DE2036875C3 - Process for the biochemical isolation of 1 menthol - Google Patents

Description

separates or can on the other hand directly in the form of the cell body or culture media, the carboxylic acid ester hydrolase contained, are used, for which reference is made to the older German Offenlegungsschrift 1815 845 will.

Another method for the biochemical isolation of 1-menthol from dl-menthol or a mixture of dl-isomers containing dl-menthol has been proposed. The process consists in that an organic carboxylic acid ester of dl-menthol or a mixture of this ester with organic carboxylic acid esters of dl-menthol isomers which contain dl-menthol, the organic carboxylic acid used for the esterification of the same as mentioned above, with an enzyme, specifically the carboxylic acid ester hydrolase, which is treated by the action of microorganisms of the classes Candida, Saccharomyces, Hanseula, Rhodotorula. Toi. ^ Psis, Schizosacchoromyces, Pichia, Sporobolo-IJ ₁ Y ₁ ^ Debaryomyces, Nadosoma, I richosporon, Brett..nomyces and Cryptococcus. These enzymes are separated from their cell bodies or their culture medium or can be used in other parts, directly in the form of cell bodies or culture media which reveal the enzyme, namely the carboxylic acid ester hydrolase, for which reference is made to the older dctit. ^ Hc Offenlegungsschrift 1 958 5 * 7 will. In contrast, the process according to the invention / for the biochemical isolation of 1-menthol consists in the fact that a formic acid ester or ester of ul-monthol, optionally in a mixture with other dl-menthol isomers, and a carboxylic acid of the general formula RCOOH, in which R is alkyl groups or alkenyl groups each having 1 to 21 carbon atoms means treated at a temperature of about 25 to about 45 ^r C with a carboxylic acid ester hydrolase obtained by inoculating a nutrient medium with microorganisms of the strains Sarcina aurantiaca, Nocardia gardinen, Micrococcus varians and lysodeikticus, Alcaligenes faecalis and viscolactis, Pseudomonas crudiviae, fluorescens, fragi and spec. No. 001-003,

Brevibacterium helvorum tnd spec. No. 004, Aerobacter cloacae and aerogenes, Agrobacterium tumefaciens, Achromobacter spec. No. 005, Lactobacillus casei and helveticus, Streptococcus lactis and iaecalis, 5 Clostridium caproicum and acetobutyricum. Flavobacterium esteroaromaticum and flavescens, Mycobacterium phlei, Arthrobacter simplex and ureafaciens, and the optically active 1-menthol separates from the enzyme reaction mixture in the usual way »o.

Other species of the strains mentioned are also effective in the same way.

The inventive method is achieved that on an industrial scale from a Menthoigemiseh the 1-menthol of interest can be produced in very satisfactory yields. On an industrial scale, yields of this order of magnitude are not possible due to stereospecific separation of isomers to achieve.

The attempt at the selective hydrolysis of the 1-menthyl ester by the above microorganisms which are used in the context of the invention carried out in the following way:

10 ml of a medium composed of 10 g of peptone, 4 g of meat extract, 4 g of yeast extract and 1 g of glucose in 1 l of water were placed in a test tube with an internal diameter of 18 mm and a length of 180 mm. This medium was inoculated with the test organism after sterilization, and the test tube is shaken at 30 ° C. for 48 hours. Dl-menthyl acetate 55.2 ° / _0, dl-Neomenthylacelat 34.3 ° / "dl-Isomeiithylacetal 15.5 ° / _o: Then, an emulsified Menthylacetatgemisch consisting of 80 ° / ₀ of a Menthylacetatgemisches with the composition of the following isomers was and a trace of dl-neoisomenthylacetate was added to the medium in the amount shown in Table 1 and the total amount shaken for 48 hours. The culture medium was extracted with ethyl ether, which was removed from the extract. The residue obtained was analyzed by gas chromatography. The hydrolysis ratio

