JP2002253289A - Method for producing optically active lactone - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily obtain an optical isomer of γ-lactones having various flavors or threshold values and to provide a useful material discriminable from a conventional material for foods and cosmetics by using the intact optical isomers or formulating the specimen in a suitable R/S ratio. SOLUTION: Lactones are subjected to deta-oxidation using a hydroxy acid or a methyl ester thereof as a substrate with a yeast and biosynthesized. If the yeast used is a yeast for brewing, an R-isomer lactone is produced when the substrate is the R-isomer and S-isomer lactone is produced when the substrate is the S-isomer. The R-isomer hydroxy acid to be the substrate can be obtained by biosynthesis using a usual lactic acid bacterium used for producing foods. The methyl ester of the S-isomer hydroxy acid can be separated from the racemate. The yeast for brewing is added to a culture medium containing the optically active hydroxy acid or the methyl ester thereof and aerobically cultured to produce the lactone having the same optical purity as that of the substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention uses an optically active hydroxy acid or its methyl ester as a substrate and has a sweet aroma that can be used for alcoholic beverages, foods, flavors and the like by treating with yeast for brewing. The present invention relates to a method for producing a gamma-lactone compound, which is a component, for each enantiomer, that is, by separating it into an R-form and an S-form.

[0002]

2. Description of the Related Art Among lactones which are cyclic esters,
Gamma decalactone and gamma decalactone have a sweet scent similar to peach and have been used as a component for preparing fragrances. Most of these are used in racemic form, but it is known that each optical isomer has a different fragrance and threshold. The threshold value of gamma-decalactone is 1.5 ppm for the R-form and 5.5 for the S-form.
6 ppm (ACS Symposiu)
m, 258, 1995). Regarding the characteristics of the scent, although both are sweet scents, the R-form is strong (stron
g), caramel-like slightly fatty scent,
On the other hand, the S-form is soft and reported to have a coconut-like aroma. The same applies to gammad decalactone, where the R-form is said to have a strong fruity scent, and the S-form is said to have a soft, milky scent (J. of Agri. Food Che).
m. 413, 37, 1989). Therefore, if these optical isomers are obtained and a lactone mixed at an appropriate R / S ratio is prepared, it will be a useful material that can be differentiated from conventional materials as a material for blending foods and cosmetics. The structural formula of gamma-decalactone is shown in Chemical formula 1, and the structural formula of gamma-decalactone is shown in Chemical formula 2.

[0003]

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[0004]

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[0005] It has been revealed that high purity R-form lactones are also contained in lactones contained as aromatic components in natural products such as fruits (J. of C).
Chromatography, 498.396 (199)
0)). However, since the content is generally very small,
It is extremely difficult to separate and purify lactones having high optical purity from natural products and use them.

[0006] As a method for producing gamma-lactones by a chemical synthesis method, a method of reacting a primary alcohol and an acrylate ester with an organic peroxide in the presence of a mineral acid and / or an organic acid to synthesize them (Japanese Patent Laid-open Publication JP-A-55-133371), from a primary alcohol, a secondary alcohol and a 2-alkenoic acid ester in the presence of an organic peroxide and a nitrogen-containing compound or 1,1-bis-t-butylperoxycyclohexane. Synthesis method by heating reaction in the presence (JP-A-4-275282 and JP-A-4
-275283), but there is no description about the optical purity of the obtained lactone.

Furthermore, a chemical synthesis method (A) in which optically active gamma-lactone is synthesized by optical resolution using phenethylamine which is a phthalic acid ester of acetylene alcohol.
gri. Biol. Chem. 43,1571,197
9) and a chemical synthesis method (Tetrahedron Lett. 2), which is synthesized by an asymmetric synthesis reaction using a metal complex.
2,247, 1981), but all of these chemical synthesis methods use expensive reagents, have complicated processes, and are not suitable for mass production. In addition, since it is necessary to use a large amount of an organic solvent, there are many problems in application to foods and the like.

