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| LICENSOR | This data is not available for free |
| LITERATURE REF. | This data is not available for free |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | January 5, 1999 |
| PATENT TITLE |
Method of producing conjugated fatty acids |
| PATENT ABSTRACT | A method for producing a cis-9, trans-11 fatty acid from a fatty acid containing double bonds in the cis-configuration at positions 9 and 12, includes the step of combining a Lactobacillus microorganism with free fatty acids in a fermentation process. The conjugated fatty acid products can include, for example, cis-9, trans-11 conjugated linoleic acid (CLA). |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | December 3, 1996 |
| PATENT REFERENCES CITED |
Chin et al., "Synthesis of CLA by Intestinal Microorganisms," FASEB J. 7, A169 (1993). Chin et al., "Synthesis of CLA by Intestinal Microorganisms," Food Research Institute, 1992 Annual Report, pp. 139-140. Eyssen et al., "Biotransformation of Linoleic Acid and Bile Acids by Eubacterium lentum," Applied and Environmental Microbiology, 47:39-43 (1984). Fairbanks, et al., "Octadeca-9-11-Dienoic Acid in Diagnosis of Cervical Intraepithelial Neoplasia," Lancetp. 329 (1988). Fujimoto et al., "Biohydrogenation of Linoleic Acid by Anaerobic Bacteria Isolated from Rumen," Biosci. Biotech. Biocem.57:1026-1027 (1993). Haumann, B., "Conjugated Linoleic Acid Offers research promise," Inform. 7:152-159 (1996). Thompson et al., "Measurement of the Diene Conjugated Form of Linoleic Acid in Plasma by High Performance Liquid Chromoatography: A Questionable Non-Invasive Assay of Free Radical Activity?," Chem. Biol. Interactions, 55:357-366 (1985). Uchida, K., "Occurrence of Conjugated Dienoic Fatty Acids in the Cellular Lipids of Pediococcus homari," Agr. Biol. Chem., 39(2):561-563 (1975). Verhulst et al., "Isomerization of Polyunsaturated Long Chain Fatty Acids by Propionibacteria," System. Appl. Microbiol. 9:12-15 (1987). Verhulst et al., "Biohydrogenation of Linoleic Acid by Clostridium sporogenes, lostridium bifer,entans, Clostridium sordellii and Bacteroides sp.,"FEMS Microbiology Ecology, 31:255-259 (1985). Jack et al., "Serum octadeca-9,11 dienoic acid--an assay of free radical activity or a result of bacterial production?," Clinica Chimica Acta, 224:139-146 (1994). Kemp et al., "The Hydrogenation of Unsaturated Fatty Acids by Five Bacterial Isolates from the Sheep Rumen, Including a New Species," Journal of General Microbiology 901:100-114 (1975). Kepler et al., "Biohydrogenation of Unsaturated Fatty Acids," The Journal of Biological Chemistry 245:3612-3620 (1970). Lee et al., "Conjugated linoleic acids and artherosclerosis in rabbits," Atherrosclerrosis 108:19-25 (1994). Mills et al., "Hydrogenation of C.sub.18 Unsaturated Fatty Acids by Pure Cultures of a Rumen Micrococcus," Aust. J. Biol. Sci., 23:1109-1113 (1970). Nicolosi et al., "Dietary Conjugated Linoleic Acid Reduces Aortic Fatty Streak Formation Greater than Linoleic Acid in Hypercholesterolemic Hamsters," Circulation, 88(suppl):24-58 (1993). |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. A method of producing cis-9, trans-11 fatty acids, the method comprising the step of: combining a fatty acid having a dissociated carboxyl group and unconjugated double bonds in the cis-9, cis-12 configuration with a Lactobacillus strain that can convert linoleic acid into conjugated linoleic acid, in a physiological buffer for a time sufficient to produce conjugated fatty acids wherein at least 50% of the conjugated fatty acids comprise conjugated double bonds in the cis-9, trans-11 configuration. 2. A method as claimed in claim 1, the method comprising the step of combining the free fatty acid with a Lactobacillus reuteri strain that can convert linoleic acid into conjugated linoleic acid. 3. A method as claimed in claim 1, the method comprising the step of combining the fatty acid with Lactobacillus reuteri PYR8 (ATCC 55739) . 4. A method as claimed in claim 1 wherein the fatty acid is selected from a group consisting of linoleic acid (18:2.DELTA..sup.9,12), gamma-linolenic acid (18:3.DELTA..sup.6,9,12), alpha-linolenic acid (18:3.DELTA..sup.9,12,15) and octadecatetraenoic acid (18:4.DELTA..sup.6,9,12,15). -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
