invention, or one which results from a process of the first aspect, can be used for various purposes without further processing, or can be additionally subjected to one or more refining steps. The oil can be used as an additive or a supplement, for example in food compositions, such as an infant formula. It may however also be used in cosmetic or pharmaceutical compositions. The invention in a further aspect therefore relates to a composition, such as a food stuff, feed or pharmaceutical composition or a cosmetic composition, which comprises, or to which has been added, an oil of the invention. Preferred compositions are foods, such as infant formula or a nutritional supplement.
The oil of the invention can therefore have a low sterol and optionally alow diglyceride content. Optionally, it may also have a high triglyceride content. This makes the oil particularly suitable for nutritional purposes, and can be used as a nutritional supplement. The oil may be supplied as an oil, or it may be encapsulated, for example, in a gelatin capsule. The oil can thus be incorporated in foods, feeds or foodstuffs, suitable for human or animal consumption. Suitable examples are health drinks and bread. Particularly contemplated is the use in infant formula, or in cosmetics.
Preferred features and characteristics of one aspect of the invention are equally applicable to another aspect mutatis mutandis.
The invention will now be described, by way of example, with reference to the following Examples which are provided merely for means of illustration, and are not to be construed as being limiting on the invention.
500 1 of broth obtained after Mortierella alpina fermentation was filtered in a membrane filter press (cloth type: propex 46K2). The broth was filtered with a pressure difference of 0.2 bar. Within 21 minutes 500 1 broth was filtered over a total filter area of 6.3 m2 which resulted in an average flow of about 230 l/m2h. The filter cake was washed in 30 minutes with 10 cake volumes of tap water at an average flow rate of 320 l/m2h.
The cake was squeezed at 5.5 bar for 30 minutes which resulted in a dry matter content of the recovered biomass of about 45%.
Extrusion was performed on the resulting biomass cake using a single screw extruder with a profiled barrel and a universal screw. The dieplate used for extrusion had holes of diameter 2mm.
Drying of the extrudate was performed in a fluidized bed dryer with air (8000 Nm3/m2h). The setpoint of the bed temperature was 80°C. The diameter of the dried extruded biomass was 2mm and its dry matter content after drying was about 96%.
A crude arachidonic acid-containing oil (ARA oil) was then extracted from the extrudate using hexane as a solvent.
5ml of crude ARA oil was extracted from the extrudate of Example 1 with a volume of 100% ethanol for 1 minute by hand-shaking. Subsequently, the bottom and toplayers were separated by centrifugation for 5 minutes at 5000 rpm. The samples were analyzed by means of (600 Mhz) NMR (for tri- and di-glycerides, sterols (only desmosterol content measured) and antifoaming agent).
Extraction of crude ARA oil with 9 volumes of 100% ethanol at two different temperatures resulted in an oil with a decreased level of sterol and diglyceride (DG) and in an increased level of triglyceride (TG, see Table 1). The yield of TG is the percentage of triglyceride remaining in the oil after solvent extraction. Also antifoaming agent was removed and found in the ethanol after extraction. However, the yield of triglycerides was low due to the fact that some of the TG dissolved (and was thus removed in) the ethanol.
TG: triglycerides
Examples 2 and 3 were repeated except using 97% ethanol at varying volumes relative to the oil.
Extraction of crude ARA oil with 1, 3 and 9 volumes of 97% ethanol resulted in an oil with a decreased level of sterol and diglyceride and in an increased level of triglyceride (see Table 2).
The yield of triglycerides was above 92% due to the fact that not much oil dissolves in 97% ethanol. At ambient temperature (about 20°C), a higher yield of triglycerides and a better removal of diglycerides and sterols was observed. Remarkably no ethanol was found in the treated oil.
TG: triglycerides
The ethanol phase was also analyzed after extraction and a significant increase in sterols was observed. Also the antifoam agent (polypropylene glycol) was extracted and found in the ethanol phase (see Table 3).
TG: triglycerides