JP2010202662A - Conjugated linoleic acid composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a process for providing large amounts of defined material in the development of a defined commercial source of CLA for both therapeutic and nutritional application. <P>SOLUTION: New compositions are provided containing conjugated linoleic acids efficacious as animal feed additives and human dietary supplements. Linoleic acid is converted to its conjugated forms in which the resulting composition is low in the contents of certain unusual isomers compared to conventional conjugated linoleic products. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to the field of human and animal nutrition, and in particular to certain novel compositions of conjugated linoleic acid (CLA). These compositions are prepared according to a novel method that controls the isomerization of 9,12-linoleic acid.

  In 1978, at the University of Wisconsin, researchers discovered the presence of substances in cooked beef that seem to inhibit mutagenesis. The material was found to be a mixture of structural isomers of linoleic acid (C18: 2) with conjugated double bonds. The c9, t11 and t10, c12 isomers are present in the greatest number, but it is uncertain which isomer is responsible for the observed biological activity. From labeling uptake studies, it appears that the 9,11 isomer is somewhat preferentially taken up and incorporated into the phospholipid fraction of animal tissue, and the 10,12 isomer appears to be in lower amounts. It is noted. (See Ha. Et al., Cancer Res., 50: 1097 (1991)).

  The biological activities associated with conjugated linoleic acid (named CLA) are diverse and complex. Currently, little is known about the mechanism of action, but several preclinical and clinical trials are increasingly appearing to shed new light on physiological and biochemical modes of action. The anti-carcinogenic properties of CLA are well documented. Administration of CLA inhibits rat breast tumor formation, as demonstrated by Ha. Et al., Cancer Res., 52: 2035s (1992). Ha. Et al., Cancer Res., 50: 1097 (1991) reported similar results in a mouse forestomach neoplasia model. CLA has also been identified in vitro as a potent cytotoxic agent against target human melanoma, colorectal and breast cancer cells. A recent major review has established conclusions drawn from individual studies (Ip, Am. J. Clin. Nutr., 66 (6 supplements): 1523s (1997)).

  Although the mechanism of CLA action remains unclear, there is evidence that certain component (s) of the immune system may be included, at least in vivo. US Pat. No. 5,585,400 (Cook et al., Incorporated herein by reference) describes a method for attenuating allergic reactions in animals mediated by type I or TgE hypersensitivity by administering a diet containing CLA. Is disclosed. CLA concentrations of about 0.1-1.0% have also been shown to be effective adjuvants in leukocyte storage. US Pat. No. 5,674,901 (Cook et al., Incorporated herein by reference) is associated with cellular immunity by administering CLA orally or parenterally in either the free acid or salt form It was disclosed that it resulted in an increase in CD-4 and CD-8 lymphocyte subpopulations. The adverse effects arising from pretreatment with exogenous tumor necrosis factor can be indirectly reduced by increasing or maintaining the levels of CD-4 and CD-8 cells in animals administered CLA. Finally, US Pat. No. 5,430,066 (incorporated herein by reference) describes the effect of CLA in preventing weight loss and anorexia by immune stimulation.

  Apart from the potential therapeutic and pharmacological applications of CLA as described above, there has been great excitement about the nutritional use of CLA as a dietary supplement. CLA has been found to have an enormous and comprehensive effect on body composition, especially redirecting the distribution of fat and lean body mass. US Pat. No. 5,554,646 (Cook et al., Incorporated herein by reference) discloses a method of utilizing CLA as a dietary supplement, where pigs, mice, and humans have 0.5% CLA. Was given a meal including. In each species, a significant decline in fat content was observed simultaneously with an increase in protein mass. In these animals, it is interesting that the increase in dietary fatty acid content with the addition of CLA did not result in weight gain and was accompanied by redistribution of fat and lean in the body. Another objective dietary phenomenon is the effect of CLA supplementation on feed conversion. US Pat. No. 5,428,072 (Cook et al., Incorporated herein by reference), incorporating CLA into animal (avian and mammalian) feed increases the efficiency of feed conversion and is supported by CLA. Data were provided indicating that it led to greater weight gain in the treated animals. The potential beneficial effects of CLA supplementation on food animal breeders are clear.

  Another important source of purpose in CLA, and what emphasizes its initial commercial potential, is that it occurs naturally in food and feed consumed by humans and animals. In particular, CLA is abundant in products derived from ruminants. For example, several studies have been conducted where CLA is surveyed in various dairy products. Aneja et al., J. Dairy Sci., 43: 231 (1990) observed that processing milk into yogurt resulted in CLA enrichment. (Shanta et al., Food Chem., 47: 257 (1993)) showed that the combination of increased processing temperature and whey addition increased the CLA concentration during the preparation of processed cheese. In another study, Shanta et al., J. Food Sci., 60: 695 (1995) did not observe any increase, although processing and storage conditions did not appreciably reduce CLA concentrations. Reported that. In fact, some studies have suggested that seasonal or inter-animal variability may account for as much as a three-fold difference in milk CLA content. (See, eg, Parodi et al., J. Dairy Sci., 60: 1550 (1977)). Also, as noted by Chin et al., J. Food Camp. Anal., 5: 185 (1992), dietary factors are related to variability in CLA content. Because of this variability in CLA content in natural sources, the intake of defined amounts of various foods is optimal to ensure that an individual or animal achieves the desired nutritional effect. There is no guarantee that the dose will be received.

  Linoleic acid is an important component of biological lipids and contains a significant proportion of triglycerides and phospholipids. Linoleic acid is known as an “essential” fatty acid, which means that because linoleic acid cannot be self-synthesized, animals must obtain it from an exogenous dietary source. Incorporation of the CLA form of linoleic acid can result in direct substitution of CLA to the lipid site to which the unconjugated linoleic acid would have migrated. However, this has not been proven and some of the observed effects that are very beneficial but not explained are that unconjugated linoleic acid in the lipid structure would otherwise not migrate. It can even result from rearrangement of CLA at the likely site. One source of animal CLA, especially in dairy products, is the biochemistry of certain ruminant bacteria on natural linoleic acid, first isomerizing linoleic acid to CLA and then secreting it into the rumen cavity. It is now clear that it comes from the sexual effects. Kepler et al., J. Nutrition, 56: 1191 (1966) isolated a ruminant bacterium, Butyrivibrio fibrisolvens, that catalyzes the formation of 9,11-CLA as an intermediate in the biohydrogenation of linoleic acid. Chin et al., J. Nutrition, 124: 694 (1994) further found that CLA found in rodent tissues is associated with bacteria, since rats without corresponding bacteria do not produce CLA.

  In the development of defined commercial sources of CLA for both therapeutic and nutritional applications, a process for preparing large quantities of defined materials is required. The problem with most CLA products made by conventional approaches is their heterogeneity and substantial variability in batch-to-batch isoforms. Considerable attention has been directed to the fact that ingesting large amounts of hydrogenated oil and shortening instead of animal fat results in a diet with high trans-fatty acid content. For example, Holman et al., PNAS, 88: 4830 (1991), found that rats fed with hydrogenated oils did not have unusual polymorphs that would interfere with the normal metabolism of naturally occurring polyunsaturated fatty acids in the rat liver. It was shown to cause accumulation of unsaturated fatty acid isomers. These relationships were summarized in the early editorial of Am. J. Public Health. 84: 722 (1974). Therefore, there is a strong need for biologically active CLA products with defined compositions.

SUMMARY OF THE INVENTION The present invention provides a novel composition of isomerized fatty acids derived from refined food grade seed oil. In practice, linoleic acid in seed oils selected to contain at least 50% linoleic acid is typically more than 90% of the 9,12-octadecadienoic acid isomer. During isomerization, 9,12-octadecadienoic acid is converted to a mixture of other isomers to form a composition having at least 50% CLA.

  Conjugated linoleic acid-containing compositions are intended for consumption by both humans and animals including food animals (eg, cattle, pigs, sheep, and birds), and as human medicines and nutritional supplements. It is an important object of the present invention to provide a safe and defined product for these applications. Conventional products also contain significant amounts of unknown fatty acid species and unusual isomers resulting from processing. Among the unusual CLA isomers are 11,13-octadecadienoic acid and 8,10-octadecadienoic acid isomers.

Accordingly, the present invention provides: A composition comprising at least 50% conjugated linoleic acid, wherein the composition is characterized by having less than 1% non-naturally occurring octadecadienoic acid isomers.

  2. The composition according to item 1, wherein the composition comprises a total of less than 1% 11,13-octadecadienoic acid isomer and trans-transoctadecadienoic acid isomer.

  3. The composition of claim 1, wherein the composition comprises a total of less than 1% of 8,10-octadecadienoic acid isomer and trans-transoctadecadienoic acid isomer.

