Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1998-03-13
2003-03-25
Carr, Deborah D. (Department: 1621)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C554S169000, C554S227000
Reexamination Certificate
active
06537787
ABSTRACT:
FIELD OF THE INVENTION
A subject of the present invention is new processes for the production of polyunsaturated fatty acid esters in a pure or concentrated form.
A more particular subject of the invention is processes for the production of esters of polyunsaturated fatty acids of the &ohgr;-3 series starting with glycerides of fatty acids extracted from fish oils, phospholipids, or 1,2-dialkyene glycerols, using an enzymatic treatment.
A specific subject of the invention is a process for the production of glycerides of polyunsaturated fatty acids in a pure or concentrated form from fish oil or other sources, characterized in that it allows, by enzymatic treatment, a mixture to be obtained containing a high content of docosahexaenoic acid (DHA) and/or of eicosapentaenoic acid (EPA), which can reach, in the case of fish oils, 60%.
Also a subject of the invention is a process for the production of glycerides of polyunsaturated fatty acids, characterized in that phospholipids whose EPA and/or DHA content represents about 50% of the total fatty acids are obtained from natural phospholipids, in the presence of polyunsaturated fatty acids, by enzymatic treatment.
The invention also relates to a process for the production of synthetic glycerides, characterized in that a 1,2-dialkylene glycerol is subjected to an enzymatic action in the presence of concentrated or pure polyunsaturated fatty acids, in order to obtain a monoacylglyceride whose EPA and DHA content represents at least 70% of the total fatty acids.
The invention relates in particular to a process for obtaining polyunsaturated fatty acid concentrates which consists of subjecting a fish oil containing DHA and EPA to a selective enzymatic hydrolysis, in position
1
,
3
or
2
, in order to obtain a mixture of free fatty acids, monoglycerides and diglycerides, separating the constituents of this mixture, collecting the free fatty acids which are purified by crystallization from urea, in order to increase the content of EPA and/or DHA, decomplexing the isolated fatty acids, carrying out an inter-esterification between the free fatty acids, concentrated into polyunsaturated fatty acids, and the crude oil, in the presence of a lipase specific for position or steric hindrance, in order to obtain a mixture enriched with polyunsaturated fatty acid glycerides, which is separated and freed from the free fatty acids.
In a preferred manner, sardine oil is used which is obtained by pressing fresh sardines caught in cold waters. The sardine offers the advantage of an easy and constant supply. On the contrary, tuna vagina bulbi oil, which is industrially exploited, although it has a higher EPA and DHA content, has the disadvantage that the supply of raw material is limited.
The effect of the initial enzymatic hydrolysis is to split the ester function of the glycerol esterified by a polyunsaturated acid and to leave intact the other ester functions according to Diagram A.
It is also possible to hydrolyze the triglycerides present in sardine oil by a non-specific lipase so as to obtain a mixture of free fatty acids in which the polyunsaturated fatty acids represent about 30% of the total mixture. This mixture of fatty acids is fractionated by physical means in order to give a mixture in which the polyunsaturated fatty acids and in particular EPA and DHA predominant, which can be up to 70-80% of the mixture of free fatty acids.
The free polyunsaturated fatty acids are reesterified in the presence of an enzyme and in particular in the presence either of a non-specific lipase or a lipase specific for position
2
.
If a non-specific lipase is used, the glycerol will be reacted with the mixture of polyunsaturated free fatty acids and a triglyceride is obtained whose polyunsaturated fatty acid content is of the order of 60%.
If, using a lipase specific for position FA.
2
, a mixture of already-concentrated polyunsaturated fatty acids and a glyceride esterified in a single position by a polyunsaturated fatty acids in inter-esterified, a 1,2-diglyceride can be obtained in which only two positions are esterified by a polyunsaturated fatty acid.
It is also possible to hydrolyze a triglyceride, position
2
of which is esterified by a polyunsaturated fatty acid using a specific enzyme and in particular by a lipase of SN.2-specific type, in order to obtain a mixture of free polyunsaturated fatty acids, monoglycerides and diglycerides having a polyunsaturated fatty acid content comprised between 80 and 100%.
After fractionation of this mixture, the free fatty acids are purified by cold crystallization in the present of urea, in order to increase the EPA and DHA content. This concentrated mixture is then subjected to an inter-esterification by a triglyceride, position
2
of which is occupied by a polyunsaturated fatty acid, and positions
1
,
3
of which are occupied by a saturated fatty acid, in the presence of a 1,3-specific lipase, so as to obtain a triglyceride at least two hydroxyls of which are esterified by a polyunsaturated fatty acid.
The invention also relates to a synthesis process for triglycerides enriched with non-saturated fatty acids, which consists of saponifying fish oil by chemical route or by enzymatic route, in order to obtain a mixture of saturated and non-saturated fatty acids, converting the saturated fatty acids into lower alkyl esters in the presence of a selective lipase in order to obtain a mixture of alkyl esters of saturated fatty acids and of non-saturated fatty acids, separating the alkyl esters of the saturated fatty acid, collecting the free non-saturated fatty acids and reacting these fatty acids with glycerol in the presence of a specific lipase in order to obtain a mixture of triglycerides enriched with polyunsaturated fatty acids.
The attached diagrams illustrate these different variants of the process.
The enrichment of the crude sardine oil, by extraction of the polyunsaturated fatty acids (PUFA), from an average content of 30% to a fatty acid content corresponding to 50-60% of the total can be brought about according to one of the two schematized processes (A and B) according to the invention.
Until recently, the majority of the production of fish oil, considered to be a by-product of processing, was intended, after hydrogenation, for the production of magarine. The low added value of the product and the use of physico-chemical treatments for extraction, deodorization, decolouration and hydrogenation involving destruction of the properties of the oil, explains the lack of interest of manufacturers in this type of process.
The principle characteristic of fish oils is their high natural content (20-30%) of polyunsaturated fatty acids, of &ohgr; 3 types, and in particular of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). At present, fish oil is the only commercially-exploitable source of long chain polyunsaturated fatty acids, despite production attempts using microbiological processes.
Since the epidemiological studies reported by Bang et al in 1971, and the physiological studies on this subject, it has become apparent that the essential fatty acids are the principle constituents of the phospholipids of the retina, of grey matter, of the epidermis; they play an important role in the central nervous system and have significant pharmacological properties in cardio-vascular diseases, as antithrombotics. Consequently, the interest shown by the pharmaceutical, cosmetic and parapharmaceutical industries has given rise to a fast-expanding market, in the USA, in JAPAN and in EUROPE. The PUFA (polyunsaturated fatty acids) market, based on the quality of the oils and therefore the added value, has 3 areas of use:
crude oils, up to 30% of PUFA, for uses in the agricultural and food industry (AFI), oleochemistry, biopolymers, animal fodder.
enriched oils, with 30 to 60%, for pharmaceutical and cosmetic uses.
purified PUFA's, with 80 to 98%, for pharmacology.
At present, the grounds for objection of the fish oil industry reside at different levels, taking into account these new markets.
quality of the raw mate
Bierman, Muserlian and Lucas
Carr Deborah D.
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