On the other hand, production of gamma-lactones by fermentation using microorganisms has been attempted. JP-A-59-
No. 820090 and JP-A-61-238708 disclose that castor oil (caster oil) can be obtained from Saccharomyces genus, Hansenula (Ha).
nsenula), Candida
Fermentation using genus and Pichia yeasts to produce gamma-decalactone from ricinoleic acid, which accounts for 90% of the constituent fatty acids in castor oil, to improve skin feel, remove unpleasant odors, Are disclosed. Also, JP-A-3-117494 discloses that
Japanese Patent Application Laid-Open No. 3-198787 discloses a method for producing gamma-lactone using yeast with a hydroxy fatty acid as a substrate, wherein gamma-decalactone is produced from 10-oxystearic acid by a microorganism having beta-oxidizing ability. ing. Further, Japanese Unexamined Patent Application Publication No.
No. 86 and JP-A-7-327689 disclose a method for producing gamma dedecalactone or gamma decalactone from oleic acid or palmitooleic acid. However, there is no description about optical purity anywhere in these.

As an attempt to synthesize optically active gamma-lactone using enzymes and microorganisms, a synthesis method using lipase as an enzyme catalyst has been disclosed (Tetrahe).
dron, 47, 6223, 1991) has a problem in application to food because the process is complicated and the reaction needs to be performed in an organic solvent. Attempts have also been made to produce optically active lactones using yeast. It has been reported that R-form lactones are produced using yeasts of the genus Sporobolomyces (J. of Organic Chem., 57,
1954, 1992), but since this yeast is not a food-grade yeast, there are many problems in applying it to food. Moreover, this method cannot produce S-form lactone.
Furthermore, it has been reported that an optically active lactone is produced by asymmetric reduction with baker's yeast (J. of O.
rganic Chem. , 52, 4363, 198
7) However, also here, only R-form lactone is produced, and S-form lactone cannot be produced.

[0010]

The optically active gamma-lactone can be added to foods as a flavor or used to produce foods, beverages, alcoholic beverages, etc. containing a high concentration as an aromatic component, but the safety as foods Considering
It is desirable not to include complicated chemical synthesis steps. Furthermore, it has been found that it has been used for foods for a long time and no harmful by-products are produced, and there is a strong demand for the development of a method using food-grade microorganisms whose safety has been established.

[0011]

Means for Solving the Problems The present inventors have prepared a process for producing optically active gamma-lactone by using an optically active hydroxy acid and / or its methyl ester as a raw material and treating it with a yeast having beta-oxidizing ability. Established.

As the hydroxy acid and / or methyl ester thereof used as a substrate, any hydroxy acid having a structure that can be lactone-cyclized by yeast can be suitably used, even if it is not a linear fatty acid. Hydroxystearic acid and its methyl ester, 10-hydroxypalmitic acid and its methyl ester, and ricinoleic acid (12-hydroxy-9-octadecenoic acid) can be particularly preferably used. Although there is no particular limitation on the number of carbon atoms of the hydroxy acid, hydroxy acids having 16 or 18 carbon atoms can be suitably used. These hydroxy acids may be used not only in a purified form but also in a composition containing these hydroxy acids in a high content. Next, a method for producing an optically active hydroxy acid will be described.

An R-form hydroxy acid having high optical purity can be easily obtained by treating an unsaturated fatty acid with a lactic acid bacterium. As unsaturated fatty acids, oleic acid or palmito oleic acid having one double bond and linoleic acid having two double bonds can be suitably used, but can be hydroxylated by lactic acid bacteria and further lactone cyclized by yeast. Any unsaturated fatty acid having a structure that can be used can be suitably used, whether it is a branched-chain fatty acid whose aliphatic chain contains a branched chain or a straight-chain fatty acid. Further, a composition containing a high concentration of unsaturated fatty acid may be used. Examples of such a composition include a suspension of yeast cells disclosed in JP-A-11-127886. An unsaturated fatty acid or a composition containing an unsaturated fatty acid is suspended in a suitable medium for lactic acid bacteria to grow. Any concentration of unsaturated fatty acid can be used as long as it is suspended in the medium without being separated, but 50 ppm to 500 pp
m is desirable, and 100 ppm to 200 ppm is more desirable. An appropriate surfactant may be added so that the unsaturated fatty acid is easily suspended. The surfactant is desirably used at a concentration that does not inhibit the growth of lactic acid bacteria. As a medium in which lactic acid bacteria can grow, in addition to wort made from malt, a YP medium composed of 2% peptone and 1% yeast extract,
Commercially available MRS medium, GYP medium and the like are preferably used. A composition containing a large amount of unsaturated fatty acids and a component capable of growing lactic acid bacteria can also be used as a medium. An example of such a composition is a residue obtained by distilling a mash obtained by alcohol-fermenting corn using yeast as a raw material (referred to as a maize distillation waste liquid). The maize distillation effluent is produced as a by-product in the production of bourbon whiskey and Glen whiskey.
Such a composition may be used alone, or may be mixed with a medium for lactic acid bacteria.