  4). Item 2. The conjugated linoleic acid-containing composition of item 1, wherein the composition has a total level of less than 1% t9, t11-octadecadienoic acid and t10, t12-octadecadienoic acid.

  5. Item 5. The linoleic acid-containing composition according to items 1 to 4, wherein the composition is an isomerized product seed oil.

  6). Item 6. The linoleic acid-containing composition according to item 5, wherein the product seed oil is selected from the group consisting of sunflower oil and safflower oil.

  7). A biologically active conjugated linoleic acid composition comprising a mixture of free fatty acid conjugated linoleic acid isomers, the mixture comprising at least about 30% t10, c12 octadecadienoic acid, at least about 30% c9, A composition comprising t11 octadecadienoic acid and a total of less than about 1% 8,10-octadecadienoic acid, 11,13 octadecadienoic acid, and trans-transoctadecadienoic acid.

  8). 8. A composition according to item 7, further comprising an edible product incorporating said t10, c12 octadecadienoic acid.

  9. 9. A composition according to item 8, wherein the edible product is for human consumption.

  10. Item 9. The composition of item 8, wherein the edible product is a feed formulated for animal consumption.

  11. Biologically active acylglycerol compositions comprising multiple acylglycerol molecules of the following structure:

ここで、R₁、R₂、およびR₃は、ヒドロキシル基およびオクタデカジエン酸からなる群より選択され、該組成物は、少なくともほぼ30％のt10,c12オクタデカジエン酸、少なくともほぼ30％のc9,t11オクタデカジエン酸、および全部で約１％未満の8,10オクタデカジエン酸、11,13オクタデカジエン酸、およびトランス-トランスオクタデカジエン酸を位置R₁、R₂、およびR₃において含むことに特徴付けられる。

Wherein R ₁ , R ₂ , and R ₃ are selected from the group consisting of a hydroxyl group and octadecadienoic acid, and the composition comprises at least about 30% t10, c12 octadecadienoic acid, at least about 30% C9, t11 octadecadienoic acid, and a total of less than about 1% 8,10 octadecadienoic acid, 11,13 octadecadienoic acid, and trans-transoctadecadienoic acid at positions R ₁ , R ₂ , and characterized in that comprises at R _3.

  12 Item 12. The composition according to Item 11, further comprising an edible product incorporating said t10, c12 octadecadienoic acid.

  13. 13. A composition according to item 12, wherein the edible product is for human consumption.

  14 13. A composition according to item 12, wherein the edible product is a feed formulated for animal consumption.

  15. A biologically active conjugated linoleic acid composition comprising: a mixture of esters of conjugated linoleic acid isomers, the mixture comprising at least about 30% t10, c12 octadecadienoic acid, at least about 30% c9, t11 octadecadienoic acid and a total of less than about 1% 8,10 octadecadienoic acid, 11,13 octadecadienoic acid, and trans-trans octadecadienoic acid.

  16. 16. A composition according to item 15, further comprising an edible product incorporating said t10, c12 octadecadienoic acid.

  17. 18. A composition according to item 16, wherein the edible product is for human consumption.

  18. Item 18. The composition of item 16, wherein the edible product is a feed formulated for animal consumption.

19. Process for producing conjugated linoleic acid comprising:
Providing an alkali that is miscible with linoleic acid-containing seed oil, propylene glycol, and a non-aqueous medium;
Forming a reaction mixture formulated with the seed oil, the propylene glycol, and an alkali miscible with the non-aqueous medium;
Isomerizing the linoleic acid contained in the seed oil by heating to form conjugated linoleic acid; and aquefying to release glycerol.

  20. 20. The process of item 19, wherein the heating is performed at 130-165 ° C for about 2-6.5 hours.

  21. Item 20. The process of acidifying and releasing glycerol, combining vacuum drying to remove water, and molecular distillation to remove unconjugated linoleic acid impurities, and a further step of deodorization. process.

  22. Item 22. The process according to Item 21, wherein the heating is performed at 130-165 ° C for about 2-6.5 hours.

  23. 20. The process of item 19, further comprising the step of treating the free fatty acid conjugated linoleic acid with a lipase to form a triglyceride.

24. Process for producing low-impurity biologically active conjugated linoleic acid, including:
Providing an alkali that is miscible with linoleic acid-containing seed oil, propylene glycol, and a non-aqueous medium;
Treating the linoleic acid-containing seed oil to form an alkyl ester of the linoleic acid;
Forming a reaction mixture formulated with the alkyl ester, the propylene glycol, and an alkali miscible with the non-aqueous medium;
Isomerizing the alkyl ester by heating to form conjugated linoleic acid; and aquefying to release glycerol.

  25. 25. The process of item 24, wherein the heating is performed at 130-165 [deg.] C. for about 2-6.5 hours.

  26. Isomerized formulation reaction mixture comprising 30-60% processed seed oil, 10-40% alkali, and 30-60% propylene glycol.

  27. Animal feed synthesized from conventional ingredients in biologically active concentrations of conjugated linoleic acid alkyl esters in proportions typical for animal species and age.

  28. 28. The animal feed of item 27, wherein the concentration of the conjugated linoleic acid alkyl ester in the food is about 0.05 to 3.5% by weight.

  29. The conjugated linoleic acid alkyl ester is composed of c9, t11-octadecanoic acid alkyl ester and t10, c12-octadecanoic acid alkyl ester together with less than 1% 11,13-octadecanoic acid alkyl ester and trans-transalkyl ester. 28. The animal feed of item 27, consisting of at least 50% to about 99% by weight of octadecanoic acid alkyl ester isomer selected.

30. A process for producing conjugated linoleic acid alkyl esters for use in livestock feed, food ingredients, or human dietary supplements:
Providing a crude alkyl linoleic acid ester having a phosphatidyl residue in the range of about 0.1 to about 0.5%;
Treating with alkali alcoholate at low temperature in the presence of a monovalent low molecular weight alcohol to cause isomerization of at least 50% of the linoleic acid alkyl ester to a conjugated linoleic alkyl ester at low temperature;
A process comprising acidifying by adding an aqueous acid and separating the linole-conjugated linoleic acid alkyl ester from the aqueous acid without distillation.

  In this composition, less than 1% 11,13 isomer, less than 1% 8,10 isomer, less than 1% double trans species (t9, t11 and t10, t12 isomer), and 1% A high proportion of linoleic acid is first converted to conjugated c9, t11 and t10, c12 isomers in a carefully controlled reaction so that less than all unidentified linoleic acid species are present compared to conventional compositions. Yielding more than 90% of these isomers. In many individual product operations, the final composition has levels that are virtually undetectable by GC analysis of these species. 11,13, and 8,10, and the 1% limit on the concentration of trans-trans isomers serve as a convenient and practical quality assurance standard for purity for food grade products manufactured on a commercial scale .

The present invention also provides a new process for making new conjugated linoleic acid-containing compositions of the required purity and defined composition. The process involves the addition of an alkali (eg, potassium hydroxide, cesium hydroxide, cesium carbonate) or organic alkali (eg, tetraethylammonium hydroxide) miscible with a non-aqueous medium to a specific non-aqueous solvent (propylene glycol). Dissolving in the absence of the base isomerization catalyst system, formulating seed oil in alkaline propylene glycol, inert gas atmosphere and atmospheric pressure, in the range of 130-165 ° C, preferably about 150 ° C. A step of heating to a temperature without refluxing, a step of separating the fatty acid fraction by acidification, and a step of purification and dehydration by vacuum molecular distillation and / or centrifugation as necessary. If desired, the process flow can be suspended after the reaction mixture is prepared, either before or after the heating step. The mixture can then be stored for further processing in successive acidification and distillation steps and / or further processed elsewhere. After heating to effect isomerization, the isomerized formulated reaction mixture contains 30-60% processed seed oil, 10-40% alkali, and 30-60% propylene glycol. In this process, it is important to utilize propylene glycol due to the heating performance of the propylene glycol and the resulting isomerization pattern. The components of the dissolved fatty acid reaction mixture are present as follows:
30-60% seed oil
10-40% alkali
30-60% propylene glycol.

