Patent Application
20080044861
The accompanying drawings, which are incorporated in and form a part of the specification, illustrate the embodiments of the present invention and together with the description, serve to explain the principles of the invention. In the drawings:
The following detailed description of the invention is provided to aid those skilled in the art in practicing the present invention. Even so, the following detailed description should not be construed to unduly limit the present invention as modifications and variations in the embodiments discussed herein may be made by those of ordinary skill in the art without departing from the spirit and scope of the present invention.
A variety of microorganisms drawn from the order of gram positive Actinomycetes are useful in the practice of this invention. Certain Actinomycetes are characterized herein as having the ability to produce reversibly modified isoflavone biotransformation products in fermentations and are favored for use in this invention. Streptomycetes or Saccharopolyspora species are particularly favored sources of microorganisms for use in the methods of this invention. Actinomycetes that can reversibly modify isoflavone biotransformation products include Streptomyces rimosus, Streptomyces fradiae, Streptomyces hygroscopicus, Streptomyces cinnamonensis, Streptomyces peucetius, Saccharopolyspora erythraea, Streptomyces avermitilis, and Streptomyces glaucescens.
However, it is further understood that the use of Actinomycetes other than those specifically listed here that can reversibly modify isoflavone biotransformation products is also contemplated herein. In particular, it is envisioned that one of ordinary skill in the art could simply test any number of different Actinomycetes by performing the methods described herein to identify other Actinomycetes that have the ability to produce reversibly modified isoflavone biotransformation products.
Another common feature of the Actinomycetes used in the practice of this invention is that they are capable of converting the isoflavone glucosides that are present in the legume derived plant material in the growth media to isoflavones (i.e., the aglycone form). This initial isoflavone glucoside biotransformation reaction that occurs in certain Actinomycete fermentations has been described (U.S. Pat. No. 5,554,519; Hessler et al., 1996).
It is further recognized that certain microorganisms produce irreversibly modified isoflavone glucosides that cannot be converted to isoflavones. Examples of such irreversibly modified isoflavones include isoflavones that have been methylated, methoxylated, hydroxylated or chlorinated. Microorganisms that catalyzed one or more of the irreversible isoflavone biotransformations include Nocardia species and Mortierella isabellina (Maatooq and Rosazza, Phytochemistry 66(9):1007-1011, 2005). The use of microorganisms that catalyze irreversible biotransformations of isoflavones is not favored in the practice of this invention.
A key feature of the method is the use of growth media that contains material from leguminous plants. Leguminous plants are known to contain isoflavones and isoflavone glucosides (see Kaufman et al., Journal of Alternative and Complementary Medicine, 3(1):7-12, 1997). As described here, the certain microorganisms will produce reversibly modified isoflavone biotransformation products that can be converted to isoflavones when cultured on growth media that contains, amongst other ingredients, material from leguminous plants. Leguminous plants that can serve as a source of isoflavone glucosides and isoflavones include soybean (Glycine max), lupine (Lupinus species), fava bean, (Vicia faba), bean (Phaseolus species), clover (Trifolium species), lentil (Lens species), Baptisia (Baptisia species), kudzu (Pueraria species), pea (Pisum species), Vigna species, garbanzo bean (Cicer species), alfalfa (Medicago species), and psoralea (Psoralea corylifolia). A variety of distinct parts of the leguminous plant can be used to obtain the material used in the growth media. For example, the material used in the growth media can be derived from the leaves, stems, roots, seed pods, or seeds of the leguminous plant, or from a combination of those plant parts. Stover, or plant leaves and stems left in the field post-harvest, is another potential source of leguminous plant material that could be used in the growth media. In particular embodiments of the invention, soybean grits, soybean flour or soybean meal is used as the leguminous plant material in the fermentation.
Any type of fermentation vessel can be used to practice this invention. Fermentation vessels aerated by shaking, stirring or any other result effective method that provides for growth of the Actinomycete and efficient production of important fermentation products such as drugs and reversibly modified isoflavone biotransformation products can be used. The large 100,000 L fermentation vessels typically used by in batch mode production of antibiotics by microorganisms are particularly contemplated by this invention.
The method of obtaining the reversibly modified biotransformation product for conversion is typically tailored to permit separation of the isoflavone biotransformation product from other desired fermentation products such as drugs. This also permits the subsequent conversion reactions that may be deleterious to the other fermentation products to be focused on the reversibly modified biotransformation product. Separation of the isoflavone biotransformation product from other desired fermentation products is also important as it can reduce or eliminate contamination of the isoflavones that are ultimately produced by the methods of this invention. In some instances, the Actinomycetes cells in the fermentation are simply lysed in the fermentation broth to obtain the spent growth media that contains the reversibly modified isoflavone biotransformation product. In other instances, the Actinomycetes cells in the fermentation are removed by techniques including, but not limited to, sedimentation, centrifugation or filtration to obtain the spent growth media that contains the reversibly modified isoflavone biotransformation product. Spent growth media obtained either by lysing the cells in the fermentation media or by separating the cells from the fermentation media can be used in the methods described herein. Finally, spent fermentation beer that contains whole Actinomycete cells can also be used.