1-menthol (f-1-isomenthol)
1-menthol (-f 1-isomenthol) + undecomposed acetic acid ester

was calculated from the peak areas of the gas chromatogram. The results are shown in Table I. The names of the microorganisms, with the exception of those of the Japanese Federaiion of Culture Collections of Microorganisms, based on Bergeys Manual od Determinative Bacteriology, 7th edition, 1957.

table

Microorganism Amount added
of the mixture of

Acetic acid ester
of the dl-menthol isomers

Hydrolysis ratio

Sarcina auranliaca IAM 1059

Nocardia curtains IAM 0105

Micrc'coccus lysodeiklicus IAM 1056

Micrococcus varians IAM 1314

Alcaligenes faecalis IAM 1061

Alcaligenes viscolactis IAM 1517. . Pseudomonas crudiviae IAM 1048.. Pseudomonas fluorescens IAM 1057 4 4 4 4 4 4 4 4

6.8
3.9

1.2

■ & 2. ·
8.9

11.5
10.2

12.5

Amount of formed

Isomeiitho!

0.0

0.0

0.0

, 0.0

1.3 1.3 0.3 1.8

continuation

Microorganism Granted amount
of the mixture of
Acetic acid ester
the dl menthol
isomers
7th hydrolysis
ratio
% Amount of
educated
Isomuuho!
V. Pseudomonas fragi IAM 1650
Pseudomonas sp. No. 001
Pseudomonas sp. No. 002
Pseudomonas sp. No. 003
Brevibacterium helvomm IAM 1637
Brevibacterium sp. No. 004
Aerobacter aerogenes IAM 1338
Aerobacter cloacae IAM 1134
Aerobacter aerogenes IAM 1539
Agrobacterium tumefaciens IAM 1625
Achromobacter sp. No. 005
Lactobacillus casei AHU 1055
Lactobacillus casei IFO 3425
Lactobacillus helveticus IFO 3219
Streptococcus lactis AHU 1089
Streptococcus fascalis AHU 1086
Clostridium acetobutyricum IFU 3346
Clostridium caproicum IAM 19228
Flavobacterium esteroaromaticum IFO 3751
Flavobacterium flavescens IFO 3085
Mycobacterium phlei IFO 3158
Arthrobacter simplex IAM 1660
Arthrobacter ureufaciens IAM 1658 4th
8th
8th
8th
4th
8th
2
2
2
2
4th
2
2
2
2
2
4th
4th
4th
4th
2
4th
2 7.8
21.8
26.9
24.6
6.3
19.4
0.9
1.5
4.8
7.3
13.1
2.8
14.5
11.2
1.6
4.7
7.9
19.4
9.9
6.4
3.6
7.1
7.8 2.1
9.7
5.9
7.2
0.0
5.2
0.0
0.0
1.8
0.5
2.7
0.0
4.3
2.8
0.0
0.0
1.2
2.8
1.2
0.0
0.0
0.5
0.5

IAM: Tokyo University, applied Microorganism Laboratory IFO: Incorporated Foundation Fermentation Laboratory AHU: Hokkaido University, Agricultural Department, applied Mycology Roem

The carboxylic acid esters of dl-menthol used derive from formic acid and organic carboxylic acids of the general formula RCOOH, where R represents an alkyl or alkenyl group having 1 to 21 carbon atoms. Examples of such acids are Acetic acid, propionic acid, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, Palmitic acid, stearic acid, acrylic acid, oleic acid and erucic acid. In general, however lower organic acids such as formic acid, acetic acid, propionic acid and butyric acid are preferred, since the hydrolysis activity of the organic carboxylic acid has a tendency to decrease in terms of both Speed as well as selectivity shows as the length of the carbon chain increases. acetic acid is the most advantageous acid for use as the esterification component and is therefore particularly preferred.