  Thus, in some embodiments, the process comprises forming a formulated reaction mixture comprising linoleic acid-containing seed oil, propylene glycol, and a non-aqueous medium miscible alkali, the above-mentioned seed oil comprising Isomerizing by heating linoleic acid to form conjugated linoleic acid, aquefying to release glycerol. Toxicity, such as that caused when other undesirable organic solvents (eg, ethylene glycol) are used, is avoided. Under non-reflux conditions, the processing temperature can be varied over a range to obtain the desired result with oils of different fatty acid compositions. Temperature is important because as the temperature increases, the proportion of trans, trans species, and other undesirable and unidentified species increases. Processing time requires about 2-6.5 hours and gives an isomerization yield greater than 90%, often as high as 99.5%. In some embodiments, the linoleic acid-containing seed oil can be first treated to produce an alkyl ester (eg, methyl ester or ethyl ester) of linoleic acid. In yet another embodiment, the conjugated linoleic acid produced can be incorporated into triglycerides by treatment with lipase in the presence of glycerol. In another embodiment, the present invention provides a low impurity CLA preparation produced by the process described above.

  In this process, due to the high natural 9,12 linoleic acid content of sunflower and safflower oil, but also low levels of sterol-contaminated phospholipids and fouling processing equipment and yielding a lower purity end product For other residues that tend, the use of sunflower and safflower oil is preferred. Other seed oils (e.g., corn, soybean, and linseed oil) can also be used, but the final product is not compositionally defined and the impurity level approaches the quality control thresholds contemplated above. The isomerization process itself becomes less predictable. In order to optimize the yield per processing unit, as a practical matter, seed oils containing at least 50% linoleic acid are desirable for industrial isomerization, but with lower or higher linoleic acid content. There is no process limitation to starting with the linoleic acid containing material that you have. Lower linoleic acid content can occur in situations where oil from different sources is formulated or when the oil is combined with non-oil components prior to isomerization. Similarly, the linoleic acid content of the isomerization fluid is much higher than the level present in natural seed oil, as in the situation where purified or synthesized linoleic acid is to be isomerized. possible.

  In some embodiments, the low impurity CLA described above can be provided as an acylglycerol or an alkyl ester. Accordingly, in some embodiments, acylglycerol compositions are provided that comprise a number of acylglycerol molecules of the following structural formula:

ここで、R₁、R₂、およびR₃は、ヒドロキシル基およびオクタデカジエン酸からなる群より選択され、組成物は、少なくともほぼ30％のt10,c12オクタデカジエン酸、少なくともほぼ30％のc9,t11オクタデカジエン酸、および全体で約１％未満の8,10オクタデカジエン酸、11,13オクタデカジエン酸、およびトランス-トランスオクタデカジエン酸を位置R₁、R₂、およびR₃において含むことに特徴付けられる。同じく、他の実施態様において、共役リノール酸異性体エステルの混合物を含む共役リノール酸組成物が提供され、混合物は、少なくともほぼ30％のt10,c12オクタデカジエン酸、少なくともほぼ30％のc9,t11オクタデカジエン酸、および全体で約１％未満の8,10オクタデカジエン酸、11,13オクタデカジエン酸、およびトランス-トランスオクタデカジエン酸を含む。

Wherein R ₁ , R ₂ , and R ₃ are selected from the group consisting of a hydroxyl group and octadecadienoic acid, and the composition comprises at least about 30% t10, c12 octadecadienoic acid, at least about 30% c9, t11 octadecadienoic acid and a total of less than about 1% 8,10 octadecadienoic acid, 11,13 octadecadienoic acid, and trans-transoctadecadienoic acid at positions R ₁ , R ₂ , and R Characterized for inclusion in ₃ . Similarly, in another embodiment, a conjugated linoleic acid composition comprising a mixture of conjugated linoleic acid isomer esters is provided, the mixture comprising at least about 30% t10, c12 octadecadienoic acid, at least about 30% c9, t11 octadecadienoic acid, and a total of less than about 1% 8,10 octadecadienoic acid, 11,13 octadecadienoic acid, and trans-trans octadecadienoic acid.

  In an alternative embodiment, the CLA free fatty acids, acylglycerols, and alkyl esters of the present invention can be formulated with edible products including animal feed and food for human consumption. In other embodiments, the CLA compositions of the present invention can be formulated with a physiologically acceptable carrier or oral delivery vehicle. In other embodiments, the biological effects of low impurity CLA may be utilized.

  In the present invention, feed or food safety conjugated linoleic acid alkyl esters are isomerized to the desired 10,12 and 9,11 isomers while limiting the formation of 8,10; 11,13; and trans, trans species. It is manufactured under conditions that preferentially control. Such conditions can be met by using an alkali alcoholate catalyzed reaction in which the seed oil breaks up to release free fatty acids from the glycerol backbone and then esterified prior to isomerization. The key to adapting this process to a commercially viable product is reducing the process steps that add cost. Typically, residues from non-oil components of seed oil (eg, sterols and phosphatides) foul equipment and reduce palatability for feed or food use. In the case of typical seed oils (eg, soy or corn), these residues are present in an amount sufficient to prevent the CLA-ester product from being used in the consumer product.

  In the compositions of the present invention, non-oil residues are not refined and removed from the oil component, but rather the oil source is selected to maintain such residues at acceptable levels. By selecting sunflower or safflower oil as the oil source, the definitive residue level is between 0.1 and 0.5% phosphatides, without extensive degumming and distillation processing steps, and It can be controlled to an unsaponifiable sterol fraction containing between 5 and less than 20% campesterol and stigmasterol, respectively. The resulting linoleic acid alkyl ester is octadecane representing a combination of various possible individual proportions of c9, t11-octadecanoic acid alkyl ester and t10, c12-octadecanoic acid alkyl ester, at least from 50% to about 99% by weight. Consists of acid ester isomers. In the alkaline alcoholate catalyzed process, approximately the same amount of each of these ester isomers is produced, but the relative proportions are altered by adding one or the other composition that is rich in one isomer. Can be done. CLA esters are then synthesized from conventional ingredients in feeds representative of animal species and age, and they are combined with conjugated linoleic acid alkyl esters in biologically active concentrations, generally from about 0.05 to 3.5. It can be incorporated into animal feed by formulating in weight percent.

The CLA-ester products of the present invention are obtained by direct isomerization of raw linoleic acid, for example a linoleic acid source that has not been subjected to a purification step. The CLA-ester composition comprises at least 50% by weight (substantially up to 100%) of ester isomers of a mixture of ester isomers of c9, t11-octadecanoate and t10, c12-octadecanoate. A second portion comprising less than about 10% aggregate 8,10-octadecanoic acid ester, 11,13-octadecanoic acid ester, and trans-trans-octadecanoic acid ester isomers, and total composition It has a third part containing phosphatidyl residue that is between 0.1 and 0.5% of the product weight. The alkyl group can be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and the like. Adjustment of the concentration of the c9, t11 and t10, c12 isomers can be made by adding a composition enriched in one or the other isomer, where c9, t11 or t10, c12 contained in the first composition part is Each yields an ester composition comprising more than 60% of all isomers of octadecanoic acid ester.
In a process embodiment of the present invention that results in a food grade composition suitable for animal feed, food ingredients, or human dietary supplements, unpurified CLA-esters having a phosphatidyl residue of less than 0.5% are monovalent Treated with alkali alcoholate in the presence of a low molecular weight alcohol (e.g. methyl or ethyl alcohol) and continued at low temperature (about 90-145 ° C) until at least 50% of the ester is converted to CLA-ester, aqueous It is acidified by adding an acid and then the CLA-ester is separated from the aqueous acid without a distillation step.

FIG. 1 is a flow diagram of the procedure used to generate CLA.

Detailed Description of the Invention Definitions:
As used herein, “conjugated linoleic acid” or “CLA” refers to any conjugated linoleic acid or free fatty acid of octadecadiene. This term is intended to encompass and suggest all positional and geometric isomers of linoleic acid having two conjugated carbon-carbon double bonds anywhere in the molecule. CLA differs from normal linoleic acid in that normal linoleic acid has double bonds at carbon atoms 9 and 12. Examples of CLA include the cis- and trans isomers of the following regioisomers (“E / Z isomers”): 2,4-octadecadienoic acid, 4,6-octadecadienoic acid, 6,8 -Octadecadienoic acid, 7,9-octadecadienoic acid, 8,10-octadecadienoic acid, 9,11-octadecadienoic acid, and 10,12-octadecadienoic acid, 11,13-octadecadiene acid. As used herein, “CLA” is a single isomer, a selected mixture of two or more isomers, and a non-selective mixture of isomers obtained from a natural source. And synthetic and semi-synthetic CLA.

  As used herein, a “triglyceride” of CLA is intended to include CLA at any or all three positions of the triglyceride backbone. Thus, triglycerides containing CLA can include any positional and geometric isomers of CLA.

  As used herein, an “ester” of CLA is an alcohol or any other chemical group (a physiologically acceptable, naturally occurring alcohol (e.g., methanol, ethanol, Including, but not limited to, any and all positional and geometric isomers of CLA. Thus, an ester of CLA or esterified CLA can include any positional and geometric isomer of CLA.