A useful property of the reversibly modified biotransformation products is that they are polar and will remain in the aqueous phase when extracted with polar or non-polar solvents. Consequently, one method of obtaining these biotransformation products is to extract the spent growth medium with a polar or non-polar organic solvent which is immiscible with water to obtain an aqueous fraction that contains the reversibly modified isoflavone biotransformation product. In certain instances, such as in fermentations that produce erythromycin or other non-polar drugs, these drugs will be partitioned into the organic phase of that solvent extraction and thus separated from the reversibly modified isoflavone biotransformation product. The polar or non-polar organic solvent can be C2-C10 organic solvent. This polar or non-polar organic solvent can also be selected from the group consisting of 1-butanol, 2-butanol, t-butanol, pentanol, hexanol, heptanol, octanol, ether, ethyl acetate, tetrahydrofuran, hexane, heptane, octane, isohexane, diethylether, methyl ethyl ketone, diisopropylether propanol, isopropyl alcohol, isobutyl alcohol, butanol, ethyl acetate, acetonitrile, acetone, methylene chloride, chloroform, carbon tetrachloride, and mixtures thereof.
Solid phase extraction is another method for obtaining isoflavone biotransformation products from the spent growth media. Solid phase extraction of the isoflavone biotransformation products from the treated (i.e., converted) material can be accomplished by any number of different methods of solid phase extraction such as column-based methods or batch-based methods. The solid phase extraction is effected with a sorbent selected from the group consisting of C18, divinylbenzene, polystyrene-divinylbenzene, non-polar polystyrene, anion exchange resin, and modified divinylbenzene. Once the isoflavone biotransformation product is adsorbed to the solid phase sorbent, the adsorbed material is typically washed with aqueous solutions that do not displace the isoflavone biotransformation product from the sorbent to remove other undesired or contaminating materials. Alternatively, certain admixtures of alcohol and water that do not displace the isoflavone biotransformation product from the sorbent, or admixtures of an organic solvent and water that do not displace the isoflavone biotransformation product from the sorbent, can be used to remove other undesired or contaminating materials. Once the adsorbed material containing the isoflavone biotransformation product has been washed, the isoflavone biotransformation product can be released by using either an alcohol, an organic solvent, an admixtures of alcohol and water that displaces the isoflavone biotransformation product from the sorbent, or admixtures of an organic solvent and water that displace the isoflavone biotransformation product from the sorbent.
One method of effecting the solid phase purification of an isoflavone biotransformation product uses an anion exchange resin and releases the isoflavone(s) by treating said resin with an alcohol, an organic solvent, an admixture of alcohol and water, or an admixture of an organic solvent and water. Similar methods have been used to effect purification of glycosylated isoflavones or isoflavone conjugates from plant materials (U.S. Pat. Nos. 6,703,051 and 6,020,471) and can be adapted to the purification of the isoflavone biotransformation products obtained from the spent fermentation beer. In practicing this method, the anionic exchange resin is typically conditioned prior to use by first converting the resin to a hydroxide form, next converting the resin to a chloride or sulfate form, and then finally converting at least some strong base sites of the resin to a carbonate form. Useful anionic exchange resins include type II macroporous strong base anion exchange resin as well as weak base anion exchange resins. Type II strong base anion exchange resin comprise a quaternary ammonium type of resin in which the four substituents of the nitrogen atom are an ethanol group, two methyl groups, and a polymeric benzyl group. Useful type II strong base anion exchange resins include IRA 910 available from Rohm & Haas, Independence Mall West, Philadelphia, Pa. 19105; Dowex 22 available from Dow Chemical U.S.A., 2040 Willard H. Dow Center, Midland, Mich. 48674; and Ionac A651 available from Sybron, Sybron Chemical Division, Birmingham Road, Birmingham, N.J. 08011. Useful weak base anion exchange resins include Duolite A-7 from Rohm & Haas. The isoflavone biotransformation product is adsorbed to the conditioned anionic exchange resin, washed with an aqueous solution, and then eluted with an alcohol, an organic solvent, an admixture of alcohol and water, or an admixture of an organic solvent and water. Useful alcohols or organic solvents for elution of the isoflavone biotransformation product from the anion exchange resin include, but are not limited to, methanol, ethanol, propanol, isopropyl alcohol, isobutyl alcohol, butanol, ethyl acetate, acetonitrile, acetone, aqueous mixtures of the foregoing solvents, methylene chloride, chloroform, carbon tetrachloride, or a mixture of any of the foregoing solvents.
Other methods, including those found in U.S. Pat. No. 5,679,806, for the solid phase extraction of glycosylated isoflavones from plant materials can also be adapted to the solid phase extraction of isoflavone biotransformation products.
Having obtained reversibly modified isoflavone biotransformation products, the next step of the method of the invention is to convert that product to an isoflavone (i.e., an isoflavone aglycone). Without being limited by theory, the reversibly modified isoflavone biotransformation products are believed to be isoflavone derivatives or conjugates that are glycosylated or esterified. Preferably, the reversibly modified isoflavone biotransformation products that are O-glycosylated or esterified at some or all of the available positions (i.e., C-7, and C-4′ for daidzein, and C-7, C-4′ and C-5 for genistein). It has been determined that one of the primary isoflavone biotransformation products produced in Saccharopolyspora fermentations is an isoflavone rhamnoside (
In the context of this invention, conversion of a reversibly modified isoflavone biotransformation product to an isoflavone comprises the conversion of any amount of the reversibly modified isoflavone biotransformation product to any isoflavone. In this context, “any amount of conversion” is taken to be an amount of conversion that would exceed the amount of “background” conversion of reversibly modified isoflavone biotransformation products that might occur in a spent fermentation broth or derived reversibly modified isoflavone biotransformation product sample held at up to 40° C. at pH3 or greater. Preferably, at least 50% or greater of the available (i.e., convertible) reversibly modified isoflavone biotransformation product is converted to an isoflavone. The amount of available (i.e., convertible) reversibly modified isoflavone biotransformation product can be determined by applying any one of the conversion methods described here, removing aliquots at various time points, and determining the time point at which the maximal amount of conversion of isoflavone biotransformation product to isoflavones has occurred. This maximal amount of conversion thus defines the amount of available (i.e., convertible) reversibly modified isoflavone biotransformation product. For example, the isoflavone biotransformation product is treated with about 3N to 4N HCl at about 80° C. for about 1, 2, 3, 4 and 5 hours to determine the maximal amount of conversion of reversibly modified isoflavone biotransformation product to isoflavones. The amount of conversion can be determined by thin-layer chromatography or any other suitable combination of separative (i.e., HPLC) and analytic techniques (UV absorption, fluorescence, Evaporative Light Scattering, mass spectrometry), where purified isoflavones (i.e., known amounts of genistein, daidzein, biochanin, formononetin, and/or glycitein) are used as qualitative and quantitative standards. In more preferred embodiments, 70% or greater of the available (i.e., convertible) reversibly modified isoflavone biotransformation products are converted to an isoflavones. In the most preferred embodiments, 95% or more of the available reversibly modified isoflavone biotransformation products are converted to an isoflavones.