According to the invention, the hydrolysis of the esters of 1-menthol is not only achieved by shaking in contact carried out with the growing organisms or the intact cells of the same, but also through Mix the esters with a culture medium free of living cells and with the cell-free extract of the microorganisms listed above. For example, the microorganisms listed above in a liquid culture medium for the accumulation of the esterases in the cell bodies or be incubated in the medium, and then an organic carboxylic acid ester of dl-menthol or a mixture of organic carboxylic acid esters of dl-methyl isomer which contain dl-methyl.

can be added to the liquid culture medium. On the other hand, after the separation of the cell body the cell bodies from the liquid culture medium

be suspended in a suitably buffered liquid and this suspension to those listed above Esters are added. Furthermore, all materials, such as extracts, which the carboxylic acid ester hydrolase contain concentrated liquid mass

sen from it, crude and purified carboxylic acid ester hydrolase, those from the cell bodies of the microorganisms listed above or their culture medium has been isolated and purified by conventional methods. It can also be raw or cleaned Carboxylic acid ester hydrolases obtained by combination

with any high molecular weight compounds

insolubilized can be used.

When performing this procedure, the

Reaction temperature 25 to 45 ° C, preferably 30 to

40 ⁰ C, for economical operation. The reaction time is about 5 to 96 hours at 30 to 40 ° C. However, this time can be shortened or lengthened by adjusting the concentration of the organic carboxylic acid esters of dl-menthol. The amount of these esters or a mixture of isomeric esters which contain dl-menthol is preferably in the range from about 2 to 20 percent by weight of the culture medium from the microorganisms listed above. If cell bodies or the carboxylic acid ester hydrolase itself are used, the amount of the ester to be used can be determined from the calculated amount of the microorganisms in the culture medium. The organic carboxylic acid esters of

dl-Menlhol or a mixture of the organic carboxylic acid esters of dl-menthol isomers, the dl-menthol can be added as such or in emulsified form with polyvinyl alcohol or other emulsifiers will. The hydrolysis can be carried out fairly quickly if the starting materials are emulsified Form.

In the method according to the invention using the microorganisms listed above formed carboxylic acid ester hydrolases on a mixture of organic carboxylic acid esters of dl-menthol isomers, which contain organic carboxylic acid esters of dl-menthol, is subject to many Only cases of the organic carboxylic acid ester of i-menthol of the hydrolysis, so that only 1-menthol is formed. In other cases, however, 1-isomenthol is formed in addition to 1-menthol. The other's esteem Menthol isomers are not subject to hydrolysis.

When the degree of hydrolysis is 80 to 100%, the reaction is terminated and the reaction product, the 1-menthol and occasionally besides it Contains 1-isomenthol, isolated from the reaction liquid. If this isolation is due to the application of an emulsifier becomes difficult, the reaction product with an organic solvent becomes extracted or polished by steam distillation. 1-menthol and 1-isomenthol can not be used by those converted organic carboxylic acid esters by utilizing the different physical and chemical properties of these materials, for example by absorption chromatography under Application of aluminum oxide, separated by solvent extraction or fractional distillation will. If the 1-menthol obtained also contains 1-isomenthol, both mentholes can be used easily by rectification via the cyanoacetic acid esters, monochloroacetic acid esters and phthalic acid monoesters, Separate recrystallization and chromatography.

The microorganisms to be used in the context of the invention form the desired carboxylic acid esterhydroiase easy because they have a high growth rate own and grow in cheap culture media. The carboxylic acid ester hydrolase used has advantageous properties high activity and stability.

The following examples serve to illustrate preferred embodiments of the invention, wherein all percentages are by weight. The following abbreviations are used in the examples applied:

IAM: Institute of Applied Microbiology,
University of Tokyo. Tokyo, Japan
HO: Institute for Fermentation, Osaka, Japan
AHl): Faculty of Agriculture, Hokkaido University, Sapporo. Japan

example 1

In a piston 100 of a culture medium consisting of 0.5 ° / ₀ glucose, 0.5 ° / ₀ potassium hydrogen phosphate were ml. Introduced 0.7% of peptone and 0.5 ° / ₀ yeast extract, sterilized, and seeded with Sarcina aurantiaca IAM 1059th After incubation with shaking (vibration amplitude 6 cm, rotation

15 rev / min. at 30 C within 24 hours an emulsion of 2 g of a mixture of Anieiscnsäurccstern of dl-methyl isomers (55.2% dl-methyl formate, 15.3% dl-isomcnlhyirormial.

