LYBERG, Ann-Marie (Vallkärravägen 21, Lund, SE-226 51, SE)
LYBERG, Ann-Marie (Vallkärravägen 21, Lund, SE-226 51, SE)
1. A PUFA enriched marine oil being substantially free from heavy metals, PCB and dioxins comprising at least 40 mol % eicosapentaenoic acid and docosahexaenoic acid and at least 50 mol % of mono and diglycerides. 2. The oil according to claim 1, comprising at least 50 mol % eicosapentaenoic acid and docosahexaenoic acid.
3. The oil according to claims 1 or 2, comprising at least at least 70 mol % of mono and diglycerides.
4. The oil according to claim 3, comprising at least 80 mol % of mono and diglycerides.
5. The oil according to any of preceding claims, wherein the molar ratio between eicosapentaenoic acid and docosahexaenoic acid is below 1.
6. The oil according to any of preceding claims, wherein the molar ratio between the mono and diglycerides is below 1. 7. The oil according to any of preceding claims, comprising from about 5 to about 30 mol % of triglycerids.
8. The oil according to any of preceding claims, wherein said oil extract is from squid.
9. A method of treating a marine oil having an eicosapentaenoic acid and docosahexaenoic acid content of at least 25 mol % comprising the steps of; a. Providing an immobilised lipase, b. Transesterifying said oil with a C1-C6 alcohol in the presence of said immobilised lipase and obtaining a product with EPA and DHA enriched in the glyceride fraction and a large part of the other fatty acid being in the form of esters of the alcohol used or as free fatty acids c. Removal of simple fatty acid esters and free fatty acids by distillation d. Obtaining a PUFA enriched marine oil being substantially free from heavy metals, PCB and dioxins comprising at least 40 mol % eicosapentaenoic acid and docosahexaenoic acid and an at least 50 mol % of mono and diglycerides.
10. The method according to claim 9, wherein said lipase is selected from the group donsisting oϊRhizomucor miehei (RM), Thermomyces lanuginosus (TL), Pseudomonas cepacia and Pseudomonas fluorescens. 11. The method according to claim 10, wherein said lipase is the one from Thermomyces lanuginosus (TL).
A polyunsaturated fatty acid (PUFA) enriched marine oil comprising eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) , and a process of production thereof
FIELD OF INVENTION
The invention relates to a PUFA enriched marine oil being substantially free from heavy metals, PCB and dioxins comprising at least 40 mol % eicosapentaenoic acid and docosahexaenoic acid and at least 50 mol % of mono and diglycerides as well as a method to produce such a PUFA enriched marine oil.
BACKGROUND OF INVENTION Long chain n-3 fatty acids (omega-3 fatty acids) have well documented positive health effects. The most important fatty acids in this group are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The dominating source of these fatty acids is various kinds of marine oils. Large efforts have been made to enrich EPA and DFIA from marine oils to obtain more concentrated products for use as food supplements or food ingredients. Most commonly the EPA and DHA containing triglycerides from the marine oil are hydrolysed to free fatty acids, followed by fractionation of the fatty acids using urea as complexation agent. More recently, the fatty acid specificity of lipases has been utilised to enrich EPA and/or DHA from marine oils. Most lipases have an ability to discriminate against the n-3 fatty acids and especially docosahexaenoic acid (DHA). Therefore lipases are utilised for enrichment of DHA and eicosapentaenoic acid (EPA) in marine oils, in free fatty acids or in simple esters of fatty acids. Furthermore, the lipases are able to operate at mild conditions, which is preferable since EPA and DHA are prone to oxidation. The choice of lipase and raw material depends on the desired lipid structure and ratio of EPA and DHA in the final product. The fatty acid specificity of the lipase should also be considered. If the fatty acids are located in triglycerides, the regiospecificity and triglyceride specificity also have an effect on the enrichment. Thus the positional distribution of the fatty acids and the glyceride molecule structure may have an impact on the ability of the lipase to enrich DHA and/or EPA in either the substrate or product and a product containing DHA and EPA as mono and di-glycerides will give rise to additional health benefits as well as emulsifying properties.