A lactic acid bacterium is inoculated and cultured in a medium in which unsaturated fatty acids are suspended. Lactic acid bacteria are a general term for bacteria that decompose carbohydrates and mainly produce lactic acid, and include genus Lactobacillus, genus Streptococcus, genus Leuconostoc, and genus Pediococcus. And the like. For example, Lactobacillus case
i JCM1163, Lactobacillus p
lantarum IAM1041, Pediococ
cus pentosaceus IAM1215, L
euconostoc mesenteroides
IAM1233 and the like. These strains can be obtained as known freely available strains such as the Fermentation Research Institute in Osaka and the RIKEN in Saitama.

The lactic acid bacteria are generally precultured in a medium used for culturing lactic acid bacteria, for example, an MRS medium or a V8 medium, until the number of cells reaches 10 ⁶ to 10 ¹⁰ per 1 ml of the culture solution. The lactic acid bacterium to be added may be added to the pre-culture solution as it is or after being concentrated by centrifugation, membrane treatment, or the like.
Inoculate so as to have ⁴ to 10 ⁸ cells. As it is, about 10
12 ° C. to about 40 ° C., preferably 20 ° C. to 30 ° C.
The culture is performed aerobically or statically by shaking, stirring, etc. for 48 hours. During this time, the unsaturated fatty acids contained in the medium are changed to the corresponding hydroxy acids, and the hydroxy acids obtained here are R with high optical purity.
It is the body's hydroxy acid.

Further, the R-form hydroxyacid is prepared by a known method, a chemical synthesis method for synthesis by an asymmetric synthesis reaction, a separation method by optical resolution or a method using an enzyme catalyst, and a method combining these methods. Alternatively, the R-form hydroxy acid may be separated from the racemic hydroxy acid. In this case, the racemic hydroxy acid may be methylated by a known method, and the R-form hydroxy acid methyl ester obtained by optical resolution may be used as it is, but is converted to the hydroxy acid again. May be.

The R-form hydroxy acid obtained as described above may be methylated by a known method. Methyl esters of hydroxy acids can be used in the same manner as hydroxy acids as substrates for lactone production.

On the other hand, the S-form hydroxy acid can be prepared by a known method (Appl. Env. Microbiol., 5).
9, 281, 1993) using microorganisms. Alternatively, the S-form hydroxy acid may be prepared by a known method, a chemical synthesis method for synthesizing by an asymmetric synthesis reaction, a method using an enzyme catalyst, or a method combining these methods. Furthermore, it may be separated from a racemic product produced by chemical synthesis or the like as it is, or after methylation by a known method, using a resin or the like that can be optically resolved, or after methylating the R-form hydroxy acid, Racemization may be performed to separate the S-form hydroxy acid methyl ester. Similarly to the R-form, the obtained hydroxy acid may be methylated before use.
The S-form hydroxy acid methyl ester thus obtained can be used in the same manner as the hydroxy acid as a substrate for lactone production.

The thus obtained optically active hydroxy acid and / or its methyl ester is purified or once added to a medium in which yeast can grow. The optically active hydroxy acid and / or methyl ester thereof formed in the organic solvent is distilled off by a known method. Further, it may be used after further increasing the purity by using an appropriate column resin. The solution containing the hydroxy acid produced by the culture method may be used as a medium in which yeast can grow as it is, or an appropriate amount thereof may be added to a medium in which yeast can grow as it is.

Examples of a medium in which yeast can grow include commercially available mixtures of peptone, yeast extract, casamino acid and the like with sugars such as glucose and maltose, raw materials for fermented beverages, and raw materials for fermented beverages and mixtures thereof. Alternatively, a medium comprising a mixture of molasses and urea can be exemplified. Alternatively, a synthetic medium such as a modified Wickerham medium may be used.