29.3% dl-neomenthyl formate and a trace of dl-neoisomenthyl formate) in 8 ml of a 2% aqueous Polyvinyl alcohol solution was added and the mixture was shaken for a further 96 hours. Then became the reaction mixture extracted with hexane. A yield of 1.73 g of the reaction product was obtained obtained which, according to gas chromatographic analysis, contained 14.8% 1-menthol and 0.4% 1-isomenthol. 1.5 g of the reaction product were in 30 ml

ίο η-hexane and dissolved in menthol (0.21 g) and not converted esters divided by chromatography on an aluminum oxide column. The menthol was recrystallized from nitromethane. This gave Q.13g of 1-menthol, melting point 41 C, [\]?

^ -49.7 ° (r 1.5, ethanol).

Example 2

100 ml of the medium described in Example 1 was loaded with the Micrococcus variants IAM 1314

ao inoculates and after incubation by shaking at 30 C within 24 hours with 2 g of a mixture of acetic acid esters of dl-methylene isomers (dl-menylethyl acetate 55.2%, dl-isomenthylacetate 15.3%, dl-neomenthylacetate 29.3% , and dl-neoisomethyl acetal, trace) added. After shaking at the same temperature for 72 hours, the reaction product was separated off from the culture medium by centrifugation. The yield of the reaction product was 1.81 g, and gas chromatographic analysis showed 13.4% 1-menthol and 0.09% 1-isomenthol. The reaction product was treated in the same manner as in Example 1. This gave 0.2 g of 1-menthol, [\] f 49.8

(c 5, ethanol).

Example 3

1 liter of a culture medium composed of 1.5% soybean powder, 0.1% glucose and 0.2% yeast extract was placed in a flask and sterilized. The nutrient medium was inoculated with Akaligenes viscalactis IAM 1061 and incubated for 24 hours at 37 ^[C. After the insoluble material in this culture material had been removed using a filler cloth, the cell bodies were centrifuged off and suspended in 500 ml of a 0.1 M phosphoric acid buffer solution (pH 7.5). 50 g of a mixture of the acetic acid esters of dl-menthol isomers with the same composition as in Example 2 were then added. After shaking the mixture * at 37 C for 72 hours, the reaction product was collected by centrifugation.

The yield of the reaction product was 44.3 j and the gas chromatographic analysis showed 23.4 " 1-menthol and 6.8% 1-isomethyl. The reaction «, product was then converted into 1-menthol, l-lsomcnthn (13.2 g) and unreacted esters by chromatography on an aluminum oxide column The menthol was esterified with monochloroacetic acid and recrystallized from methanol. where 11.5 Crystals of the monochloroacetic acid chain of 1-Mci thol, which was then hydrolyzed to give 8.1 g of crystals of! -menthol (\] f 49.9 (r 5th ethanol).

B c i s ρ i c I 4

In a basin fermenter, 51 were a Culture medium from 30% Klciccxtrakt (obtained dun I xlraction of 1 part bran with 5 parts hot

309641/31

Water), 0.5 "/" peptone and 0.1% glucose and sterilized. 250 ml of a preculture medium were inoculated with Pseudomonas fluorescens IAM 1057 and incubated at 37 ° C (stirring 250 rev / min., aeration 2.5 l / min.). After the 24 hour Incubation, 500 ml of the propionic acid ester of dl-menthol were added and the mixture continued at 0.5 l / min. Incubated for 96 hours. Then the stirring was stopped and the floating on top Separated reaction product. The yield of the reaction product was 418 g, of which I-menihol Was 37.6 per cent. The reaction product was converted into I-menthol and unreacted ester by chromatography divided on an aluminum oxide column. The yield of! -Menthol was 161 g, [\] f - -49.3 ° (c ■ = 5, ethanol).