  “Non-naturally occurring isomers” of CLA include octadecadienoic acid c11, t13; t11, c13; t11, t13; c11, c13; c8, t10; t8, c10; t8, t10; c8, c10; Including but not limited to trans-trans isomers, and not including the t10, c12 and c9, t11 isomers of octadecadienoic acid. “Non-naturally occurring isomers” can also be indicated as “minor isomers” of CLA, since these isomers are generally produced in small amounts when CLA is synthesized by alkaline isomerization.

  As used herein, “low impurity” CLA comprises a total of less than 1% 8,10 octadecadienoic acid, 11,13 octadecadienoic acid, and trans-transoctadecadienoic acid. Figure 2 shows a CLA composition containing free fatty acids, alkyl esters, and triglycerides.

  “Prepared edible product” means any pre-packaged food approved for human consumption.

  As used herein, “c” includes a chemical bond in the cis orientation and “t” indicates a chemical bond in the trans orientation. Where CLA positional isomers are represented without "c" or "t", the representation includes all four possible isomers. For example, 10,12 octadecadienoic acid includes c10, t12; t10, c12; t10, t12; and c10, c12 octadecadienoic acid, whereas t10, c12 octadecadienoic acid or CLA is exactly that Indicates a single isomer.

  As used herein, the term “oil” refers to a free flowing liquid that contains long chain fatty acids (eg, CLA) or other long chain hydrocarbon groups. Long chain fatty acids include, but are not limited to, various isomers of CLA.

  As used herein, the term “physiologically acceptable carrier” refers to any carrier or excipient normally used with oily pharmaceuticals. Such carriers or excipients include but are not limited to oil, starch, sucrose, and lactose.

  As used herein, the term “oral delivery vehicle” refers to any means of orally delivering a pharmaceutical product, including but not limited to capsules, pills, tablets, and syrups.

  As used herein, the term “edible product” refers to any food or feed suitable for consumption by humans, non-ruminants, or ruminants. An `` edible product '' is a prepared and packaged food (e.g., mayonnaise, salad dressing, bread, or cheese food) or animal feed (e.g., extruded and crushed animal feed, Or a rough mixed feed).

  The compositions of the present invention result from a highly controlled isomerization process and from using a suitable starting material, sunflower or safflower oil. This composition has not heretofore been obtained for industrial scale applications. This is because traditional processes historically produce conjugated linoleic acid for a completely different purpose, as a so-called drying oil in the paint industry. There was also no correct assessment of the implications of the isomer content of the final product. This is because analytical methods for characterizing fatty acids have not been widely available.

In older isomerization processes, some of them are still used in a more modern appearance, and the production of conjugated fatty acids is aqueous at high temperatures above 200 ° C and usually at superatmospheric pressures. Performed in alkali (generally NaOH). For example, U.S. Pat.No. 2,350,583 (Bradley) discloses an aqueous alkaline process utilizing treated soap that has undergone both conjugation and polymerization under fairly severe conditions of 200-250 ° C. for several hours. ing. A fraction of dry oil was obtained by distillation starting with linseed oil (see also GB 558,881 for a very similar process). In a variation of the process, U.S. Pat. No. 4,381,264, for obtaining a double bond conjugation of various polyunsaturated fatty acids, low water content reaction zone (0.5% water) the presence of SO _2, the stoichiometry Teaches a process involving a target base. The aqueous alkaline process was adapted in US Pat. No. 4,164,505 to a continuous flow process in which alkali metal hydroxide and water were continuously charged into a flow zone maintained between 200 ° C. and 370 ° C. At those temperatures, the reaction time should be greatly shortened, but relatively little control over isomerization is possible. At the upper end of the temperature range, it is expected to be almost completely converted to a double trans species.

  Methods for producing CLA using various non-aqueous solvents and catalysts have been described in the literature. Burr (US Pat. No. 2,242,230) discloses the use of solvents such as methanol, butanol, ethanol, and glycol in combination with various catalysts. These reaction parameters are summarized in Table 1. With the exception of glycol, the reaction was carried out either under reflux conditions or in a sealed tube. These reaction conditions result in inaccurate control of two important reaction parameters—temperature and pressure—identified by the inventors. Inaccurate control of these reaction parameters appears to lead to more incomplete conjugation and formation of undesirable isomers.

同様に、Baltesら(米国特許第3,162,658号)は、脂肪酸の共役化のための触媒として、非水性溶媒および種々の金属塩基の使用を開示している。Baltesらにより記載される方法の種々の反応パラメーターは、表２に要約される。Baltesらはまた、種々の低沸点溶媒の使用を開示している。これらの反応のほとんどが、用いられる溶媒の沸点より高い温度で行われたことから、反応は加圧下で行われ、圧はオクタデカジエン酸異性体の形成に影響する独立した要因であることが明らかである。従って、これらの反応に由来する生成物は、望ましくない異性体を含む。

Similarly, Baltes et al. (US Pat. No. 3,162,658) disclose the use of non-aqueous solvents and various metal bases as catalysts for the conjugation of fatty acids. The various reaction parameters of the method described by Baltes et al. Are summarized in Table 2. Baltes et al. Also disclose the use of various low boiling solvents. Since most of these reactions were conducted at temperatures above the boiling point of the solvent used, the reaction was carried out under pressure, and pressure was an independent factor affecting the formation of octadecadienoic acid isomers. it is obvious. Therefore, the products derived from these reactions contain undesirable isomers.

本発明のCLAは、8,10異性体、11,13異性体、および種々のトランス-トランス異性体のような異性体を欠いている。この組成物は、図１のフローダイヤグラム様式に示される厳密に制御された非水性アルカリ異性化プロセスにより生成された。好ましくは、ヒマワリ油またはベニバナ油が、不活性ガス雰囲気下、大気圧で、高沸点溶媒、すなわちプロピレングリコール中、溶媒の沸点より低い温度で、過剰のアルカリと反応させられる。これらの反応条件は、共役化プロセスの温度(および一定の大気圧)の正確な制御を可能にする。好ましくは、アルカリは、水酸化カリウム、水酸化セシウム、炭酸セシウムのような無機アルカリ、またはテトラエチルアンモニウムヒドロキシドのような有機アルカリである。好ましくは、触媒は、油の脂肪酸含有量と比較して１モル過剰に提供される。溶媒はプロピレングリコールである。好ましくは、反応は、130〜165℃の温度範囲内で、最も好ましくは約150℃で行われる。反応の時間は変更され得るが、しかしながら、反応が長時間行われる場合、望ましくない異性体の形成の見込みも増加する。2.0〜6.5時間という比較的短い反応時間が優れた収率のために申し分ないことを証明している。

The CLA of the present invention lacks isomers such as 8,10 isomers, 11,13 isomers, and various trans-trans isomers. This composition was produced by a strictly controlled non-aqueous alkaline isomerization process shown in the flow diagram format of FIG. Preferably, sunflower oil or safflower oil is reacted with excess alkali in an inert gas atmosphere at atmospheric pressure and in a high boiling point solvent, ie propylene glycol, at a temperature below the boiling point of the solvent. These reaction conditions allow for precise control of the temperature (and constant atmospheric pressure) of the conjugation process. Preferably, the alkali is an inorganic alkali such as potassium hydroxide, cesium hydroxide, cesium carbonate, or an organic alkali such as tetraethylammonium hydroxide. Preferably, the catalyst is provided in a 1 molar excess compared to the fatty acid content of the oil. The solvent is propylene glycol. Preferably, the reaction is conducted within a temperature range of 130-165 ° C, most preferably about 150 ° C. The time of the reaction can be varied, however, if the reaction is carried out for a long time, the likelihood of formation of undesirable isomers also increases. A relatively short reaction time of 2.0 to 6.5 hours proves to be satisfactory for an excellent yield.

  It will be appreciated by those skilled in the art that the reaction conditions described above can be varied depending on the oil to be conjugated, the source of alkali, and the equipment to produce the desired composition. Pre-analysis of a particular oil can suggest that conditions must be changed to obtain the desired composition. Therefore, temperature ranges, pressures, and other reaction parameters represent starting points for individual process plans and are intended as guides only. For example, the temperature ranges described are not meant to be the only ranges that can be used. The basic aspect is to provide accurate temperature control. However, care must be taken because an increase in pressure can lead to incomplete isomerization and formation of undesirable isomers. Finally, the length of the conjugation reaction can be varied. In general, an increase in the amount of undesired isomers occurs with an increase in the length or reaction. Therefore, the optimal reaction time will allow the reaction to proceed almost or essentially to completion, but will not result in the formation of undesirable isomers.