A medium containing an optically active hydroxyl acid and / or a methyl ester thereof is inoculated with a yeast having beta-oxidizing ability. As the yeast used here, for example,
Saccharomyces genus, Pichia genus, Hansenula (Hansenu)
la), yeasts of the genus Candida, genus Kluyveromyces, genus Zygosaccharomyces, and the like. Such strains include Candida krus which can be obtained as known freely available strains such as the Fermentation Research Institute in Osaka and the RIKEN in Saitama.
ei JCM1609, Hansenula anom
ala JCM3585, Candida membr
anaefaciens IFO10215, Zygo
saccharomyces bailii IFO1
098 etc., commercially available baker's yeast of the genus Saccharomyces,
Sake brewing yeast (commercially available from Japan Brewing Association) or S
accharomyces cerevisiae S2
Known free strains such as 88C (American Yeast Genetic Conservation Center strain) can be exemplified. Although yeast strains other than the genus Saccharomyces may be used, yeasts of the genus Saccharomyces, which have been used safely for the production of fermented products since ancient times, can be particularly preferably used.

These yeasts are used in an amount of 10 per ml of culture solution.
⁴ -10 was inoculated so as to ^eight, from about 10 ° C. to about 40
Cultivation is carried out at a temperature of 20 ° C, preferably 20 ° C to 30 ° C. The culture method is performed according to the culture method of general microorganisms,
Aerobic culture methods are advantageous. This is generally due to the fact that beta oxidation is an aerobic reaction, but also in a static culture method, optically active lactone is produced by dissolved oxygen dissolved at the start of culture. Aerobic culture can be performed by shaking, stirring, aeration stirring, or the like.
The culturing time can be appropriately set according to the medium composition, the culturing temperature, and the like. However, it is preferable that the culturing is performed until about 10 ⁸ yeast cells per 1 ml of the culture solution, and generally requires 24 to 72 hours. At this time, if the hydroxy acid used as a raw material is an R-form, an R-form lactone is generated,
If it is an S-form, an S-form lactone is produced. In other words, the yeast maintains the optical activity of the substrate hydroxy acid and / or its methyl ester during beta oxidation and lactone cyclization. Also,
In the case of a culturing method, the beta oxidation step can be performed simultaneously with the hydroxylation step described above. That is, it is also possible to produce optically active lactones by simultaneously inoculating a yeast having a beta-oxidizing ability and a lactic acid bacterium having a hydroxylating ability and culturing them under appropriately set conditions.

Steps such as inoculation of the strain and collection of the culture solution are as follows.
It can be performed according to a method generally performed in the past. As described above, the R-form and the S-form lactone have different flavors and threshold values, and therefore, they can be mixed and used at an appropriate ratio so as to obtain a desired flavor.
In addition, an optically active lactone can be isolated from a liquid composition produced using a method generally used for isolating a substance from a culture solution of a microorganism. Since optically active lactone is accumulated in the culture solution, extract and purify the optically active lactone after separating the cells from the culture solution by centrifugation or while the cells are suspended in the culture solution. You can also. The optically active lactone is dissolved in an organic solvent and has a relatively high boiling point, so it is purified by extraction, distillation, column chromatography, or other means using an organic solvent. Optically active lactones can also be provided for various uses.

A liquid containing ethanol may be added to the liquid composition containing the optically active lactone, or the liquid composition may be distilled after the addition. Examples of the liquid containing ethanol include a fermentation liquid brewed for producing an alcoholic beverage, an aqueous solution containing ethanol, and the like. The fermented liquid may be a brewed liquid during the production of whiskey, beer, wine, shochu, sake and the like. When these are used, a corresponding distilled liquor can be produced.

[0025]

The present invention will be described in more detail with reference to the following examples.
The present invention is not limited by these examples. The analysis of optically active lactones in the following examples was performed by HPLC using a CHIRALCEL OD-H column (manufactured by Daicel Chemical). Further, the optical purity ee (%) was obtained by the calculation formula shown in Expression 1.