One method of converting an isoflavone biotransformation product to an isoflavone is to treat the isoflavone biotransformation product with a sufficient amount of an acid and a sufficient degree of heat to result in conversion of said isoflavone biotransformation product to an isoflavone. In the context of this invention, sufficient acid and sufficient heat are defined as the amount and acid and heat required to convert any amount of the reversibly modified isoflavone biotransformation product to any isoflavone. In this context, “any amount of conversion” is taken to be an amount of conversion that would exceed the amount of “background” conversion of reversibly modified isoflavone biotransformation products that might occur in a spent fermentation broth or derived reversibly modified isoflavone biotransformation product sample held at up to 40° C. at pH3 or greater. Preferably, at least 50% of the available reversibly modified isoflavone biotransformation products are converted to isoflavones. As noted above, conversion of 70% or greater of the available reversibly modified isoflavone biotransformation products to isoflavones is more preferred, and a 95% or greater conversion rate is most preferred. Sufficiency of the acid and heat treatment are easily determined by comparing the percent conversion that is observed in the test acid and heat conditions to the amount of conversion that occurs in the conversion methods taught herein (i.e., treatment of the reversibly modified isoflavone biotransformation product with about 3N to 6N HCl at a temperature of about 50° C. to 80° C. for about 3 hours or with about 3N to 4N HCl at about 80° C. for about 3.5 hours). A variety of acids can be used to effect this conversion. For example, boric acid, benzoic acid, butyric acid, carbonic acid, citric acid, hydrobromic acid, hydrochloric acid, hydrofluoric acid, hydroiodic acid, lactic acid, malic acid, mandelic acid, nitric acid, propionic acid, sulfuric acid, oxalic acid, perchloric acid, phosphoric acid, phosphonic acid, pyrophosphoric acid, pyruvic acid, valeric acid, acetic acid and formic acid can be used in the conversion reaction. Another variable in the conversion method is time. A period of time sufficient to complete the conversion reaction in the presence of sufficient heat and acid is used. As described above, time course determinations would permit one skilled in the art to determine if sufficient time has been allowed for conversion of 50% or greater of the available reversibly modified isoflavone biotransformation products.
Another method of converting the obtained isoflavone biotransformation product to an isoflavone entails a first step where the obtained isoflavone biotransformation product is treated with an acid and heat followed by a second step where the acid and heat treated biotransformation product from the first step is treated with at least one enzyme selected from the group consisting of a cellulase, a hemicellulase, a pectinase, an arabinosidase, or any combination of those enzymes. In this method, the amount of acid and heat in the first step and the amount of enzyme in said second step are sufficient to result in conversion of said isoflavone biotransformation product to an isoflavone. Sufficiency of the amount of acid, heat, and enzyme treatment are defined as the amount and acid, heat and enzyme required to convert any amount of the reversibly modified isoflavone biotransformation product to any isoflavone In this context, “any amount of conversion” is taken to be an amount of conversion that would exceed the amount of “background” conversion of reversibly modified isoflavone biotransformation products that might occur in a spent fermentation broth or derived reversibly modified isoflavone biotransformation product sample held at up to 40° C. at pH3 or greater. Preferably, at least 50% of the available reversibly modified isoflavone biotransformation products are converted to isoflavones. Conversion of 70% or greater of the available reversibly modified isoflavone biotransformation products to isoflavones is more preferred, and a 95% or greater conversion rate is most preferred. As previously described, the amount of available (i.e., convertible) reversibly modified isoflavone biotransformation product can be determined by applying any one of the conversion methods described here (i.e., acid and heat or acid and heat followed by one or more enzymes), removing aliquots at various time points, and determining the point at which the maximal amount of conversion of biotransformation product to isoflavones has occurred.