  Following the conjugation reaction, the resulting CLA-containing composition can be further purified according to FIG. To separate the fatty acids from the conjugated reaction mixture, the reaction mixture is cooled to approximately 95 ° C, an excess of 50 ° C water is added, and the mixture is slowly cooled down to about 50 ° C to 60 ° C. Stir. When adding water, a fatty acid soap is formed and glycerol is formed as a by-product. Next, a 1 molar excess of concentrated HCl is added with stirring. The aqueous and non-aqueous layers are then separated at about 80-90 ° C. The lower layer containing water and propylene glycol is then extracted. Residual propylene glycol is removed by vacuum dehydration at 60-80 ° C.

The dry CLA composition can then preferably be degassed with a degassing unit with a cold trap to remove any residual propylene glycol. The CLA is then distilled at 190 ° C. in a molecular distillation plant with a vacuum of 10 ⁻¹ to 10 ⁻² mbar. The advantage of this purification system is that the time during which CLA is kept at an elevated temperature is short (less than 1 minute). Conventional batch distillation procedures should be strictly avoided because they contain elevated temperatures of approximately 180-200 ° C. for up to several hours. At these elevated temperatures, undesirable trans-trans isomer formation occurs. Nearly 90% of the feed material is recovered as a slightly yellow distillate. The CLA can then be deodorized by heating at about 120 ° C. to 170 ° C., preferably about 150 ° C. for 2 hours to improve odor and taste. Excessive heating can lead to the formation of trans-trans isomers. These procedures produce CLA compositions having a solvent level of less than about 5 ppm, preferably less than about 1 ppm. This process removes toxic trace levels of solvent so that the resulting composition is essentially free of toxic solvent residues.

  The process described above is readily adaptable to both experimental and commercial scales. For example, 400 kg safflower oil can be conjugated at 400C for 5 hours with 200 kg KOH added as a catalyst in 400 kg propylene glycol. The resulting CLA can then be purified as described above. Furthermore, commercial scale batch systems can be easily modified to produce the desired CLA composition. For example, stainless steel reactors should preferably be lined with glass to prevent corrosion due to PH levels below 3.0. However, it should be noted that conjugation processes utilizing non-aqueous solvents are generally not as corrosive as those performed with water.

  Preferred oils for conjugation are sunflower and safflower oil. Compared to soybean oil, these oils have lower concentrations of undesirable components such as phosphatides and sterols. These undesirable components can contribute to the formation of gums and other undesirable polymers that foul the conjugation device. The various properties of these oils are summarized in Tables 3, 4, and 5.

続く実施例において、最適条件に及ばない条件下または先行技術の水性アルカリ方法に従ってのいずれかで作られたCLA組成物と比較して、本CLA組成物の鍵となる性質を強調するために、いくつかの比較実験が行われた。実施例１において、CLAは、本方法に従い調製された。実施例２において、CLAは、従来の水性アルカリ方法により調製された。実施例３において、高温においてという以外、実施例１の反応が実質的に繰り返される。最後に、実施例４において、実施例２の反応と実質的に同一である水性アルカリ反応が低温で行われる。各実験の正確な条件および詳細は、実施例において記載される。CLA異性体含有量の分析の概略は、表１〜４に記載される。

In the examples that follow, to highlight the key properties of the present CLA compositions as compared to CLA compositions made either under suboptimal conditions or according to prior art aqueous alkaline methods, Several comparative experiments were conducted. In Example 1, CLA was prepared according to this method. In Example 2, CLA was prepared by a conventional aqueous alkaline method. In Example 3, the reaction of Example 1 is substantially repeated except at high temperatures. Finally, in Example 4, an aqueous alkaline reaction that is substantially identical to the reaction of Example 2 is performed at low temperatures. The exact conditions and details of each experiment are described in the examples. A summary of analysis of CLA isomer content is given in Tables 1-4.

  Referring to the data in Table 5, for each of the four experiments, a relative range percentage is given for each identified peak corresponding to an individual isomer. The GC plot gave a number of peaks for each sample tested. The range under each of these peaks was integrated to obtain the overall value. The identification of the peak was determined by its relative position from published standard elution profile and chemical literature. The top row represents the residual value of the unconjugated starting material, 9,12-linoleic acid. Both the low and high temperature reactions in propylene glycol gave extremely high conversions in excess of 99% of the total starting material.

  Referring to column 1, unlike any of the control compositions of Example 1, the peak corresponding to the 11,13 isomer mixture, in particular the peak corresponding to c11, c13, the peak for any of the 8,10 isomers, It is clearly evident that the peaks for the unidentified isomers have completely disappeared. In the case of the C9, t11 isomer, the peaks for both the 8,10 and 9,11 isomers in the GC overlap, and the example was determined by subtracting the portion of the peak identified as 8,10 by NMR studies. Only one material was elucidated here. Since this was not done in other experiments, row 3 gives the combined values 8, 10, and 9, 11, for Examples 2-4. In general, for 8, 10, 11, 13, and unidentified isomers, values less than 1% undetectable are of therapeutic and nutritional values. This is because contaminants that are potentially harmful to the body and mind, especially those known to be suspected of absorption pathways in lipogenesis, are reduced to trace levels. In non-ruminants, for example, adding 0.25-2.5% CLA to the diet can increase the range of CLA in tissues in ruminants, so other animals have alternative isomers. Can be a source of CLA provided.

  Example 2 provides a sample of a typical aqueous alkaline product of CLA produced conventionally. The conversion is less efficient both overall and in the generation of the c9, t11 and t10, c12 isomers. A high proportion of 11,13 isomers are suspected, and an obvious proportion of unidentified material is also noted.

  Example 3 illustrates the critical importance of the temperature parameter. The upward shift in temperature in the propylene glycol medium sharply increases the amount of contaminating isomers expense the c9, t11 and t10, c12 isomers. Also of interest is a dramatic increase in trans, trans species, due to the desire for double bond rearrangements that produce more stable electron conjugation at higher temperatures and increased energy stress levels.

  Example 4 illustrates that a decrease in temperature in an aqueous alkaline system actually reduces the amount of some contaminating isomers. However, the formation of this electron conjugation is explained by the overall mechanical energy of the system, since there is a dramatic drop in yield and the level of the 11,13 isomer group remains very high. Rather, it is suggested that the effect of the base in the aqueous medium is more greatly influenced. It is also noted that extremely long reaction times of 22.5 hours; too long for more efficient industrial scale batch processes.

  Table 6 only converted the relative isomer proportions in the various reactions to their corresponding peak ratios as a function of peak range. This process produced a virtually complete conversion of 9,12-linoleic acid to approximately equal amounts of each of the two desired CLA isomers. At higher temperatures, even in propylene glycol, the 11,13-isomer generation remains below 1/3 that of the low temperature aqueous alkaline process.

  In some embodiments, the present invention also provides a method for producing alkyl esters of CLA. After fat splitting and dehydration, the free fatty acids are combined with methanol or other monohydric low molecular weight alcohol and heated to the temperature at which the alcohol boils. Esterification proceeds under reflux conditions while removing reaction water through a condenser. After adding an additional amount of the same or another monohydric alcohol, the alcoholate catalyst is formulated into the ester mixture. Typical alcoholate catalysts are sodium or potassium ethoxide, or their methyl, butyl, or propyl counterparts.

  In esterification, methanol or ethanol is preferred, but other branched or straight chain monohydric alcohols can be used. The longer the aliphatic chain of the alkyl group, the more lipid-compatible the material. Also, the viscosity tends to increase. For different types of feeds or foods, their consistency varies and products of different viscosities can obtain the desired flow or synthetic characteristics without affecting the therapeutic or nutritional properties arising from the CLA part. Can be used. The theory and practice of esterification is conventional. A basic description of the most common methods is given in McGraw-Hill Encyclopedia of Science & Technology, McGraw-Hill Book Co., N. Y .: 1996 (5th edition). Since the animal and human bodies have various esterases, CLA-esters are cleaved and readily release free fatty acids. Tissue uptake may have different dynamics depending on the tissue involved and the benefits required.

  In the isomerization process, it has been found that the alcoholate catalyst produces a product that is much superior to aqueous alkali-mediated isomerization. The latter process always produced undesirable isomers even under mild reaction conditions. The milder conditions give the undesired isomers in lower amounts but are very uneconomical in yield as shown in the examples. In most systems, the appearance of the c9, t11 and t10, c12 isomers dominates and they are formed in approximately equimolar amounts. It has never been possible to control the isomerization of one isomer by excluding the other. Increasing the proportion of one or the other isomer (depending on the physiological effect to be achieved) is desirable, but currently this is often a rich source of the desired isomer. Must be done by adding

  The present invention contemplates the use of pure preparation derivatives of CLA. For example, CLA can be provided free or linked through an ester linkage, or provided in the form of an oil-containing CLA triglyceride as described in Examples 5 and 6. In these embodiments, the triglyceride can consist of CLA partially or completely connected to the glycerol backbone. CLA can also preferably be provided as a methyl ester or ethyl ester as described in Examples 8 and 9. Still further, CLA can be in the form of a non-toxic salt such as a potassium or sodium salt (eg, by reacting the same amount of free acid with an alkali hydroxide at about 8-9). Salt formed).