CROSS-REFERENCE TO RELATED APPLICATIONS Not applicable STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT BACKGROUND OF THE INVENTION Linoleic acid is an 18 carbon molecule that contains double bonds in the cis-9, cis-12 configuration. Conjugated linoleic acid (CLA) is a general term for a set of positional and geometric isomers of linoleic acid that possess conjugated double bonds, in the cis or trans configuration, at positions 9 and 11 or at positions 10 and 12. CLA occurs naturally in a wide variety of foods, especially in foods such as cheese that are derived from ruminant animals. Ha, Y. L., N. K. Grimm and M. W. Pariza, Carcinogenesis, Vol. 8, No. 12, pp. 1881-1887 (1987); Ha, Y. L., N. K. Grimm and M. W. Pariza, J. Agric. Food Chem., Vol. 37, No. 1, pp. 75-81 (1987). CLA is now recognized as a nutritional supplement and an effective inhibitor of epidermal carcinogenesis and forestomach neoplasia in mice, and of carcinogen-induced rat mammary tumors. CLA can prevent adverse effects caused by immune stimulation in chicks, mice and rats, and can decrease the ratio of low density lipoprotein cholesterol (LDL-cholesterol) to high density lipoprotein cholesterol (HDL-cholesterol) in rabbits fed an atherogenic diet. CLA also reduces body fat in mouse, rat and chick models. The effective behavior of CLA in such animal systems suggests similar benefit when provided in the human diet. In contrast, linoleic acid, the precursor of CLA is associated with enhancing mammary cancer in rodents. Methods of using CLA are described in issued U.S. Pat. Nos. 5,017,614; 5,070,104; 5,208,356; 5,428,072; 5,430,066; 5,504,114, and 5,554,646. Linoleic acid can be converted to CLA by chemical methods (American Oil Chemists' Society Official Method Cd 7-58, pages 1-11, American Oil Chemists' Society, Champaign, Ill. (1973), or by enzymatic isomerization. The non-toxic salts of the free CLA acids may be made by reacting the free acids with a non-toxic base. Natural CLA may also be prepared from linoleic acid by the biological action of .DELTA..sup.12 -cis, .DELTA..sup.11 -transisomerase from a harmless microorganism such as Butyrivibrio fibrisolvens, a strictly anaerobic rumen bacterium, that produces both a cis-9, trans-11 CLA isomer and a cis-9, trans-11, cis-15 linolenic acid isomer as intermediates in the biohydrogenation of linoleic acid and linolenic acid, respectively. Kepler, C. R., et al., J. B. C. 245:3612-3620 (1970); see also Kemp, P. et al., J. Gen. Microbiol. 90:100-114 (1975), Mills, S. C. et al., Aust. J. Biol. Sci. 23:1109-13 (1970), Verhulst, A. et al., FEMS Microbiol. Ecol. 31:255-259 (1985), Fujimoto, K. et al., Biosci. Biotech. Biochem. 57:1026-1027 (1993), and Eyssen H. and A. Verhulst, Appl. Environ. Microbiol. 47:39-43 (1984). Jack, C. I. et al., Clinica Chimica Acta 224:139-146 (1994) purport to identify a large number of bacteria that convert linoleic acid into CLA. However, no reliable analytical methodology was presented. It is more likely that linoleic acid was oxidized rather than converted to CLA. Harmless microorganisms in the intestinal tracts of rats and other monogastric animals may also convert linoleic acid to CLA (S. F. Chin, W. Liu, K. Albright and M. W. Pariza, 1992, FASEB J.6:Abstract #2665). The presence in rumen of bacteria that possess linoleate isomerase, such as B. fibrisolvens, could explain the high concentration of CLA in tissues obtained from ruminant animals. B. fibrisolvens is not commercially useful to produce CLA from its free fatty acid precursor because it is a very strict anaerobe that is difficult to grow, even under laboratory conditions. In addition, B. fibrisolvens has an undesired reductase activity that converts the active products to other compounds. Other rumen bacteria are known to hydrogenate cis, cis linoleic acid isomers having double bonds at positions 5 and 8, 8 and 11, 11 and 14 to octadecanoic acid (Kemp, P. and Lander, D. J., Brit. J. Nutr. 52:171 (1984)). Nicolosi, R. J. et al, "Dietary Conjugated Linoleic Acid Reduces Aortic Fatty Streak Formation Greater than Linoleic Acid in Hypercholesterolemic Hamsters" Circulation 88 (suppl.), 2458 (1993), and Lee, K. L. et al. "Dietary Conjugated Linoleic Acid and Atherosclerosis in Rabbits" Atherosclerosis 108:19-25 (1994) established that CLA inhibits atherosclerosis in rabbits and hamsters. Fairbank, J. et al., The Lancet p. 329, Aug. 6, 1988, reported detecting cis, trans isomers of 18:2(9,11) in cultures of Lactobacillus. However, this paper presented no standards, and, given the difficulties inherent in sorting out bacterial metabolism, thus included no credible evidence for making CLA using a bacterial system. This report was refuted by Thompson, S. and M. T. Smith, Chem. Biol. Interactions J. 55:357-366 (1985), which showed that a method described by Fairbank did not produce authentic CLA, but rather oxidized linoleic acid. Uchida, K., Aqr. Biol. Chem. 39:561-563 (1971) describes a strain of Pediococcus that synthesizes certain unspecified CLA isomers de novo and deposits them in its membrane. The strain does not use linoleic acid as a substrate for a linoleate isomerase enzyme. Moreover, the strain produces numerous isomers. Even if one is active, it represents too small a fraction of the total production of conjugated fatty acids to make the strain commercially practical. Verhulst, A. et al., System. Appl. Microbiol. 