Example 5

500 ml of the culture medium with the composition according to Example 1 were placed in a flask and sterilized. It was inoculated with Brevibacterium helvorum IAM 1637 and incubated by shaking at 37 ° C. for 24 hours. The cell bodies were then centrifuged off and suspended in 100 ml of a 0.1 M phosphoric acid buffer solution. The suspension was then subjected to ultrasound treatment (22 KCS, 200 watts, 30 minutes) while cooling with ice water. The liquid was centrifuged on the ultracentrifuge to remove insoluble matter and mixed with an emulsion of 5 g :; Mixture of Amciscnsäurcestern of dl-Mentholisomeren with the same composition as in Example 1 emulsified with 25 ml of an aqueous 2 ° / _o polyvinyl alcohol solution, is added. After the reaction for 48 hours at 30 ^° C., the reaction product was extracted with ether. The yield of the reaction product was 4.8 g, and the gas chromatographic analysis showed 13.4% I-menthol and 3.4% 1-isomenthol. After chromatography over an aluminum oxide column to separate I-menthol and 1-isomenthol from the unreacted esters, 0.5 g of a mixture of I-menthol and 1-isomenthol were obtained. This was converted into the monochloroacetic acid ester, which was then cooled to −20 ° C., thereby obtaining 0.41 g of the monochloroacetic acid ester of I-menthol, which was hydrolyzed to give 0.23 g of I-menthol. Melting point = - 42.7 C, [x] f = - = -50.2 ° (c = 1.5, ethanol).

IU

attempt yield 1 meal content (G) (%) 1 0.87 17.6 2 0.88 17.8 3 0.87 17.4 4th 0.90 17.4 5 0.92 16.8 6th 0.90 16.3 7th 0.92 16.3 8th 0.88 16.3 9 0.93 15.1 10 0.94 15.0

2 g of the reaction product obtained according to this example were chromatographed over a Aluminum oxide column according to Example 1

, ^hande '· 0.3 g of I-menthol were obtained, [«]?

= _ -49.9 (c = -1.5, ethanol).

Example 7

100 ml of the culture medium was placed in a flask

- <sTrT- ^Z ₍ ^usan l ^regulations introduced according to example 1,

fm £ μ υ " ^{0 with Lact} ° bacillus casei IFO 3425

Fumes After incubation by shaking at 37 ⁰ C

dor S «L ^V - ^{On unden were 2} 8 of ^a mixture«., «,, ... o _{ester uer j in example} 2 given

"eren added. Shaking at the same temperature for 72 hours then continued the reaction product with ether i- The yield of the reaction product was ^ rf-ses enihiclt according to the gas regulation -n analysis 17.4% I-menthol and

the

η η ιΤ, ^{WIC were} treated in Beis Piel3. 0 2g menthol were obtained, [. *]? - 49.7 ° (c>, ethanol). Melting point 41.9 "C.

Examples
Following the procedure of Example 7, however

fraud

, f

Example 6

50 ml of the ultrasound-treated liquid according to Example 5 were on a column with a cross-linked dextran gel (diameter 3.5 cm, length cm) for collecting the carboxylic acid ester hydrolase which was then concentrated by the semi-permeable membrane method and made insoluble by combining with 5 g of "DEAE" cellulose (diethylaminoethyl cellulose). 2 g of the insoluble product were in 50 ml of a Suspended 0.1 M phosphoric acid buffer solution and added the ester emulsion and in the same Wise implemented 24 hours. The same operation was repeated eight times, the reaction mixture being determined in each case by gas chromatography was analyzed with the following results:

^{On LacI} ° bacillus casei IFO 3425, ^deS ^ action product 1.83 g, Ä; ⁸¹ ?? ¹ »^ Analy« yielded 6.7% oroduki »,. T- ">'-'somenthol. The reaction

treat H Γ '" ^ ^glC ' ^{dlen Wcise as in the} example P ^{iel 3} bchandeH. Fs 0.09 g! -menthol were obtained, hjf