Acids useful for the first step include, but are not limited to, boric acid, benzoic acid, butyric acid, carbonic acid, citric acid, hydrobromic acid, hydrochloric acid, hydrofluoric acid, hydroiodic acid, lactic acid, malic acid, mandelic acid, nitric acid, propionic acid, sulfuric acid, oxalic acid, perchloric acid, phosphoric acid, phosphonic acid, pyrophosphoric acid, pyruvic acid, valeric acid, acetic acid and formic acid. The obtained isoflavone biotransformation product is treated in said first step with acid sufficient to obtain a pH of about 3.0-5.0 at a temperature from about 80° C. to about 100° C. Either one enzyme or a mixture of enzymes is used in the second step. Alternatively, only one enzyme such as a cellulase, a hemi-cellulase, or a pectinase is used in said second step. A mixture of cellulase, a hemi-cellulase, a pectinase and an arabinosidase can also be used in said second step. As used here, a cellulase can comprise either an exo-beta-1,4-glucanase activity (i.e., a cellulase that preferentially hydrolyses terminally linked sugars of polysaccharides) or a combination of both an exo-beta-1,4-glucanase activity and an endo beta-1,4-glucanase activity (i.e., enzymes that hydrolyze interior beta-1,4-bonds in polysaccharides to release dextrins and oligosaccharides from beta-glucans). Beta glucanases that comprise an endo beta-1,4-glucanase activity (i.e., enzymes that hydrolyze interior beta-1,4-bonds in polysaccharides to release dextrins and oligosaccharides from beta-glucans) cannot be used as a sole enzyme in this method to effect conversion of the reversibly modified isoflavone biotransformation products. However, a beta glucanase comprising an endo beta-1,4-glucanase activity can be used in the second step of this method with any one of a cellulase comprising an exo-beta-1,4-glucanase activity, a hemi-cellulase, a pectinase or an arabinosidase. Alternatively, a beta glucanase comprising an endo beta-1,4-glucanase activity can be used in the second step of this method in combination with a hemi-cellulase, a pectinase or an arabinosidase. As used here, a hemicellulase is any enzyme that catalyzes the degradation or hydrolysis of hemicellulose. Examples of types of hemicellulose include but are not limited to xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan. As used herein, a pectinase is any enzyme that catalyzes the degradation or hydrolysis of pectin. Pectins include, but are not limited to, homogalacturonan, rhamnogalacturonan I (which comprises alternating L-rhamnose and D-galacturonic acid subunits with α-(1-5)-L-arabinan and β-(1-4)-D-galactan side chains), and Rhamnogalacturonan II. Pectinases include, but are not limited to, polygalacturonase enzymes and rhamnogalacturonidase enzymes. As used herein, an arabinosidase is any enzyme that degrades or hydrolyses an arabinan, an arabinoxylan, or an arabinogalactan. Arabinosidases include, but are not limited to, arabinan endo-1,5-alpha-L-arabinosidases, beta-L-arabinosidases, and alpha-N-arabinofuranosidases. As used herein, an arabinase is an arabinan endo-1,5-alpha-L-arabinosidase.
Another variable in the two step conversion method using acid and heat and then one or more enzyme(s) is time. A period of time sufficient to complete the conversion reaction in the presence of sufficient heat and acid (in the first step) and then in the presence of sufficient enzyme(s) is used. As described above, time course determinations would permit one skilled in the art to determine if sufficient time has been allowed for conversion of 50% or greater of the available reversibly modified isoflavone biotransformation products.
This method can further comprise the step of cooling the acid and heat treated isoflavone biotransformation product from the first step to a temperature from about 40° C. to about 60° C. before adding at least one enzyme in a subsequent step. Conversion of 80% or greater of the available reversibly modified isoflavone biotransformation products to isoflavones is preferred, and a 95% or greater conversion rate is most preferred. However, the use of thermostable enzymes (i.e., enzymes that are active at temperatures of greater than 60° C.) is further contemplated. Thermostable enzymes are typically obtained from thermophilic organisms or by mutagenesis and selection of thermostable variants of enzymes. Use of thermostable enzymes could obviate completely or lessen the amount of cooling necessary after completion of the first step of this method.
Furthermore, the acid and heat treated biotransformation product from the first step can also be adjusted to a pH that matches the pH optima of the enzyme or enzymes used in the second step. This is accomplished by neutralizing the acid present in the sample containing the acid and heat treated isoflavone biotransformation product with either an appropriate base or an appropriate buffer. Appropriate bases include, but are not limited to, sodium, ammonium or potassium hydroxide. Appropriate buffers include, but are not limited to, Tris buffers, sodium phosphate buffers, sodium citrate buffers and sodium succinate buffers.
Having converted the reversibly modified isoflavone biotransformation product to an isoflavone, the final step of the claimed method entails isolation of the isoflavone from other compounds present in the treated (i.e., converted) material that has been subjected to any of the previously described conversion methods. As defined earlier, “isolating an isoflavone” refers to any process whereby an isoflavone is separated from another component that is mixed with the isoflavone. In this regard, isolation of the isoflavone may be either partial or complete. In other words, isolation of the isoflavone may represent nothing more than a partial purification whereby the isoflavone present in the converted material is separated from at least one component that is present in the original mixture. For example, it is anticipated that various saccharides (i.e., mono-, di-, oligo-, and poly-saccharides such as glucose, fructose, rhamnose, sucrose, cellulose, etc.) will be present in the treated (i.e., converted) material that also contains the isoflavone that has been released from the reversibly modified biotransformation products. Any step or method, such as a solvent extraction, that separates the isoflavone from those saccharides would thus constitute an isolation of an isoflavone in the context of this invention, even though other plant compounds that happen to copurify with the isoflavone are also present in the fraction that contains the isoflavone. The isolated isoflavone obtained by this method would thus be substantially free of saccharides. In this regard, it is possible that the isolated isoflavone, though substantially free of some compound originally present in the treated (i.e., converted) mixture, can also contain other compounds such as flavanoids, flavones, or flavanones.
Concentration of an isoflavone such that the isoflavone is not separated from any other component that is mixed with the isoflavone does not constitute “isolating an isoflavone”. For example, if a treated (i.e., converted mixture) containing the isoflavone released from the reversibly modified biotransformation products and other compounds is simply subjected to a distillation or evaporation step such that all of the components originally present in that mixture are still present in roughly the same ratios (i.e., molar ratios or mass ratios), then this distillation or evaporation step would not constitute an isolation step as described herein.