EPA and/or DHA of marine origin have been concentrated by different strategies using lipases. Most lipases discriminate against DHA more than against EPA such as Candida rugosa (formerly Candida cylindraced) and Rhizomucor miehei (Mukherjee et al. 1993). Nevertheless, there are lipases that discriminate against EPA more than against DHA such as porcine pancreas, Chromobacterium viscosum, Pseudomonas sp., Pseudomonas cepacia and Pseudomonas fluorescens
(Breivik et al. 1997, Halldorsson et al. 2004, Mukherjee et al. 1993). The lipase from Rhizomucor miehei was utilised for enrichment of DHA in free fatty acid from fish oil by esterification of the oil with butanol or glycerol (Halldorsson et al. 2003, Hills et al. 1990). Another approach for concentrating both DHA and EPA in the free fatty acids was to esterify free fatty acids from marine origin with glycerol catalysed by one of the lipases from Pseudomonas sp., Pseudomonas fluoresceins, Thermomyces lanuginosus (formerly Humicula lanuginosa) or Rhizopus oryzae (Haraldsson et al. 2000). Enrichment of both DHA and EPA in the glyceride fraction was obtained by ethanolysis or hydrolysis of fish oil catalysed by the lipase from Pseudomonas sp., Pseudomonas fluorescens, Geotrichum candidum (Breivik and Haraldsson 1995, Breivik et al. 1997, Haraldsson et al. 1997). In the alcoholysis process several lipases including those from Humicola lanuginosa (nowadays called Thermomyces lanuginosus) Geotrichum candidum, Aspergillus niger and Candida rugosa did not function well due to poor catalytic activity or poor selectivity (Breivik and Haraldsson 1995).
US 5945318 disclose a method in which a number of different lipases have been evaluated. Many of the lipases showed a low or no selectivity between different fatty acids. None of the lipases was immobilized.
Accordingly a number of methods that have been used in the production of EPA and DHA have resulted in that EPA and DHA in the final product are ethyl esters and not mono-and di-glycerides. Examples of such method are disclosed in WO 2004043894 and Liang et al., 2000).
Both EPA and DHA have health promoting effects for humans, some of which are common for the two acids. In addition each one of the fatty acids has important health benefits. EPA has pronounced anti-inflammatory effects while DHA is of crucial importance for the development and maintenance of the brain and nervous system. The inclusion of plentiful DHA in the diet improves learning ability. Decreases in DHA in the brain are associated with cognitive decline during aging and with onset of sporadic Alzheimer disease. Not only the fatty acid composition, but also the kind of lipid in which the fatty acids are included influences the health effects of dietary lipids. It has been observed that the intake of diglycerides instead of triglycerides has several positive health effects: lowering of blood glucose, improved insulin resistance, and can thus be useful in prevention of diabetes. Furthermore, the accumulation of body fat is reduced, and risk factors for arteriosclerosis are reduced.
EPA and DHA containing products available for use as food supplements and food ingredients have the fatty acids as free fatty acids, simple esters (methyl or ethyl), as triglycerides and recently also as phospholipids. Most products contain more EPA than DHA. The product of the present invention contains EPA and DHA
mainly as di- and mono-glycerides, with additional health benefits as well as emulsifying properties, useful for some applications. Furthermore the product of the present invention contains more DHA than EPA, thus strengthening the specific health benefits of DHA. In this product, the positive effects of DHA and diglycerides are thus combined in a new way.
SUMMARY OF THE INVENTION
The invention relates to a PUFA enriched marine oil being substantially free from heavy metals, PCB and dioxins comprising at least 40 mol % eicosapentaenoic acid and docosahexaenoic acid and at least 50 mol % of mono and diglycerides.
By providing such a new PUFA enriched marine oil an improved oil is available providing EPA and DHA mainly as di- and mono-glycerides, with additional health benefits as well as emulsifying properties. Furthermore the product of the present invention contains more DHA than EPA, thus strengthening the specific health benefits of DHA.
It has surprisingly been found that by by use of a two step method in which an immobilised lipase is used in combination with a step of separation gives rise to an unique PUFA enriched oil, wherein the EPA and DHA are substantially mono- and di-glycerides. A product which is substantially free from ethyl esters. The invention also relates to a method of treating a marine oil having an eicosapentaenoic acid and docosahexaenoic acid content of at least 25 mol % comprising the steps of; providing an immobilised lipase, transesterifying said oil with a C1-C6 alcohol in the presence of said immobilised lipase and obtaining a product with EPA and DHA enriched in the glyceride fraction and a large part of the other fatty acid being in the form of esters of the alcohol used or as free fatty acids, removal of simple fatty acid esters and free fatty acids by distillation and obtaining a PUFA enriched marine oil being substantially free from heavy metals, PCB and dioxins comprising at least 40 mol % eicosapentaenoic acid and docosahexaenoic acid and at least 50 mol % of mono and diglycerides. BRIEF DESCRIPTION OF THE DRAWINGS
FIG 1 shows Mol% DHA in the glyceride fraction (TG + DG + MG) against loss of DHA from this fraction during ethanolysis of squid oil catalysed by RM, TL, PC and PF.