9:12-15 (1987) describes the production of trans-10, cis-12 CLA by Propionibacterium acnes. This organism is a pathogen that includes a reductase that reduces the useful trans-10, cis-12 product to other non-CLA fatty acid products. Of the various positional and geographic isomers present in CLA, the cis-9, trans-11 isomer is believed to be an active form, at least for the anticarcinogenic activity of CLA. Between 76 and 92% of the CLA isomers in uncooked meat is in the cis-9, trans-11 configuration. In processed plant oils, about 42% of the isomers were in the cis-9, trans-11 configuration and another 42% were in the trans-10, cis-12 configuration, as was the case in CLA prepared chemically from linolic acid. The trans-10, cis-12 CLA isomer may also have biological activity of the type described, but it has not been possible to prepare quantities of this isomer enzymatically for testing. It is also thought that CLA is not unique in this ability, but rather that cis-9, trans-11 and trans-10, cis-12 isomers of other fatty acids will have comparable biological activities. It is known that linoleic acid can be efficiently converted to CLA when the linoleic acid is provided in the form of free fatty acid, rather than as a triglyceride. It would be desirable to obtain a microorganism capable of producing the active isomer or isomers of CLA and other conjugated fatty acids, where the organism is easily grown and used and is food-safe. BRIEF SUMMARY OF THE INVENTION We have discovered a method for forming fatty acids that have conjugated double bonds in the cis-9, trans-11 configuration, and may have other double bonds as well. In the method, an unconjugated free fatty acid having a pair of double bonds in the cis-configuration at carbon positions 9 and 12 is exposed to a Lactobacillus (or membrane preparation therefrom) that is shown herein to contain a linoleate isomerase activity that can convert free linoleic acid into conjugated linoleic acid. The cis-9, trans-11 fatty acids thus produced can have biological activities of the type recognized in the art for cis-9, trans-11 CLA. The Lactobacillus is readily grown and maintained in anaerobic culture. Conditions for directing production of the preferred cis-9, trans-11 isomers are described herein. The linoleate isomerase of the organism described herein requires the fatty acid to have a dissociated carboxyl group and unconjugated double bonds at positions 9 and 12 (e.g., 18:2.DELTA..sup.9,12, where 18 refers to the number of carbons in the fatty acid, 2 refers to the number of double bonds, and 9,12 indicate the positions of the double bonds). The activity of the organism is reduced by the presence of an extra double bond at position 6 (see 18:3.DELTA..sup.6,9,12) , but is promoted by an extra double bond at position 15 (see 18:3.DELTA..sup.9,12,15). A fatty acid having double bonds at both positions 6 and 15 (octadecatetraenoic acid) was consumed faster than gamma-linolenic acid, but slower than linoleic acid. A suitable strain is a Gram positive aerobic Lactobacillus organism that can convert linoleic acid mainly to a cis-9 trans-11 CLA isomer without producing the trans-10, cis-12 isomer or the cis-10, cis-12 isomer, thereby indicating that the linoleate isomerase of the organism acts specifically on cis-12, rather than on the cis-9 double bond of the fatty acid. It will be apparent to those skilled in the art that the forementioned objects and other advantages may be achieved by the practice of the present invention. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS Not applicable DETAILED DESCRIPTION OF THE INVENTION In one aspect, the present invention is a method for converting a fatty acid having unconjugated double bonds at positions 9 and 12 into a fatty acid having conjugated cis-9, trans-11 double bonds. In a second aspect, the present invention is an organism that can accomplish the conversion. In the method of the present invention, an unsaturated free fatty acid containing cis double bonds at the 9 and 12 positions and a dissociated carboxyl group is combined with a linoleate isomerase to produce primarily an isomer of the fatty acid having conjugated double bonds and a cis-9, trans-11 configuration. The conjugated fatty acids thus produced are stable and can be extracted from the cell culture medium. The linoleate isomerase, a membrane-bound enzyme, can be provided in the form of whole bacterial cells or as a cell membrane preparation obtained from whole bacterial cells. The cells are preferably grown to early stationary phase before being harvested for use in the method. The reaction between the linoleate isomerase of the cells and the free fatty acids can be accomplished at a temperature ranging from about 4.degree. C. to about 60.degree. C., preferably 4.degree. C. to 12.degree. C., and for a time (e.g., 1-24 hours, preferably 1-5 hours, more preferably about 3 hours) sufficient to produce a conjugated fatty acid product. The Examples, below, provide guidance on the amount of time to accomplish the conversion. Neither the enzyme activity, nor the ratio of isomers produced, is affected by the presence of oxygen. The conversion reaction medium can be any that permits maintenance of the organism and its membrane bound linoleate isomerase activity, and is preferably a buffer having a pH in the range of 7.4 to 8.8, and more preferably between 8.0 and 8.8, most preferably about 8.5. Between pH 5.5 and 7.4, less conjugated fatty acid is produced, although slightly more fatty acid is produced at the low end of that range. Under preferred conditions, and in the presence of sufficient free fatty acid, 7.8 mg of conjugated fatty acid has been produced per gram of cells. It is envisioned that the enzyme may be obtained in purified form, either from the membrane, or by production using the available techniques of molecular biology and recombinant DNA. The production in the method of a fatty acid isomer having a single conjugated double bond is characterized by an absorption peak at 233-234 nm. Isomers produced in the method can be distinguished from one another by gas chromatography. At least 50%, preferably at least 80%, more preferably at least 90%, still more preferably at least 95%, and more preferably 98-99%, of the fatty acid isomers initially produced by the enzyme from 18 carbon fatty acids are in the cis-9, trans-11 configuration. The negligible remainder of the isomers produced are cis-9, cis-11and trans-9, trans-11, the latter being a conversion product of less than 10%, preferably less than 5%, of the major cis-9, trans-11 isomer. Trans-10, cis-12 and cis-10, cis-12 fatty acid isomers are not detected, nor are trans-9, cis-11 and cis-9, cis-11 isomers. Cis-9, trans-11 isomers of C-18 fatty acids are biologically active. Other isomers may also have biological activity and are found in commercial preparations of CLA. The high level of the biologically active cis-9, trans-11 isomer in preparations made according to the present invention can be advantageous for certain applications such as anticarcinogenic activity, in that it raises the specific biological activity of the conjugated fatty acid isomer preparation produced in the method. In view of the demonstrated functional benefits of the cis-9, trans-11 CLA isomer, it is anticipated that the cis-9, trans-11 isomers of other 18 carbon fatty acids will have similar activities. The same holds true for other fatty acids of other lengths, except insofar as the cis, trans conjugated bonds will be in positions other than 9 and 11, because of the different fatty acid chain lengths. The conjugated fatty acids obtained by the practice of the described method of preparation may be free or bound chemically through ester linkages. The product is heat stable and can be used as is, or dried and powdered. The conjugated fatty acids are readily converted into a non-toxic salt, such as the sodium or potassium salt, by reacting chemically equivalent amounts of the free acid with an alkali hydroxide at a pH of about 8 to 9. It is also envisioned that advantageous conjugated fatty acids can be supplied in milk or milk products naturally enriched with the fatty acids by adding a source of free linoleic acid and the harmless bacteria described herein to milk and incubating the mixture for about 1 hour at 37.degree. C. or until the linoleic acid is converted into CLA. The conjugated fatty acids and their non-toxic derivatives, such as the