Of course, isolation of an isoflavone can also comprise further purification of one or more isoflavones from the treated (i.e., converted mixture) from the previous step of the method of this invention. For example, any one of the isoflavones produced by the method of this invention (i.e., genistein, daidzein, biochanin, formononetin, or glycitein) can be purified to a level of at least about 70%, 80%, 90%, 95% or 99% or greater purity on a mass basis using methods described herein or known to those of skill in the art. Alternatively, two isoflavones, such as genistein, daidzein, can be co-purifed to a level of at least about 70%, 80%, 90%, 95% or 99% or greater purity on a mass basis using methods described herein or known to those of skill in the art.
A variety of methods for isolating isoflavones can be used. Particularly contemplated are methods involving solvent extraction and solid phase extraction with sorbents. It is of course understood that a combination of isolation methods can also be used, especially when isolating isoflavones in more purified forms. It is also understood that other techniques, such as preparative column chromatography or high-performance liquid chromatography can be used either alone or in combination with methods such as solid phase or solvent extraction to isolate isoflavones from the treated mixture.
With respect to isolation of isoflavones by solvent extraction of the treated (i.e., converted material), isolation of the isoflavone can be effected by extracting said isoflavone from an aqueous phase comprising the treated material with a polar or non-polar solvent that is immiscible in water and recovering the isoflavone in the solvent phase. This solvent can be propanol, isopropyl alcohol, isobutyl alcohol, butanol, ethyl acetate, acetonitrile, acetone, methylene chloride, chloroform, carbon tetrachloride, or a mixtures of these solvents. Once the isoflavone is isolated in the solvent, it can be recovered by evaporating the solvent.
A variety of methods for extracting isoflavones from plant tissue with solvents have been described that can also be adapted for use in this particular method for isolating isoflavones from treated (i.e., converted) materials obtained from reversibly modified biotransformation products produced in fermentations. For example, U.S. Pat. No. 7,015,339 describes a method for isolating phenolic compounds such as isoflavone aglycones comprising the steps of: (a) providing an aqueous plant extract at a first pH greater than 10, the aqueous plant extract comprising a plurality of phenolic compounds; (b) washing the aqueous plant extract with an organic solvent; (c) adjusting the pH of the aqueous plant extract to a pH of less than 9; and (d) isolating the phenolic compounds from the aqueous plant extract. In the context of this invention, this method of U.S. Pat. No. 7,015,339 can be adapted to provide for isolation of isoflavones by adjusting the aqueous treated (i.e., converted) material containing the isoflavone aglycones to a pH greater than 10, washing with an organic solvent, adjusting the aqueous phase to a pH of less than 9, and then isolating the isoflavones from the aqueous fraction. That same patent also provides a similar method for isolating plant phenolics that include isoflavone aglycones comprising (a) providing an aqueous plant extract at a first pH less than 10, the aqueous plant extract comprising a plurality of phenolic compounds; (b) extracting the aqueous plant extract with a first organic solvent to yield a first organic extract; (c) extracting the first organic extract with an aqueous phase of pH greater than 10 to yield a phenol rich aqueous phase; (d) adjusting the pH of the phenol rich aqueous phase to a pH of less than 9; and (e) isolating the phenolic compounds from the phenol rich aqueous phase. In the context of this invention, this method of U.S. Pat. No. 7,015,339 can be adapted to provide for isolation of isoflavone aglycones from the aqueous treated (i.e., converted) material by providing the aqueous treated (i.e., converted) material at a pH of less than 10, extracting with an organic solvent to yield a first organic solvent containing the isoflavone aglycone, and then extracting that organic solvent phase with an aqueous phase of pH greater than 10 to yield an aqueous phase containing the isoflavones, adjusting this aqueous phase back to a pH of less than 9, and then isolating the isoflavones from that aqueous phase. Other examples of methods for isolating isoflavones from plant materials or other sources with solvents that can be readily adapted and applied to isolating the isoflavones produced by treatment of spent fermentation beer are described in U.S. Pat. Nos. 5,554,519 and 6,517,840.
U.S. Pat. No. 7,033,621 is especially notable in that it describes a process for the production of isoflavones from plants which comprises simultaneously contacting plant material with water, an enzyme which cleaves isoflavone glycosides to the aglucone form, and a C.sub.2-C10 organic solvent, to form a combination and incubating the combination for a time sufficient to allow isoflavones of the aglucone form to partition into the organic solvent component, and thereafter recovering isoflavones from the organic solvent component. This method could be adapted to the methods of this invention where the conversion step comprises treatment with an acid and heat followed by an enzyme treatment by coincubating the aqueous material containing the enzyme(s) with the organic solvent. The isoflavones can then be isolated from the organic solvent fraction at the conclusion of the reaction. The advantage of this particular process is that it represents a “one pot, single stage” process that can simplify the isoflavone isolation procedure.
Solid phase extraction is another method for isolating isoflavones from the treated (i.e., converted) material produced by this method. Solid phase extraction of the isoflavones from the treated (i.e., converted) material can be accomplished by any number of different methods of solid phase extraction such as column-based methods or batch-based methods. The solid phase extraction is effected with a sorbent selected from the group consisting of C18, divinylbenzene, polystyrene-divinylbenzene, non-polar polystyrene, anion exchange resin, and modified divinylbenzene. Once the isoflavone is adsorbed to the solid phase sorbent, the adsorbed material is typically washed with aqueous solutions that do not displace the isoflavone from the sorbent to remove other undesired or contaminating materials. Alternatively, certain admixtures of alcohol and water that do not displace the isoflavone from the sorbent, or admixtures of an organic solvent and water that do not displace the isoflavone from the sorbent, can be used to remove other undesired or contaminating materials. Once the adsorbed material containing the isoflavone has been washed, the isoflavone can be released by using either an alcohol, an organic solvent, an admixture of alcohol and water that displaces the isoflavone from the sorbent, or admixtures of an organic solvent and water that displace the isoflavone from the sorbent.