FIG 2 shows the reaction profile for the lipase from Thermomyces lanuginosus.
FIG 3shows Mol% EPA in the glyceride fraction (TG + DG + MG) against loss of EPA from this fraction during ethanolysis of squid oil catalysed by RM, TL, PC and PF.
DETAILED DESCRIPTION OF THE INVENTION
In the context of the present application and invention the following definitions apply:
The term "emulsifying properties" is intended to mean that emulsions can be formed easily when oil and water are vigorously mixed.
All fatty acid compositions are expressed using the term "mol %". One mol % of fatty acid X means that 1 % of the fatty acid residues in that fraction is fatty acid X.
Contents of different lipid classes (triglycerides, diglycerides, monoglycerides, free fatty acids and fatty acid ethyl esters) are expressed as mol %. One mol % diglycerides means that the number of moles of diglycerides constitutes 1 % of the total number of moles of the different lipid classes. The term "substantially free from" is intended to mean that the analysed values of contaminants are far below the limits set by the EU Commission according to heavy metals, PCB:s and dioxins in fish oil. ( < 0.1 ppm of each one of the elements: arsenic, cadmium, mercury and lead.)
The term "PUFA (polyunsaturated fatty acids)" is intended to mean docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).
OIL EXTRACT The invention relates to an improved PUFA enriched marine oil being substantially free from heavy metals, PCB and dioxins comprising at least 40 mol % eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and at least 50 mol % of mono and diglycerides. Said invented marine oil having improved properties such as having emulsifying properties which enables the possibility to form emulsions with water rich components to form dressings, sauces, desserts, doughs, dairy drinks, fruit and vegetable based drinks and many other kinds of food. The content of eicosapentaenoic acid and docosahexaenoic acid in the oil extract may be at least or equal to 40 mol % and the content of the mono and diglycerides at least or equal to 60 mol %. Other examples are at least 45 mol % EPA + DHA and at least 55 mol % mono and diglycerides or 50 mol % EPA + DHA and 50 % mono and diglycerides.
The ratio between eicosapentaenoic acid and docosahexaenoic acid may be lower than 1 and the molar ratio between mono and diglycerides may be lower than 1.
The content of the mono and diglycerides mat be at least 50, 60, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 ,76, 77, 78, 79, 80 mol %or even higher.
The content of the triglycerides may be from about 5 to about 30 %, such as 5-25, 5-20, 5-15 mol %. Specific examples are 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 mol %.
The oil extract may be obtained from any suitable marine oil source such as be selected from the group consisting of different kinds of fish, algae, crustaceans and for example one suitable source is squid.
METHOD OF PRODUCING AN OIL EXTRACT
The invention relates to a method of treating a marine oil having an eicosapentaenoic acid and docosahexaenoic acid content of at least 25 % comprising the steps of; providing an immobilised lipase or a mixture of lipases, transesterifying said oil with a C1-C6 alcohol in the presence of said immobilised lipase and obtaining a product in which EPA and DHA are enriched in the glyceride fraction and a large part of the other fatty acids are present as esters of the alcohol used or as free fatty acids. After distillation to remove the esters of the alcohol and free fatty acids, a PUFA-enriched marine oil substantially free from heavy metals, PCB and dioxins comprising at least 40 mol % eicosapentaenoic acid and docosahexaenoic acid and an at least 50 mol % of mono and diglycerides.
Immobilisation of the lipase or lipases provides several advantages such as higher activity and stability of the lipases. Suitable support materials are porous inorganic or organic materials, with silica and polypropylene as a couple of examples. Some immobilised lipases are commercially available, such as Lipozym RM IM from Novozymes, Denmark.
By the use of specific lipases it is possible to selectively transesterify the ester moieties of saturated and unsaturated fatty acids in marine oil triglycerides. By the use of specific lipases and/or mixtures of lipases it is possible to design different mixtures of mono-, di- and triglyceride as well as the amount of EPA and DHA.
Examples of different lipases which can be used are those from Thermomyces lanuginosus, Pseudomonas cepacia, Pseudomonas fluorescens and Rhizomucor miehei. One suitable lipase being the one from Thermomyces lanuginosus, which can be obtained from Novozymes, Denmark or Sigma-Aldrich, St Louis, MO, USA.