non-toxic salts, in addition to being added to an animal's feed or human food or formed in situ can be administered in the form of pharmaceutical or veterinary compositions, such as tablets, capsules, solutions or emulsions to the animal or the humans. The exact amount to be administered, of course, depends upon the form employed, the route of administration, and the nature of the animal's or human's condition. Generally, the amount employed as a pharmaceutical will range from about one part per million (ppm) to about 10,000 ppm in the animal's or human's diet. However, the upper limit of the amount to be employed is not critical because the products are relatively non-toxic and are normal constituents of the human diet (including human breast milk). The amounts to be added to a conventional animal feed or human's food as an additive can range from 0.01% to 2.0% or more by weight of the animal's or human's food. The preferred pharmaceutical and veterinary compositions of CLA contain the non-toxic sodium or potassium salt of CLA in combination with a pharmaceutical diluent. When the compositions are solutions or suspensions intended for oral administration the diluent will be one or more diluents, such as lactose or starch, and the product will be a tablet, capsule or liquid. When the compositions are solutions or suspensions intended for parenteral administration the preferred diluent will be Sterile Water for Injection U.S.P. In a second aspect, the invention is a substantially pure preparation of a Lactobacillus strain having the desired linoleate isomerase activity. A "substantially pure preparation" is derived from a single bacterial isolate and comprises primarily (more than about 50% preferably more than 90%) individual cells having an ability to perform the enzymatic conversion described in the method. A preparation can be considered substantially pure for purposes of this application if, when brought into contact with a free fatty acid, conversion occurs to the extent noted above. A preferred bacterial cell having the desired linoleate isomerase activity is Lactobacillus reuteri PYR8 (previously denoted PLR8), deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA and accorded Accession Number ATCC 55739. The preferred microorganism is a Gram positive, catalase-negative, bacteria which forms non-motile rods. It grows at 45.degree. C. but not at 15.degree. C., and can be distinguished from related L. fermentum by G+C content. L. fermentum has a G+C content of 52-54%, whereas L. reuteri has a G+C content of 40-42.3%. The G+C content of ATCC 55739 is 42.25%. The two can also be distinguished by differences in murein type, by differences in electrophoretic mobility of L-lactate dehydrogenase proteins, and by difference in cellular C18:1 isomer number. A cellular fatty acid profile of PYR8 was examined along with those of one strain of L. reuteri (ATCC 23272), and two strains of L. fermentum (ATCC 14931 and ATCC 23271) using GC. The final detection temperature was lowered to 180.degree. C. to separate all peaks. The two ATCC Lactobacillus species differed in their isomer number of C18:1. L. reuteri had three isomers while L. fermentum had only two isomers. PYR8 had three isomers of C18:1, further demonstrating that the isolate was properly assigned to L. reuteri. Detailed biochemical characteristics of the L. reuteri organism are set forth in Table I. With this strain, the extent of production of conjugated fatty acids is directly proportional to the cell biomass synthesis, suggesting that the isomerase is an accumulated enzyme and is not a functional enzyme for cell growth. TABLE I ______________________________________ Biochemical characteristics ______________________________________ Indole production - Cellobiose - N-Acetylglucosaminidase - Esculin pH - .alpha.-Glucosidase + Esculin hydrolysis - .alpha.-Arabinosidase - Glycogen - .beta.-Glucosidase + Lactose weak .alpha.-Fucosidase - Maltose weak Phosphatase - Mannitol - .alpha.-Galactosidase + Melezitose - .beta.