Methods for the solid phase extraction of isoflavone biotransformation with anion exchange resins or sorbents have been described previously in the preceding section of the detailed description of the invention on solid phase extractions of isoflavone biotransformation products. As disclosed in Example 1 of U.S. Pat. No. 6,020,471, these methods are also effective in the solid phase extraction of isoflavone aglycones. It is therefore anticipated that anion exchange resin based extraction methods can also be used to effect isolation of isoflavones from the treated (i.e., converted) materials produced by the methods of this invention.
Other methods of solid phase extraction of isoflavones with non-polar or slightly polar resins are described in U.S. Pat. No. 4,428,876 and can be adapted to the isolation of isoflavones in the practice of this invention. Either slightly polar, acrylic ester based adsorbent resin or non-polar, styrene-vinylbenzene based adsorbent resin can be used in this method. Methanol or aqueous methanol is typically used to elute the isoflavones from the non-polar resin.
In view of the foregoing, it will be seen that the several advantages of the invention are achieved and attained.
The embodiments were chosen and described in order to best explain the principles of the invention and its practical application to thereby enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated.
The first isoflavone related biotransformation that occurs during the erythromycin fermentation process involving S. erythrea is the hydrolysis of glucoside groups from the isoflavone core and the release of the free isoflavone aglycones (Hessler et al., Appl. Microbiol Biotechnol, 47:398-404,1997). However, this study indicated that the desired isoflavone aglycones did not accumulate in these fermentations. The object of this example was to characterize the isoflavone biotransformation products produced in these fermentations.
The erythromycin producing strain used for this example was the wild-type “white” Saccharopolyspora erythraea FL2267, which is the same strain as ATCC 11635 (American Type Culture Collection, Manassas, Va.). This strain is also referred to as FL300 in the experiments described herein. Spores of S. erythraea were produced on E20A agar (Reeves et al., Antimicrobiol. Agents Chemother, 46, 3892-3899, 2002). Seed cultures were prepared in CFMI broth (Carbohydrate-based Fermentation Medium, a variation of SCM broth described previously [12]. CFM1 is a medium designed for laboratory use where a soluble medium is desired for convenient analysis of growth and chemical analysis of erythromycin production. CFM1 per liter distilled water: Difco™ soluble starch, 60 g; Bacto™-soytone (Difco™), 20 g; CaCl2.2H2O (Sigma), 0.1 g; Bacto™-yeast extract (Difco™), 1.5 g; MOPS, 26.5 g; pH adjusted to 6.8 with 4N NaOH (Sigma, Saint Louis, Mo.). CFM1 lacks the glucose, vitamins and trace elements of modified SCM [12]; and has 60 g (4×) of soluble starch. Fermentations were performed in either CFM1 broth or OFM1 broth (Oil-based Fermentation Medium). OFM1 contains insoluble medium components and is meant to closely correlate to an industrial-type fermentation medium. OFM1 per liter: toasted nutrisoy flour (ADM, Decatur, Ill.), 22 g; Difco™ soluble starch, 15 g; CaCO3 powder (JT Baker, Phillipsburg, N.J.), 3 g; MgSO4.7H2O (JT Baker, Phillipsburg, N.J.), 0.5 g; FeSO4.7H2O (JT Baker, Phillipsburg, N.J.), 15 mg; Soy oil, 50 ml (ADM, Decatur, Ill.). Pigmentation and sporulation studies were conducted on R2T2 agar. R2T2 is R2T described in Weber et al. (J. Bacteriol., 172, 2372-2383, 1990) minus peptone.
Fermentations were performed in unbaffled 250-ml Erlenmeyer flasks with milk-filter closures. The flasks were incubated at 32.5° C.±0.2° C., and 65%±3% humidity on an Infors Multitron Shaker having 1-inch circular displacement. Seed cultures containing CFM1 broth were prepared on the same shaker and under the same growth conditions that the fermentations were performed. Seed cultures were inoculated from fresh spores prepared from E20A agar plates. Fermentations were inoculated with 1.25-ml of a seed culture in late logarithmic growth phase (40-45 h) into 25-ml of CFM1 or OFM1 broth. Consistency in the preparation of the seed culture was required for each strain to obtain and maintain optimum and reproducible fermentation performance from experiment to experiment. Fermentations were grown for 3-5 days; their volumes were then corrected for evaporation through the addition of water before being further analyzed. At the conclusion of the indicated fermentation period, the spent growth media was separated from the cells and extracted with ethyl acetate. The ethyl acetate extracts were then concentrated by evaporation to yield material for analysis by TLC or HPLC.
Three solvent systems were used for identification of the isoflavones by Thin-Layer Chromatography (TLC). First, chloroform:methanol:acetic acid (10:1:1) was used to separate isoflavone aglycones (genistein, daidzein). However, hexane:ethyl acetate:methanol (20:20:8) was used for the better resolution of the glycosylated isoflavones (“spots A, B and C”, genistin, daidzin). All reagents were HPLC grade. All samples for the TLC analyses were prepared as ethyl acetate solutions. As a standards 0.3 mg/ml ethyl acetate solutions of the pure isoflavones (Indofine, Hillsborough, N.J.) were used. The third solvent system used was chloroform:MeOH:water (80:20:2). For analytical TLC experiments, Silica Gel 60 F254 plates (Macherey-Nagel) with 0.2 mm thickness were used. Presence of the F254, a fluorescent indicator with a 254 nm excitation wavelength made possible identification of the isoflavone products that appeared as dark spots at 254 nm (UVP transilluminator).