  In one embodiment of the invention, a novel triacylglycerol is synthesized comprising a novel CLA isomer mixture disclosed herein below for non-aqueous isomerization of linoleic acid from sunflower and / or safflower oil. The Pure triacylglycerol rich in CLA (90-96%) can be confirmed by HNMR. Esterification proceeds using immobilized Candida antarctica lipase. Preferably, the CLA comprises at least 40% up to 45-48% c9, t11-octadecadienoic acid and t10, c12-octadecadienoic acid, and mixtures thereof. Less than 1% of esters 8,10; 11,13; and trans, trans isomers, or a total of less than 5%. The resulting triacylglycerol is not further purified to remove all levels of phosphatidyl and sterol residues. However, these levels remaining from the isomerization of sunflower and safflower oil are sufficient for commercial applications including safe and edible products in feed and food.

  Immobilized Candida antarctica lipase should be used in a manner similar to that described for Harraldson et al. For n-3 polyunsaturated fatty acids. The esterification reaction is carried out in the absence of any solvent at 50 ° C. to 75 ° C., preferably at 65 ° C., and a vacuum is used to remove water or alcohol (derived from the ester) that is by-produced during formation It is done. This completes the triacylglycerol product and ensures a very pure product, essentially free of any mono- and diacylglycerol, in essentially quantitative yield. A stoichiometric amount of free fatty acids can be used (ie, 3 molar equivalents based on glycerol, or 1 molar equivalent based on the number of molar equivalents of hydroxyl groups present in the glycerol moiety). Based on the total weight of the substrate, only a 10% dose of lipase is required and can be used any number of times. This is very important from the viewpoint of productivity. All of this, together with the fact that no solvent is required, makes this process highly feasible from a scale-up and industrialization perspective. This is because the reduction in volume and bulkiness is enormous. Also, a slight excess (<5/5) of free fatty acids can be used to speed up the end of the reaction and ensure the completion of the reaction.

  At the start of the reaction, 1- or 3-mono-acyl glyceride (acyglyeride) is formed first, followed by 1,3-diacyl glyceride (idacy), and finally triglyceride with more extended reaction time . Mono- and diacylglyerides are alternative molecular synthetic pathways, such as the synthesis of phospholipids or other functional lipids, although they exhibit physiological activity but have greater solubility in an aqueous cytoplasmic environment It is a useful intermediate in that it can precipitate in In contrast, triglycerides often accumulate intact in cell membranes or storage vesicles. Therefore, administering CLA in mono-, di-, or triglycerol form, rather than free fatty acids or esters, affects the mode and distribution of CLA component uptake, metabolic rate, and structural or physiological roles. obtain.

In one preferred embodiment, administration is oral. CLA can be formulated in tablets, pills, dragees, capsules, solutions, liquids, slurries, suspensions, and emulsions with a suitable carrier such as starch, sucrose, or lactose. The CLA can be provided in either an aqueous solution, an oily solution, or any other form described above. The tablets or capsules of the present invention may be coated with an enteric coating that dissolves at a pH of about 6.0 to 7.0. A suitable enteric coating that dissolves in the small intestine but not in the stomach is cellulose acetate phthalate. In some embodiments, CLA is provided as a soft gelatin capsule (Tonalin ^™ ) containing 750 mg of 80% CLA. CLA can also be provided by any of a number of other routes including, but not limited to: intravenous, intramuscular, intraarterial, intramedullary, intradural, intraventricular, percutaneous. Subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal methods. Further details on formulations for administration and techniques for administration can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).

  An effective amount of CLA can also be provided as an adjunct to various prepared edible products and drinks. For the purposes of this application, prepared edible product means any natural processed meal or non-edible edible product to which CLA has been added. CLA can be added in the form of free fatty acids or as an oil containing part of CLA or complete triglycerides. Therefore, CLA can be incorporated directly into various prepared edible products including, but not limited to: diet beverages, diet bars, supplements, prepared frozen foods, candy, snack products (eg, Chips), prepared meat products, milk, cheese, yogurt, and any other fat or oil-containing food.

  CLA is susceptible to oxidation. Therefore, for human use, wrap CLA with a suitable antioxidant, such as a lectin, tocopherol, ascorbate, ascorbyl palmitate, or a spice extract such as rosemary extract Is desirable.

Example 1
Isomerization of safflower oil using propylene glycol at low temperature Safflower oil was isomerized using KOH as a catalyst at low temperature in propylene glycol. The isomerization device consists of a two-necked flask with a thermometer in one neck, leaving a small opening to relieve excess pressure. A nitrogen supply was connected to the other neck of the flask. The solution added to the flask was stirred using a magnetic stir bar and magnetic stirrer. The temperature of the flask was controlled by placing the flask in an oil bath with automatic temperature control installed on a magnetic stirrer.

  The flask was filled with 60.27 g propylene glycol and 28.20 g KOH and immersed in an oil bath. The temperature was raised to 130 ° C. to dissolve the KOH. After KOH was dissolved, 60.09 g safflower oil was introduced into the flask. A large volume of nitrogen was circulated in the 2-neck flask for 5 minutes and then reduced to a smaller volume. The mixture was heated to 150 ° C. and maintained for approximately 40 minutes. The mixture was then reacted at 150 ° C. for 3.5 hours. Occasionally, 3 ml samples were removed for analysis.

The sample was placed directly in 6 ml of hot water and citric acid was added in excess until the free fatty acids separated as the top layer. Heating was necessary to prevent solidification while citric acid was added. To convert free fatty acids to methyl esters for analysis by gas chromatography, 0.025 g of free fatty acids, 5 ml of 4% HCl solution, and ethanol were added to the test tube. Nitrogen was added to the tube, then the tube was sealed and placed in a water bath at 60 ° C. for 20 minutes. The tube was then cooled and 1 ml purified water and 5 ml isooctane were added. Nitrogen was added to the tube and the tube was shaken for 30 seconds. The resulting upper layer was added to 1 μl of purified water in a new tube and shaken again under nitrogen. The resulting upper layer was then washed with isooctane and decanted into a third test tube. A small amount of sodium sulfate was added for moisture absorption. A 1 μl sample was then injected directly into the gas chromatography.
Gas chromatography conditions were as follows:
System: Perkins-Elmer Automatic system injector: 240 ° C without split Detector: Hydrogen ionization detector at 280 ° C Carrier: Helium column: WCOT fused silica 0.25mm x 100M, CP-SL88 for FAME,
DF 0.2
Heating program: 80 ° C (0 minutes) at 10 ° C per minute, up to 220 ° C and then 220 °
Hold for 10 minutes.

  All results are expressed as a percentage of the relative peak area. Since standards are generally not available, the eluted peaks were confirmed with other systems. GC-MS determines the number but not the position of cis and trans bonds. Therefore, NMR analysis was used to confirm the position of the bond. The main peaks were c9, t11 and t10, c12. For NMR analysis of CLA isomers, see Marcel S.F. Lie Ken Jie and J. Mustafa, Lipids, 32 (10) 1019-34 (1997) (incorporated herein by reference).

  This data, shown in Table 6 and summarized in Table 10, shows that the isomerization of safflower oil using polypropylene glycol as solvent, KOH as catalyst, and low temperature lacks the 8,10 and 11,13 isomers. It has been demonstrated to result in the production of linoleic acid. The highly polar column utilized in this experiment can be successfully used to separate the 8,10 and 11,13 isomers from the c9, t11 and t10, c12 isomers. The 8,10 isomer tends to elute simultaneously with the c9, t11 isomer or immediately after. The 11,13 isomer elutes before the t10, c11 isomer or coelutes with the t10, c12 isomer, depending on the column conditions.

  Conjugated linoleic acid produced according to this method was characterized by comparison with various produced isomers. First, the isomerization reaction proceeded to completion essentially. Completion of the reaction is obtained by dividing the total peak area by subtracting residual c9, t12 linoleic acid from the linoleic acid isomer by the total peak area. This value is 0.994. Second, the ratio of the c9, t11 and t10, c12 isomers to the total peak area can be determined. This value is 0.953. Third, the ratio of t9, t11 and t10, t12 isomers to c9, t11 and t10, c12 isomers can be determined. This value is 0.010. Fourth, the ratio of t9, t11 and t10, t12 isomers to the total peak area can be determined. This value is 0.009. Fifth, the ratio of the t10, c12 isomer to the c9, t11 isomer can be determined. This value is 1.018. These ratios are summarized in Table 11.