[0026]

(Equation 1)

(Example 1) (Method for producing R-form 10-hydroxystearic acid) Using lactic acid bacteria, 10-hydroxystearic acid was produced from oleic acid. The strain used was Leuconostoc mesenteroides purchased from the University of Tokyo.
IAM1233, Lactobacillus pl
antarum IAM1041, Lactobacillus yamanashi purchased from RIKEN
ensis JCM3821, Leuconostoc mesenteroides purchased from Fermentation Research Institute
IFO3349, Leuconostoc message
nteroides IFO3426, Leucono
stoc meenteroides IFO383
2 of 6 strains. These strains were used in MRS medium (Difc
(purchased from Company o) 100 ml and inoculated at 30 ° C. for 18 hours. The cells were collected, suspended in 100 ml of a 0.5 M (pH 6.5) phosphate buffer containing 0.01% Tween 80, added with 667 ppm of oleic acid, and reacted at 30 ° C. for 20 hours. The reaction-terminated liquid was adjusted to pH 2 with hydrochloric acid, and 10-hydroxystearic acid generated with an ethyl acetate / methanol (9: 1) solution was extracted, and the optical purity was measured. The results are shown in Table 1. In all strains, R-form 10-hydroxystearic acid having high optical purity (1 in Table 1) was obtained.
0-HSA).

[0028]

[Table 1]

(Example 2) (Method for producing R-form 10-hydroxystearic acid methyl ester) 15 g of R-form 10-hydroxystearate obtained in Example 1
Hydroxystearic acid was dissolved in 180 ml of a methanol solution containing 14% of boron fluoride and reacted at 75 ° C. for 15 minutes. After completion of the reaction, 100 ml of saturated saline was added, and the mixture was extracted three times with 100 ml of n-hexane. The extract was dehydrated, dried, dried, dissolved again in 200 ml of n-hexane, and purified by liquid chromatography using a normal phase column to obtain about 4 g of R-form 10-hydroxystearic acid methyl ester. Was.

Example 3 (Method for Preparing Racemic 10-Hydroxystearic Acid Methyl Ester) 5 ml of R-form 10-hydroxystearic acid methyl ester prepared in Example 2
Was added to 10 ml of dichloromethane in which 1.5 g of pyridinium chlorochromate was dissolved, and reacted for 1 hour and 30 minutes at room temperature to prepare a keto acid. The obtained keto acid was manufactured by Sepi-Pak 3 manufactured by Millipore.
Purified in 5 cc. Further, 500 mg of keto acid is added to 30 m
After dissolving in 1 l of methanol, 150 mg of sodium borohydride was added and reacted at 4 ° C. for 2 hours. The reaction solution was adjusted to pH 4 with hydrochloric acid and extracted with ether, whereby about 200 mg of racemic 10-hydroxystearic acid having an optical purity of ee = 0 was produced.

(Example 4) (Preparation method of optically active 10-hydroxystearic acid methyl ester)
R-form and S-form were separated from 1 g of hydroxystearic acid methyl ester as follows. Racemic sample C
HIRALCEL OD column (250mm × 20m
m, manufactured by Daicel Chemical Industries, Ltd.) and n-hexane / isopropanol (100: 1) as an eluent at a flow rate of 6 ml / min. At this time, it was not possible to completely separate by one treatment, and the eluted fraction of R-form and S-form was treated seven times by a Closed Loop type liquid chromatograph, which was treated with the same column as it was. R-form having an optical purity of ee = 100 is 42 m
g, 130 mg of S-isomer having an optical purity of ee = -100 was obtained.