The aglycones, genistein and daidzein, remain in the fermentation only transiently and their disappearance coincides with appearance of two new spots on TLC (
In a fermentation biotransformation with another erythromycin-producing bacterium, Aeromicrobium erythreum, we observed that the first (desired) step of the conversion, genistin to genistein, occurred as rapidly as it did with S. erythraea, but the second (undesired) step, conversion of genistein, did not occur at all, even after four days growth. The two organisms fermentation extracts were compared side by side (
Further evidence that spot A represented a biotransformation product of genistein was obtained when in vivo biotransformation reactions were performed in which pure genistein was added to S. erythraea cultures that were grown in a medium lacking soyflour and the same (i.e., comigrating) spot “A” observed in the original S. erythraea fermentations with soyflour appeared. To produce, purify and characterize the “spot A” biotransformation product of pure genistein, about 5 microliters of a dense spore suspension of S. erythraea was added to 40 ml of SCM broth in a 250 ml shake flask and incubated at 30° C. and 400 rpm (1 inch orbital displacement) for 2 days until cultures are thick and well grown. The entire 40 ml culture was transferred to a 2 L shake flask containing 100 ml of SCM and 100 mg of genistein (added directly to the medium from the vial), incubate the culture at 30° C. with shaking for 5 to 24 hrs. The broth was stored at 4° C. until all batches were ready for purification.
To purify the spot A isoflavone biotransformation product from the spent fermentation broth, the cells were first removed from the broth by centrifugation. The fermentation broth was then extracted twice with one-half volume of ethyl acetate and the solvent layers were combined and concentrated by evaporation under vacuum. The concentrated extracts were then applied to the top of a 12 g silica-gel flash chromatography column and processed on an Isco, Inc. Combi-Flash Companion Flash chromatography apparatus using a linear gradient elution method programmed to run from a starting condition of 25% hexane and 75% ethyl acetate to a finishing condition of 100% ethyl acetate over a 15 minute run. The individual fractions containing pure spot A isoflavone biotransformation product were identified by UV absorption profile and thin layer chromatography analysis. They were combined and dried completely in vacuo. The dried powder was then suspended in 80% ethanol, heated to 90° C. and dissolved completely, followed by cooling at 4° C. overnight. Crystals appeared overnight and were washed with cold ethanol and dried in vacuo.
The chemical analysis of spot “A” by mass spectral analysis, NMR analysis and X-ray Crystallographic analysis led to its clear structural identification as genistein 7-O-alpha-rhamnoside (
The implication of the identity of spot A as genistein 7-O-alpha-rhamnoside was that the fermentation organism, S. erythraea must therefore harbor a gene coding for a rhamnosyltransferase that is capable of transferring a rhamnose group onto the genistein aglycone converting it to the rhamnosyl derivative. A further implication of this result was that if the gene coding for the isoflavone rhamnosyltransferase could be cloned and knocked out, then the unwanted biotransformation of the isoflavone aglycones could be stopped during the erythromycin fermentation, thus allowing the recovery of the isoflavone aglycones at the end of the fermentation.
To this end, the S. erythraea rht gene was cloned and analyzed. The DNA sequence of the S. erythraea rht gene was determined and analyzed by BLAST homology searches. The strongest homology of the S. erythraea rht gene was to a putative dTDP-rhamnosyl transferase from Mycobacterium leprae (gil|13092868|emb|CAC30261.1|) to which it showed a 60% sequence identity over a region of 270 bp. The same level of sequence identity was found to a putative dTDP-rhamnosyltransferase from Nocardia farcinica IFM 10152. (accession numbers gi|54018118|dbj|BAD59488.1|).
In order to knockout the rht gene of S. erythraea, an integrative plasmid was constructed which was designed to insert into the rht gene and inactivate the gene by insertional disruption. No transformants could be obtained with the plasmid construct designed to disrupt the rht gene, but in control experiments, many hundreds of transformants could be obtained with similar plasmid constructs that were designed to knockout another gene unrelated to the rht gene. These results suggested that rht might be a gene essential for the growth of S. erythraea and therefore could not be inactivated unless a replacement gene was transferred into the cell on the same plasmid that was used for the inactivation of the rht gene. When a replacement rhamnosyltransferase gene from a Corynebacterium species was included on the insertion plasmid, then it was found that insertional disruption of the S. erythraea rht gene could be achieved, however the disruption of rht did not stop the biotransformation of genistein to rhamnosylgenistein. It is believed that the failure to block biotransformation by replacement of the S. erythraea rht gene with the Corynebacterium rht gene was due to the ability of the Corynebacterium rht gene product to catalyze biotransformation of genistein to rhamnosylgenistein.
The conclusion of this series of experiments led to the development of another molecular genetic based strategy for stopping biotransformation of isoflavones in the erythromycin fermentation. These experiments, which attempted to block the first biotransformation reaction where the isoflavone glycosides from the soybean growth media were converted to isoflavones by inactivating endogenous S. erythraea beta-glucosidases and beta-galactosidases, also met with little success.
Further analysis of thin layer chromatography plates and identification of highly polar UV-absorbing spots indicated that the final isoflavone biotransformation products may not be irreversibly degraded or modified as previously believed. To demonstrate that these final transformation products were actually reversibly modified biotransformation products, the isoflavone biotransformation products were obtained and treated in a conversion reaction. In this particular example, the conversion reaction consisted of acid and heat treatment. The products of this conversion reaction were then further purified and analyzed. Conversion of the isoflavone biotransformation products to isoflavones was thus demonstrated as described in detail below.