Example 2
Aqueous isomerization at high temperature and pressure
50 g water and 25.32 g NaOH were added to a high pressure reactor (Parr Model 450ML Benchtop Alloy 400, equipped with a pressure gauge and stirrer). NaOH was dissolved and 94.0 g safflower oil was added to the reactor. The reactor was closed and flushed with nitrogen for 2 minutes, then all valves were closed. The reactor was heated to 210 ° C. in an electric gasket and maintained at that temperature for 6 hours. The temperature was then lowered to 60 ° C., then the pressure was released and the reactor was opened. 2 g of the resulting solidified soap was removed from the reactor and dissolved in water at approximately 40 ° C. Citric acid was then added to lower the pH of the solution below 6. Samples were withdrawn from the fatty acid top layer and prepared for gas chromatography as in Example 1.

  Gas chromatographic results are presented in Table 7 and summarized in Table 10. These data indicate that this isomerization method forms relatively large amounts of 8,10 and 11,13 isomers. The ratio is shown in Table 11.

Example 3
Non-aqueous alkaline isomerization of safflower oil under high temperature and pressure
100.48 g propylene glycol and 46.75 g KOH were added to the high pressure reactor as described in Example 2. The reactor was then heated to 130 ° C. to dissolve KOH. Then 100.12 g safflower oil was added to the KOH-propylene glycol mixture. The reactor was closed, flushed with nitrogen for 1 minute, and all valves were closed. The reactor was then heated to 210 ° C. and maintained at that temperature for 1 hour. The reactor was cooled and the contents decanted into 120 g of hot water. While stirring, 35.3 g 37% HCl and 27.59 g citric acid were added sequentially to the fatty acid. The sample was removed from the top layer and dried in a vacuum flask at 60 ° C. The resulting fatty acid sample was analyzed by gas chromatography as described in Example 1.

  Results are presented in Table 8 and summarized in Table 10. This experiment shows that high temperature isomerization of safflower oil with KOH and non-aqueous solvents results in the formation of significant amounts of the 8,10 and 11,13 isomers, and the t9, t11 and t10, t12 isomers Prove that. The ratio is shown in Table 11.

Example 4
Aqueous alkaline reaction at low temperature
49.94 g water and 39.96 g NaOH were added to the high pressure reactor as described in Example 3. The mixture was heated until the NaOH was dissolved. Next, 100.54 g safflower oil was added to the high pressure reactor, the reactor was flushed with nitrogen, and all valves were closed. The high pressure reactor was heated to 179 ° C. for 22.5 hours. Samples were prepared for gas chromatography as in Example 3. Data is provided in Table 9 and summarized in Table 10. This experiment demonstrates that the conjugation reaction does not progress to completion when using low temperatures for aqueous alkaline isomerization. Furthermore, significant amounts of the 8,10 and 11,13 isomers are produced. The ratio is shown in Table 11.

* -8,10の合計の割合は、0.5未満である
na-値はc9,t11/8,10の成分として反映される

* The total ratio of -8,10 is less than 0.5
na-values are reflected as components of c9, t11 / 8,10

*c9,t11は、8,10異性体を含む
na- 11,13は検出されず
実施例５
直接エステル化によるCLAのトリアシルグリセロールの調製
一般に、H核磁気共鳴スペクトルは、溶媒として重水素化クロロホルム中、BrukerAC 250 NMR分光器で記録された。HPLC分離は、Waters 製 PrepLC(R)System
500A 機器により、Millipore製PrepPak(R)500/Silica Cartridge カラムを使用して、石油エーテル中10％のジエチルエーテルで溶出して行った。分析GLCは、Haraldssonら、ActaChem.Scanned. 45：723 (1991)に記載されるように、先に記載される手順に従って、Perkin-Elmer 8140 ガスクロマトグラフで行った。

* c9, t11 includes 8,10 isomers
na- 11,13 not detected Example 5
Preparation of CLA triacylglycerols by direct esterification In general, H nuclear magnetic resonance spectra were recorded on a Bruker AC 250 NMR spectrometer in deuterated chloroform as solvent. HPLC separation is performed by Waters PrepLC (R) System
This was done on a 500A instrument using a Millepore PrepPak® 500 / Silica Cartridge column eluting with 10% diethyl ether in petroleum ether. Analytical GLC was performed on a Perkin-Elmer 8140 gas chromatograph according to the procedure described previously, as described in Haraldsson et al., ActaChem. Scanned. 45: 723 (1991).

Immobilized Candida antarctica lipase was provided by NovoNordisk (Denmark) as Novozyme ^™ . This was used directly as provided in the esterification experiments. Analytical grade diethyl ether purchased from Merck was used without any purification, but synthetic grade n-hexane, also from Merck, was freshly distilled prior to use in extraction and HPLC chromatography. Glycerol (99%) was purchased from Sigma and Aldrich Chemical Company and used without further purification. The CLA concentrate was provided by Natural Lipids (Norway) as a free fatty acid as Tonalin ^™ . Its purity was confirmed by analytical GLC and high-field NMR spectrometer, which showed some glyceride impurities. CLA concentrate is 43.3% 9-cis, 11-trans-linoleic acid, 44.5% 10-trans, 12-cis-linoleic acid, 5.4% as determined by GLC at the Science Institute. % Other CLA isomers, 5.6% oleic acid, and 0.6% palmitic acid and stearic acid, respectively.

Example 6
Preparation of CLA Triacylglycerol by Direct Esterification Immobilized Candida antarctica lipase (1.25 g) was mixed with glycerol (1.22 g, 13.3 mmol) and CLA as a free fatty acid (M.wt. 280.3 g / mol; 11.6 g, 41.5 mmol). The mixture was gently stirred at 65 ° C. on a magnetic stirrer hotplate under a continuous vacuum of 0.01-0.5 Torr. Volatile water generated during the course of the reaction was continuously condensed into a liquid nitrogen cold trap. After 48 hours, the reaction was stopped, n-hexane was added, and the enzyme was separated by filtration. The organic phase was treated with an alkaline aqueous solution of sodium carbonate to remove excess free fatty acids (if necessary). The organic solvent (if appropriate, after drying over anhydrous magnesium sulfate) was removed under reduced pressure on a rotary evaporator followed by high vacuum processing to give a virtually pure product as a slightly yellowish oil (10.9 g Average M.wt. 878.6 g / mol; yield 93%). When stoichiometric amounts of free fatty acids are used, titration with standardized sodium hydroxide is applied to determine the free fatty acid content of the crude reaction product (ester groups corresponding to at least 99% incorporation). Free fatty acid content of less than 1%, based on the number of moles, which is equivalent to a triglyceride content of at least 97%). The crude product was directly introduced into the HPLC eluting with 10% diethyl ether in n-hexane to give 100% pure triglyceride as a colorless oil. 250MHz 1H NMR (CDC13) 8 (ppm) 6.35-6.23 (3H, ddt, Jtrans = 15.0 Hz, J = 10.9Hz, jallyl = 1.3, = CHCH = CH), 5.98-5.90 (3H, dd, Icis = 10.9, J = 10.9, -CH = CHCH =), 5.71-5.59 (3H., Dtd, Jtrans = 15.0 Hz, J = 6.9 Hz, J = 6.9 Hz, J = 2.2 Hz, = CH = CHCH2-), 5.35-5.26 (4H, m, = CH2CH = CH- and -CH2C -1CH2-), 4.33-4.26 (2H, dd, Jgem = 11.9 Hz, J = 4.3, -CH2CH2CH2-), 4.18-4.10 2H, dd, Jgem = 1.8 Hz, J = 6.0, -CH2CHCH2-), 2.37-2.31 (6H, t, J = 7.4 H2, -CH2COOR), 2.19-2.05 (12H, m, -CH2CH = CH-), 1.66-1.60 (6H, qu ., J = Hz, -CH2CH2COOR), 1.43-1.30 (18H, m, -CH2-), 0.91-0.86 (9H, t, J = 6.7 Hz, -CH3) .13C-NMR (CDC13): 8 (ppm ) 173.2, 172.8, 134.6, 130.0, 128.6, 125.5, 68.8, 62.0, 34.0, 32.9, 31.6, 29.6-28.9 (6C), 27.6, 24.8, 22.5, 14.1.