^{v (f} 1. ethanol), melting point 41.5 ° C. ^Be 'game 9

a «° K ^I Si ^{!; i C} ^ rOiciJm ' ^{AM 19228 was made in 5OOml} « do j ί e Ti T ^{S with the} rich co-

was ^{withdrawn 8} d ^ 'eh trash ¹ ' J ^{eDas GemiSCh}

Incubate for 48 hours «, ο" ³ °° ^{C in} " ^Crhalb Γ

pronsäureestem dir hi μ ^{8 Cmes} mixture of Ca-

further

spro _duct elsLh
swam De AuH »
15.7 g, and dS ίϊ ££ ^η

^to 8 ^and

^ammIun 8 ^deS ' ^{which one to the top} '

.% 1Menthr _and 4 ₉ Ä _u ^naI y ^se «« fion product η ,,? Λ ' ^{/ o Ms} ° menthol. The reaction In this Τίΐ • ^{"What falls} * rdampf distilled · I-menthol and π μ ⁰ I"^Gem> MOORISH from I-menthol

X ^hO i S ^thl I ⁹ ° / o. ^ menthol 2 ^ , as in iw <nirl 5. ae-

Thereby were and isomenthol
get, enX ^hO i

il

cleans. 2.3 g! -Menthol were obtained, [^ Jf ¹ 49.9 "(C 5, ethanol).

Example 1Ü

100 ml of cow fluid having the same composition as in Example 1 was placed in a flask and sterilized. It was inoculated with Havobaclerium esteroaromaticum IFO 375] and incubated by shaking at 37 "C within 36 hours. Then 2 g of the paimitic acid esters of dl-menthol were added and the total mass was shaken for a further 72 hours. The reaction mixture was then extracted with ether and 3 91 g of the extract were obtained. The gas chromatographic analysis showed a content of 13.2%! -Menthol of the extract. This extract was obtained as a distillate with steam and then nicely. The obtained crude! -Menthol was obtained from Niuomethane crystallized and gave 0.14 g of I-menthol, [λ Jf 49 / Γ (c I ₁ ethanol), melting point 41.2 C.

Example 11

100 ml of a culture medium with the same composition as in Example 3 were introduced into a flask, sterilized and inoculated with Mycobaeternmi phlei IFO 315b. After incubation with shaking at 37 ¹ C within \ on 30 hours, 2 g of (jemisches the Laurinsäureesier DJ M.enthohsomeren described in Example 1 was added, and the total amount Wettere 96 hours shaking. Then, the reaction mixture was extracted with ether. Ls 3.92 g of extract were obtained. The gas chromatographic analysis showed a content of the extract of 7.1% 1-Meathol and 0.0% I-somenthol. The extract was treated in the same way as in Example 10 and was 23 g 1-menthoi obtained, {*}? -49.0 ^v <r \ ethanol »," melting point --- 41.4 ¹ C

Example 12

A culture medium with the same composition as in the example! 1 was inoculated with Arthrobacter simplex IAM J660 and incubated by shaking at 37 ° C. within 4% hours. 4 g of dl-menthyl myristate were added and, after further shaking, left to stand for 96 hours. The reaction product floating above was collected. Yield 3.4 g. The reaction product was steam distilled. 0.94 g of 1-MentholkristaHe were obtained, [-x] f 50.0 '<<·' 5, ethanol).

ίο Example 33

Corresponding to the procedure of Example 7, but using Nocardia curtains JAM 0105 instead of Lactobacillus casei Jl-O 3425, the yield of the reaction product was 1, SOg, and Uk gas chromatographic analysis showed 6.5%, ■ J-Mcnlhul andÖ, -e%: i-Jsoawiithyi as. The reaction product was treated in the same way as in Example 3. 0.1 OS g of 1-M.enthol were obtained, \ -xjf 48JB ¹ - (c- 1, ethanol), melting point - 41.5 ¹ C.

Example 14

According to the method of Example 2, but using Aerobacter cloacao JAM 1134 instead of Micrococcu: variants IAM 1314, the yield of the reaction product was 1.76 g, and the gas chromatographic analysis showed 13.8%! -Menthol and 0.0% 1-isornenthol on. The reaction product was treated in the same manner as in Example 1. 0.3 g of 1-menihol were obtained, {■ *)? - 49 9 ^V (f 5, ethanol).