-Galactosidase + Raffinose weak Indoxyl-acetate + Rhamnose weak Arginine utilization + Salicin - Leucine aminopeptidase + Xylose - proline aminopeptidase - Nitrate utilization + Pyroglutamic acid - Hemolysis - arylamidase Bile green - Tyrosine aminopeptidase - utilization Arginine aminopeptidase + Fructose + (massive gas) Alanine aminopeptidase + Glucose + (acid + gas) Histidine aminopeptidase + Meat peptidase Motility - Glycine aminopeptidase - Growth at 15.degree. C. - Catalase - Growth at 45.degree. C. + ______________________________________ The L. reuteri isolate is a distinct biotype from other L. reuteri isolates in that it possesses the characteristic linoleate isomerase activity that is not present in, for example, ATCC 23272, another L. reuteri isolate from human feces. Also, this isolate does not produce reuterin. To obtain a suitable enzyme preparation, the organism can be grown to early stationary phase before being harvested (e.g., by centrifugation) and then weighed. The cells are re-suspended in a physiological buffer (e.g., Tris-maleate at 0.1 Molar, pH 5.4). Greater conversion is observed when the cells are suspended at a ratio of 1 part bacteria to at least about 10-15 parts of aqueous buffer. The yield of conjugated fatty acids decreases significantly if the system includes less than 10-15 parts water. The conjugation can be achieved by combining the cells with the free fatty acids with gentle stirring until the conjugated product is produced. The linoleate isomerase of ATCC 55739 appears to act specifically on fatty acids having double bonds at positions 9 and 12 to produce primarily products having conjugated cis-9, trans-11 double bonds, but within that constraint, appears to act upon all such fatty acids. The known fatty acids have either 12, 14, 16, or 18 carbons. The invention would also apply to fatty acids that can be produced by carbon chain elongation, which can be achieved naturally or using methods known to the art. Elongation can occur before or after enzymatic formation of the conjugated double bonds according to the present invention. Thus, products having conjugated cis, trans double bonds at positions other than 9 and 11 can be formed by practicing the method of the present invention on, for example, an 18 carbon fatty acid and subsequently altering the length of the fatty acid chain. References herein to producing cis-9, trans-11 products are to be understood to include such other products. The cellular fatty acids of the treating strain can also increase when the strain is combined with free fatty acids. It is economical to prepare free fatty acids for use in the method by hydrolysing a plant oil using, for example, lipase. The fatty acid can be hydrolyzed with or without a stabilizing emulsifier such as 2% lecithin, and can be sonicated for better distribution. Yield may be higher if an emulsifier is included or if the reaction occurs under anhydrous conditions. A working stock of the fatty acids can be prepared by diluting the fatty acid preparation in a buffer such as Tris-HCl (0.1M, pH 8.5). It is advantageous to utilize an inexpensive, edible plant oil rich in the desired fatty acid as the source of free fatty acids. For example, corn oil, rich in linoleic acid (18:3.DELTA..sup.9,12,15), sunflower oil, linseed oil or evening primrose oil, which is rich in gamma-linolenic acid (18:3.DELTA..sup.6,9,12), are all suitable. Other suitable fatty acids include octadecatetraenoic acid (18:4.DELTA..sup.6,9,12,15) and alpha-linoleic acid. The Lactobacillus shows no activity against unsaturated fatty acids lacking double bonds at both positions 9 and 12. Inactive substrates include oleic acid (18:1.DELTA..sup.9), octadecanoic acid (18:1.DELTA..sup.12), eicosadienoic acid (20:2.DELTA..sup.11,14), eicosatrienoic acid (20:3.DELTA..sup.8,11,14), eicosatrienoic acid (20:4.DELTA..sup.5,8,11,14), and arachidonic acid (20:4.DELTA..sup.5,8,11,14). Table II provides a list of substrates tested with ATCC 55739. TABLE II ______________________________________ List of the tested substrate and the major putative products of the linoleate isomerase of L. reuteri PYR8 Substrates.sup.a Major Products ______________________________________ Oleic acid (18:1.increment..sup.9) None Octadecanoic acid (18:1.increment..sup.12) None Linoleic acid (18:2.increment..sup.9,12) cis9, trans11 isomer.sup.b Gamma-linolenic acid (18:3.increment..sup.6,9,12) cis6, cis9, trans11 Alpha-linolenic acid (18:3.increment..sup.9,12,15) cis9, trans11, cis15 Octadecatetraenoic acid (18:4.increment..sup.6,9,12,15) cis6, cis9, trans11, cis15 Eicosadienoic acid (20:2.increment..sup.11,14 None Eicosatrienoic acid (20:3.increment..sup.8,11,14) None Eicosatrienoic acid (20:3.increment..sup.11,14,17) None Arachidonic acid (20:4.increment..sup.5,8,11,14) None ______________________________________ .sup.a Each substrate (0.2 mg) was reacted with the cells (20 mg) of L. reuteri PYR8, and product was analyzed by GC, as described in methods. .sup.b As a known product. |
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