In brief, a fermentation was performed in modified OFM1 media containing 2× soyflour lacking soy oil. Soy oil was omitted and extra soyflour was added to simplify the analysis of the fermentation broths by thin layer chromatography. Two sets of samples were taken at each fermentation time point. In the first set of control samples, the cells were separated from the spent fermentation broths. The aqueous spent fermentation broths were extracted with an organic solvent (ethylacetate:n-butanol, 9:1), which was then separated from the aqueous phase, concentrated and analyzed by TLC (
The strong acid and heat treatment was conducted as follows. To 500 ul of fermentation broth in a 15 ml polypropylene test tube with screw cap closure, 250 ul of concentrated (12 N) HCl was added. The tube was incubated at 80° C. for 3.5 h, cooled, and two ml of water was added to the acidified solution. The aqueous mixture was extracted once with 1.5 ml of ethylacetate:n-butanol (9:1). The solvent fraction was separated from the aqueous fraction, evaporated, and the remaining small residue was resuspended in 25 ul of ethyl acetate. Five ul of the resuspended product was spotted on thin layer silica gel plates which were then developed and visualized according to the method described in the previous Examples.
Samples taken prior to the start of the fermentation (0 hrs,
Next the extracts of untreated fermentation broth (
U.S. Pat. No. 6,616,953 describes a milder acid treatment process using sulfuric acid in combination with heat and industrial enzyme treatments of spent fermentation broths of S. erythraea. This particular treatment method is of interest as it can be practically scaled up for operation on an industrial level. In the lab scale experiments described in this example, we demonstrate that the combined treatment of spent fermentation broths with acid, heat and enzymes is also highly effective in converting the biotransformation products in the spent fermentation broth into the desired isoflavone aglycones.
In brief, 3 ml of fermentation broth sample from S. erythraea were obtained as described in the previous examples. Concentrated sulfuric acid was then added (20-40 ul) to the 3 ml fermentation broth sample to lower the solution to pH 3. The solution was then heated to 90° C. for 3.5 hr, cooled to 45° C., at which point 30 ul of Validase® BG (beta-glucanase) and 30 ul of Crystalzyme® PML-MX (pectinase/cellulase) enzyme solutions were added. Validase® BG (an endo beta 1,4 glucanase) and Crystalzyme® PML-MX (a mixture consisting of cellulases, hemicellulases, pectinases, and arabinases) enzyme solutions were purchased from Valley Research, South Bend, Ind. The enzyme treatment was performed for 17 hrs before the solutions were solvent extracted as described in the previous examples for TLC analysis.
A comparison of the two methods for converting reversibly modified isoflavone biotransformation products is shown (
To test the ability of the Validase® BG (an endo beta 1,4 glucanase) and Crystalzyme® PML-MX (a mixture consisting of cellulases, hemicellulases, pectinases, and arabinases) enzyme solutions to perform the conversion reaction independently of one another, each enzyme preparation was tested individually and in combination. In brief, 3 ml of spent fermentation broth was brought to pH to 3 with sulfuric acid and heated for 3.5 hours at 90° C. After cooling to 45° C., either 1% Crystalzyme® PML-MX (C), 1% Validase® BG (V), or both (C+V) were added to the acid treated material. This was then incubated for 20 hrs and extracted with equal volumes of ethyl acetate:n-butanol (9:1), agitated for 1 min, and then centrifuged to separate the organic and aqueous phases. Organic phases were transferred to eppendorf tubes, dried in vacuo, and resuspended in 400 ul acetonitrile. About 380 ul of the supernatant was harvested and dried in vacuo. The material was then resuspended in 40 acetonitrile and spotted 5 ul/lane on TLC plates as described previously.
The results of the experiment shown in
Ten other Streptomycetes of industrial importance were tested to demonstrate the production of reversibly modified isoflavone biotransformation products and their conversion to the desired isoflavone aglycones by acid and heat treatment. In these experiments, the erythromycin-producing organism, Saccharopolyspora erythraea was included as a positive control.
The results show that all of the Streptomycetes tested have the ability to perform the first isoflavone glucoside biotransformation reaction, namely, they are able to hydrolyze glucoside groups from isoflavone glucosides genistin and daidzin to form the isoflavone aglycones, genistein and daidzein. More importantly, the results also show that five out of ten of these other Streptomyces (numbers 3, 4, 5, 7, and 10,
Five Streptomycetes catalyze the first desirable biotransformation reaction (i.e., hydrolysis of isoflavone glucosides in the fermentation media to form the isoflavone aglycones, genistein and daidzein) but fail to catalyze the subsequent biotransformation reactions that require conversion for recovery of isoflavone aglycones. These particular Streptomycetes with the desirable property of catalyzing first biotransformation reaction to form isoflavones (i.e., isoflavone aglycones) are Streptomyces roseolus, Streptomyces antibioticus, Streptomyces griseus, Streptomyces lasaliensis, and Streptomyces parvulus. This result indicates that isoflavones could be directly recovered from the spent fermentation beer produced when any one of these five Streptomycetes are fermented on a leguminous plant based substrate such as soy without having to perform conversion treatments. Production of isoflavones by Streptomyces roseolus in soy-based fermentations has previously been described in U.S. Pat. No. 5,554,519.
erythraea ATCC 11635
cinnamonensis ATCC 15413
hygroscopicus ATCC 25253
As various modifications could be made in the constructions and methods herein described and illustrated without departing from the scope of the invention, it is intended that all matter contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative rather than limiting. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims appended hereto and their equivalents.
Each and every patent and non-patent literature reference cited herein is hereby specifically incorporated by reference in its entirety.
This invention was made with Government support under a National Cancer Institute of the National Institute of Health Grant No. R44 CA093165. The government has certain rights to this invention.