The transesterification step may be done with an alcohol or a mixture of alcohols such as methanol, ethanol, propanol, butanol, pentanol or hexanol. During the transesterification it is of importance to protect the unsaturated fatty acids from oxidation. This may be done by carrying out the reaction under nitrogen. The concentration of the marine oil extract may be done by distillation in one or several distillation steps, whereby the relatively volatile fatty acid esters and free fatty acids can be removed from the less volatile residual glyceride mixture. In the distillation step potential volatile contaminants such as PCB and dioxins are efficiently removed from the glyceride mixture. The distillation is preferably carried out at low temperature and pressure.
The invented process is especially adapted to the preparation of marine oil extracts as defined above having a specific combination and amount of mono- and diglycerides as well as EPA and DHA having healthy effects on the mammal, such as a human being.
Following examples are intended to illustrate, but not to limit, the invention in any manner, shape, or form, either explicitly or implicitly.
Materials Tridecanoic acid, albumin from bovine serum and non-immobilised lipases from Rhizomucor miehei (RM), Thermomyces lanuginosus (TL), Pseudomonas cepacia (Amano lipase PS)(PC) and Pseudomonas fluorescens (Amano lipase)(PF) were bought from Sigma (St Louis, MO, USA). The squid oil was deodorized. Accurel MPlOOO, a polypropylene support for immobilisation, was obtained from Akzo (Obernburg, Germany).
Immobilisation of Upases
The lipases were immobilised on the polypropylene support MPlOOO by adsorption. The amount of lipase added during the immobilisation corresponded to a 40 mg protein/g support. The lipase was dissolved in 20 mM sodium phosphate buffer (pH 6). The solution was centrifuged and the supernatant was added to the MPlOOO, which was prewetted with ethanol (3 ml/g preparation). The mixture containing both enzyme and support was shaken overnight at room temperature. Then the enzyme preparation was filtered and washed with sodium phosphate buffer (20 mM, pH 6). To the enzyme preparation 1 ml sodium phosphate buffer (200 mM, pH I)Ig preparation was added and this preparation was dried under reduced pressure overnight. The protein loading of the lipase preparations was determined to 22 mg protein/g support for Rhizomucor miehei, 33 mg protein/g support for Thermomyces lanuginosus, 19 mg protein/g support for Pseudomonas cepacia and 6 mg protein/g support for Pseudomonas fluorescens.
Ethanolysis of squid oil catalysed by Upases
The reaction between squid oil (2.8 g) and ethanol (0.55 ml) was catalysed by an immobilised lipase from either Rhizomucor miehei (0.25 g), Thermomyces lanuginosus (0.08 g), Pseudomonas cepacia (0.03 g) or Pseudomonas fluorescens (0.06 g) with shaking at room temperature. Nitrogen was added prior to shaking to protect the unsaturated fatty acids from oxidation. 50-μl samples from the reaction mixtures were removed and diluted with 0.94 ml tetrahydrofuran. From this solution 50 μl was withdrawn and applied on two thin-layer chromatography (TLC)
plates in order to monitor the fatty acid composition of the lipids by gas chromatography using methods described below.
Thin-layer chromatography Triglycerides (TG), diglycerides (DG), monoglycerides (MG) and free fatty acids (FFA) were separated on silica gel 60 TLC plates (Merck, Darmstadt,
Germany) using the mobile phase cyclohexane/diethyl ether/acetic acid (50:50: 1).
In order to visualise the lipids and fatty acids in UV light, the TLC plate was sprayed with 0.1% 2,7-dichlorofluorescein in ethanol. The bands of TG (start samples only), DG, MG and FFA were scraped off and placed in glass tubes for fatty acid analysis.
Since the method above did not separate the ethyl esters from the triglycerides, the former were separated from the other components using the mobile phase cyclohexane/diethyl ether/acetic acid (90: 10: 1). The TLC process was performed twice to improve the separation. The plate was visualised as described above and then the band of ethyl esters and TG were scraped off for fatty acid analysis.
Fatty acid analysis Triglycerides, diglycerides and monoglycerides were separately methylated using sodium methoxide as reagent. Methylation of free fatty acids was carried out using sulphuric acid as catalyst. Ethyl esters from the TLC plates were extracted in cyclohexane. Fatty acids in the form of either methyl esters or ethyl esters were analysed using gas chromatography.
Gas chromatography analysis
1 μl samples were injected into a Varian gas chromatograph 3400 (Varian Instrument Group, Walnut Creek, CA, USA), which was equipped with a flame- ionization detector. The column was an SP-2380 fused silica capillary column (30 m x 0.32 mm, 0.20 μm film thickness; Supelco, Bellefonte, PA, USA).