(Example 5) (Method for producing lactone from 10-hydroxystearic acid and its methyl ester by brewery yeast) By the operations of Examples 1 to 4, the R-form and the racemic 10-form were prepared.
Hydroxystearic acid, R-form, S-form and racemic 10-hydroxystearic acid methyl ester were obtained. If these are mixed in appropriate amounts, the optical purity ee = 0 to 100 and 10-hydroxystearic acid can be obtained.
With the methyl ester of hydroxystearic acid, a sample having an optical purity of ee = -100 to 100 can be easily produced. Therefore, these were mixed in appropriate amounts to prepare samples having various optical purities. These preparations were treated with brewer's yeast to produce gamma-lactone. The yeast used for brewing was Saccharomyces cerevisiae.
es cerevisiae) strain N130, the present yeast strain is an alcohol-producing yeast,
Japanese Patent No. 27886 discloses the feature. In addition, the strain has been deposited under accession number FERM P-16364. This yeast was inoculated into 50 ml of a YPD medium (1% yeast extract, 2% polypeptone, 2% glucose) and cultured with shaking at 28 ° C. overnight. This culture solution 20m
of YP medium containing 10-hydroxystearic acid and 10-hydroxystearic acid methyl ester having various optical purities at a concentration of 100 μg / ml (1%
Yeast extract, 2% polypeptone)
After culturing at 28 ° C. for 48 hours, gammad decalactone was produced. The generated gammad decalacton is n
After extraction with -hexane and concentration using a rotary evaporator, the optical purity was measured. The relationship between the optical purity of the substrate used and the optical purity of the gamma dedecalactone produced is shown in FIGS. 1 and 2, and it is clear that there is a high correlation between the optical purity of the substrate and the optical purity of the lactone. It has been shown that a lactone having a desired optical purity can be produced by preparing a hydroxy acid having a desired optical purity or a methyl ester thereof. When no yeast cells were added, almost no lactone was produced.

Example 6 Example 5 was performed using another strain.
A lactone was produced in the same manner as described above. The strain used was Saccharomyces cerevisiae.
revisiae) N130 strain, Saccharomyces IFO2114 strain purchased from the Fermentation Research Institute, and the National Collection of East Culture (Na
Tional Collection of Yeas
t Cultures) purchased from Saccharomyces
S. cerevisiae NCYC431 strain. Substrates were R-form 10-hydroxystearic acid (shown as 10-HSA in the table) with optical purity ee = 100% and its methyl ester, racemic 10-hydroxystearic acid with ee = 0% (10-HSA in the table). And its methyl ester. Table 2 shows the optical purity ee% of the produced gammad decalactone.

[0034]

[Table 2]

As shown in Table 2, even when other yeast strains were used, there was a high correlation between the optical purity of the substrate and the optical purity of the lactone.

Example 7 Commercially available ricinoleic acid (12-
Hydroxy-9-octadecenoic acid) was purchased, and a methyl ester was prepared in the same manner as in Example 2. Using ricinoleic acid and its methyl ester as substrates, gamma-decalactone was produced in the same manner as in Examples 5 and 6, and the optical purity was measured. The strain used was the N-130 strain as in Example 6. Since commercially available ricinoleic acid is in the R form, its methyl ester is also in the R form. Table 3 shows the optical purity ee% of the produced gamma-decalactone.

[0037]

[Table 3]

As shown in Table 3, it is clear that all gamma-decalactone produced when R-form ricinoleic acid and its methyl ester are used as a substrate is R-form.

[0039]

According to the present invention, optical isomers of gamma-lactones having different flavors and threshold values can be easily obtained. If a lactone is prepared by mixing these optical isomers as they are or at an appropriate R / S ratio, it is possible to provide a useful material that can be differentiated from conventional materials as a material for preparing foods and cosmetics.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the optical purity of 10-hydroxystearic acid (10-HSA) used as a substrate and the optical purity of gamma dedecalactone produced.

FIG. 2 is a graph showing the relationship between the optical purity of methyl ester of 10-hydroxystearic acid used as a substrate and the optical purity of gamma dedecalactone produced.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:85) C12R 1:85)

Claims (6)

[Claims] 1. A process for producing an optically active gamma-lactone, comprising treating an optically active hydroxy acid and / or its methyl ester with yeast. 2. The method according to claim 1, wherein the lactone is gamma-decalactone and / or
3. The method of claim 1, wherein the method is gammad decalactone. 3. The method according to claim 1, wherein the yeast is a Saccharomyces yeast. 4. The method according to claim 1, wherein the optically active gamma-lactone is in the S form. 5. The method according to claim 1, wherein the optically active R-form hydroxy acid is prepared by treating an unsaturated fatty acid as a raw material with a lactic acid bacterium. 6. The method according to claim 1, wherein the methyl ester of the optically active S-form hydroxy acid is separated from the racemic hydroxy acid methyl ester.