  Samples were collected periodically as the reaction proceeded to monitor the progress of the reaction and provide more detail about the composition of the individual glycerides during the reaction. They were analyzed with a HNMR spectrometer to provide good insight into the composition of mono-, di-, and triacylglycerols during the course of the reaction. The results are demonstrated in Table 12 below. As can be pointed out from the table, 1,3-diacylglycerol occupied the predominance of the reaction mixture during the first 2 hours of the reaction. After 4 hours, triacylglycerol predominated and reached 98% composition after 22 hours and 100% after 48 hours. As expected, 1,2-diacylglycerol reached considerably lower levels than 1,3-diacylglycerol. 1-monoacylglycerol reached a maximum during the first hour of the reaction, but 2-monoacylglycerol was not detected throughout the reaction.

実施例７
CLAの収率および組成における温度および反応継続時間の変化の影響
ベニバナ油の共役化における温度および反応継続時間の効果を測定した。水およびNaOHを、表１欄１および２に示されるように、高圧反応器(Parr Model 450 ML Benchtop Alloy 400、圧力計および攪拌機を備える)に加えた。NaOHを溶解させ、そしてベニバナ油を反応器に加えた(欄３)。反応器を閉じ、そして窒素で２分間フラッシュし、次いで全ての弁を閉じた。反応器を電気ガスケット中で所望の温度まで加熱し(欄４)、そしてその温度に所望の時間維持した(欄５)。次いで、温度を60℃まで下げて、それから圧を逃がして反応器を開けた。各反応について、得られる固体化した石鹸２ｇを、反応器から取り出し、ほぼ40℃で水に溶解した。次いで、溶液のpHを６未満に下げるためにクエン酸を加えた。サンプルを脂肪酸最上層から抜き取り、そしてガスクロマトグラフィーのために調製した。

Example 7
Effect of temperature and reaction duration changes on CLA yield and composition The effects of temperature and reaction duration on the conjugation of safflower oil were measured. Water and NaOH were added to the high pressure reactor (Parr Model 450 ML Benchtop Alloy 400, equipped with pressure gauge and stirrer) as shown in Table 1, columns 1 and 2. NaOH was dissolved and safflower oil was added to the reactor (column 3). The reactor was closed and flushed with nitrogen for 2 minutes, then all valves were closed. The reactor was heated in the electric gasket to the desired temperature (column 4) and maintained at that temperature for the desired time (column 5). The temperature was then lowered to 60 ° C. and then the pressure was released and the reactor was opened. For each reaction, 2 g of the solidified soap obtained was removed from the reactor and dissolved in water at approximately 40 ° C. Citric acid was then added to lower the pH of the solution below 6. Samples were withdrawn from the fatty acid top layer and prepared for gas chromatography.

  Gas chromatographic results are shown in column 6 (total proportion of 9,11 and 10,12 isomers), column 7 (total proportion of 11,13 isomers), and column 8 (total CLA isomers or total yield). Ratio). These data show that as the reaction duration and temperature increase, the total amount of conjugation and the proportion of the 11,13 isomer increase. Under conditions where the formation of the 11,13 isomer is low, the total amount of conjugation is also low.

実施例８
ベニバナ脂肪酸メチルエステル(FAME)の共役化
反応は、密閉した容器中で行った。以下の成分を一緒に混合した：100ｇのベニバナFAMEならびにほぼ2.8ｇのKOCH₃および2.8ｇのメタノールの混合物。２つの成分の混合中メタノールの蒸発のために、おそらくはメタノールよりもKOMeが存在した。混合物は、密閉した反応容器中窒素雰囲気下で、５時間111〜115℃で撹拌した。異性体の分配はガスクロマトグラフィーにより分析した。結果は表２に要約される。GCの生データは、表３に表される。これらのデータは、共役化ベニバナFAMEは、穏やかな条件下で達成され、かなりの量の望ましくない8,10および11,13異性体を欠く生成物をもたらし得ることを示唆する。

Example 8
The conjugation reaction of safflower fatty acid methyl ester (FAME) was carried out in a sealed container. The following ingredients were mixed together: 100 g safflower FAME and a mixture of approximately 2.8 g KOCH ₃ and 2.8 g methanol. Presumably KOMe was present rather than methanol due to the evaporation of methanol during the mixing of the two components. The mixture was stirred at 111-115 ° C. for 5 hours under a nitrogen atmosphere in a sealed reaction vessel. The distribution of isomers was analyzed by gas chromatography. The results are summarized in Table 2. GC raw data are presented in Table 3. These data suggest that conjugated safflower FAME is achieved under mild conditions and can result in products lacking significant amounts of the undesired 8,10 and 11,13 isomers.

実施例９
共役化ベニバナFAMEの大規模バッチ生成
ベニバナ共役化FAMEの生成は、２つの工程、メタノリシスおよび共役化に分けられ得る。メタノリシスのために、6,000kgのベニバナ油が密閉した反応器中へ投入された。反応器を大気圧で窒素でパージし、そして1150リットルのメタノールおよび160ｋｇのNaOCH₃(30％溶液)を加えた。混合物を撹拌しながら65℃まで加熱し、そして65℃で２時間反応させた。反応器を窒素ガスでパージしながら、得られる下層をデカントした。ついで、1000リットルの水(40〜50℃、その中に50kgのクエン酸一水和物が溶解されている)を撹拌しながら加えた。層を分離させ(ほぼ60分)、そして反応器を窒素ガスでパージしながら、下層をデカントした。得られるベニバナFAME生成物を減圧下１時間80℃で乾燥した。

Example 9
Large scale batch production of conjugated safflower FAME The production of safflower conjugated FAME can be divided into two steps, methanolysis and conjugation. For methanolysis, 6,000 kg of safflower oil was charged into a closed reactor. The reactor was purged with nitrogen at atmospheric pressure and 1150 liters of methanol and 160 kg of NaOCH ₃ (30% solution) were added. The mixture was heated to 65 ° C. with stirring and reacted at 65 ° C. for 2 hours. The resulting lower layer was decanted while purging the reactor with nitrogen gas. Then 1000 liters of water (40-50 ° C. in which 50 kg of citric acid monohydrate was dissolved) was added with stirring. The layers were allowed to separate (approximately 60 minutes) and the lower layer was decanted while the reactor was purged with nitrogen gas. The resulting safflower FAME product was dried at 80 ° C. under reduced pressure for 1 hour.

To conjugate safflower FAME, 250 kg of KOCH ₃ was dissolved in methanol to form a paste and added to the reactor. The mixture was then heated to 120 ° C. with stirring and the reaction was allowed to continue for 3 hours. The mixture was cooled to 100 ° C. and 1000 liters of water (40-50 ° C. in which 50 kg of citric acid monohydrate had been dissolved) was added with stirring. The mixture was stirred for 15 minutes and then the layers were allowed to separate in 20 minutes. The lower layer was decanted and the product was dried at 80 ° C. for 1 hour and then stored under nitrogen.

The resulting CLA was analyzed using a Perkin Elmer Autosystem XL GC under the following conditions:
Column: WCOT fused silica 100m x 0.25mm, CP SIL 88 coated carrier: He gas, 30.0 PSI
Temperature: 220 ° C
Run time: 35-90 minutes Injection: 240 ° C with no split Detector: FID at 280 ° C
GC results are summarized in Tables 15 and 16.

以下は、本発明のCLA遊離脂肪酸、トリグリセリド、およびエステルを含む代表的な動物の飼料の実施例である。

The following are examples of representative animal feeds comprising the CLA free fatty acids, triglycerides, and esters of the present invention.

Example 10
A. Pig animal feed

Ｂ．ブタのための育成飼料-仕上飼料
（40〜240 LBS[18〜109KGS]より）

B. Rearing feed for pigs-finishing feed (from 40 to 240 LBS [18 to 109 KGS])

Ｃ．ブタ育成飼料-仕上飼料
（121〜240 LBS[55〜109KGS]のブタに対して）

C. Pig breeding feed-Finished feed (for pigs with 121-240 LBS [55-109KGS])

ブタ微量ミネラル予備混合物の組成および分析

Composition and analysis of porcine trace mineral premix

ブタビタミン予備混合物の組成

Composition of porcine vitamin premix

Ｄ．ニワトリのための18％タンパク質層状飼料

D. 18% protein layered feed for chickens

Ｅ．ブロイラーのための幼動物飼料および仕上飼料

E. Infant and finished feed for broilers

Ｆ．育成／仕上七面鳥飼料

F. Training / finishing turkey feed

Ｇ．乾燥ドッグフード処方物

G. Dry dog food formulation

Ｈ．半生ドッグフード処方物

H. Half-life dog food formulation

(The invention's effect)
In developing a defined commercial source of CLA for both therapeutic and nutritional applications, a process is provided for preparing large quantities of defined materials that are low in harmful CLA. It was.

Claims (1)

The invention described herein.

Images