The response factors of the different fatty acid methyl esters were obtained from analysis of a standard mixture. These response factors were used to calculate the relative amounts of different fatty acids within a sample based on mol%. These data were compared with the internal standard (methyl tridecanoate) to determine the absolute amount (μmol) of the fatty acids in the sample.
Ethanolysis of squid oil was catalysed by lipases from Rhizomucor miehei (RM), Thermomyces lanuginosus (TL), Pseudomonas cepacia (PC) and Pseudomonas fluoresceins (PF) in order to compare their ability to discriminate against EPA and DHA.
The initial conversion rate of the triglycerides for the four lipases is presented in Table 1.
Table 1 Conversion rate of triglycerides in squid oil
Rhizomucor miehei 7.4
Thermomyces lanuginosus 4.0
Pseudomonas cepacia 2.1
Pse udomonas fluorescens 4.3
Fig. 1. shows the enrichment of DHA against the DHA loss for four lipases. The lipase that obtained the highest DHA recovery during the enrichment of DHA was that from Thermomyces lanuginosus. After 24 hours of reaction the glyceride fraction contained 37 mol%, and 1.7 mol% of DHA was lost. An even higher enrichment after 24 hours of reaction was provided by the lipase from Rhizomucor miehei. However, this lipase also showed a considerable DHA loss (6.8 mol%). The lipase from Thermomyces lanuginosus was therefore considered the most suitable lipase for enrichment of DHA in the squid oil.
The reaction profile provided by the lipase from Thermomyces lanuginosus is shown in Fig. 2. During the first hour, the triglycerides were converted to diglyceride at a high rate. No DHA ethyl esters were formed during the first 1.5 hours.
The enrichment of EPA against the loss of EPA is displayed in Fig. 3. Both Pseudomonas lipases enriched the squid oil with respect to EPA with the least EPA losses. After 24 hours of reaction these lipases enriched the glyceride fraction to 20 mol% EPA, and 0.7-1.3 mol% of EPA was lost. The lipases from Rhizomucor miehei and Thermomyces lanuginosus obtained a slightly higher EPA enrichment. However, the EPA loss was much larger.
The lipase from Thermomyces lanuginosus was chosen to catalyse ethanolysis in a larger scale. Squid oil (500 g), ethanol (108 ml) and immobilised lipase from Thermomyces lanuginosus (13.9 g) were stirred with continuous nitrogen addition for 24 hours. After 24 hours of reaction, the DHA and EPA content of the glyceride fraction had reached 35 and 17 mol%, respectively. The recovery of DHA was 97 mol% and the recovery of EPA was 79 mol%. The dominating glyceride fraction was the diglycerides (Table T).
The reaction was stopped by filtering off the enzyme after which the ethanol was evaporated.
Short part distillation
The ethyl esters were separated from the glyceride fraction after ethanolysis by using short path distillation (KD4, UIC GmbH, Alzenau-Hδrstein, Germany). The distillation was first performed at 120 0 C, and the remaining part was distilled once more at 130°C. After the first distillation the distillate contained 167.6 g and the remainder 270.3 g. After the second distillation the distillate contained 6.1 g and the remainder (the final product) contained 243.1 g.
The fatty acid composition of the original squid oil and the purified product is presented by Table 3.
Table 2 Lipid composition of squid oil before reaction, after 24 hours of reaction and after purification
Squid oil After 24 hours Purified of reaction Product mol% mol% mol%
Triglycerides 90.8 4.7 12.6
Diglycerides 7.7 19.2 51.4
Monoglycerides 0.6 11.9 33.1
Free fatty acids 0.9 0.3 0.3
Ethyl esters 0 63.9 2.6
Table 3 Fatty acid composition of squid oil (glyceride fraction) before reaction and after purification
Fatty acid Squid oil Purified
C14:0 5.7 3.4
C15:0 0.6 0.3
C16:0 22.8 13.5
C16:l 4.2 2.4
C18:0 1.7 0.9
C18: l 17.2 11.5
C18:2 1.7 1.3
C18:3 1.0 0.9
C20: l 4.4 3.4
C18:4 3.6 5.5
C20:4 1.0 1.3
C22: l 2.2 2.5
C20:5 12.4 17.2
C22:4 1.7 0.5
C22:5 0.7 1.0
C22:6 19.1 34.5
The reaction as well as the purification step was repeated and the result was a lipid composition in which there were at least 40 mo% EPA and DPA as well as at least 50 mol% of mono- and di-glycerides.
Breivik H, Haraldsson GG; (Norsk Hydro A/S, Norway), assignee. 1995 19950307.
Refined oil with high concentrations of polyunsaturated fatty acids.
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