B e 1 j> ρ j e i 15

Following the procedure of Example 12, however using Agrobacteiium tumefaciens IAM 1525 instead of Arthrobaeier simplex IAM 1660, 3.2 g of the reaction product receive. The reaction product was desuüiert with steam. There were 0.87 c 1-Mentholk.ristalk obtained, ^ Jf = 48.8 ° (r - 5, ethanol).

Claims (3)

1 2 l-menthone can be obtained, which is contained in natural claims: mint oil, by reduction with metallic sodium. 1. Process for the biochemical isolation of On an industrial scale, 1-menthol is converted from d-Citro-1-menthol, characterized in 5 nellal produced which is part of the that one represents a formic acid ester or ester of lemon oil, namely by cyclization dl-menthol, optionally mixed with an- and hydrogenation. However, since natural raw materials, their dl-menthol isomers, and a carboxylic acid such as 1-menthol and d-cilronellal, the optical activity of the general formula RCOOH, in which R are alkyl, only accessible to a very limited extent groups or alkenyl groups each having 1 to ia is a method for producing 1-menthol 21 carbon atoms means cheap industrial chemicals at a temperature. temperature of about 25 to about 45 ° C with a carbon case 1-menthol from optically inactive raw materials Treated acid ester hydrolase, which is produced by inoculation lines, is an important problem m the a nutrient medium with microorganisms separating the optically active! -menthol from the Strains Sarcina aurantiaca, Nocardia curtains, 15 mixture of the dl-menthol isomers, since the simultaneous Micrococcus varians and lysodeikticus, Alcaligenes Formation of all jl-isomers (dl-menthol, dl-lsofaecalis and viscolactis, Pseudomonas crudiviae, menthol, dl-neomenthol and dl-neoisomenlhol) unfluorescens. fragi and spec. No. 001-003, Brevi- is avoidable. There have been numerous attempts bacterium helvorum and spec. No. 004, Aerobacter undertook to separate the 1-menthol, however cloacae and aeiogencs, Agrobacterium tumefaciens, * o so far no Ver-Achromobacter has been successful in industry spec. No. 005, Lactobacillus casei drive to be realized. Because of this, will and helveticus. Streptococcus lactis and faecalis, actually racemic dl-menthol in numerous Clostridium caproicum and acetobutyricum, flavo- cases used without the remaining isomers bacterium esteroaromaticum and flavescens, having myco- seperated. bacterium phlei, Arthrohacter simplex and urea- as The most important industrial process for the manufacture of faciens has been formed, and the optically active development of dl-Menthpl consists in the hydrogenation of 1-menthol from the enzyme reaction mixture in thymol and the equilibrium setting of the usual Way. ten mixture of the dl-menthol isomers in the 2. The method according to claim 1, characterized by the following combination of heat to a product that is used as the starting material of the acetic acid 30 setting: About 70 ° / _u dl-menthol, about 20% dl-Iv> ester of dl-menthol. menthol, about 10% dl-neomenthol and a trace of 3. The method according to claim 1 or 2, characterized in dl-neoisomenthol. The proportions of these isomers characterized in that the dl-menthol ester vary in a manner corresponding to the reaction conditions. Amount of from about 2 to about 20 percent by weight so that the composition of the product is extremely high based on the culture medium is used. 35 is variable. Another process for dl-menthol isomers is that a mixture of the dl-menthol isomers. its components mainly dl-menthol (approx 70%), dl-isomenthol and dl-neomenthol are through <o Cyclization and hydrogenation of dl-citronellal produced and the dl-menthol is separated therefrom. The subject of the invention is a method for bio- The known method for optical separation chemical isolation of 1-menthol from dl-menthol- the dl-menthol obtained thereby consist in the opcarbonsäurtester using carbonic isolation of this compound from the phthalic acid ester hydrolase, which by microorganisms acid half-esters, 1-menthyl acetate and dl-camphor earth Classes Sarcina, Nocardia, Micrococcus, Alealic acid esters. These procedures are very expensive and vice versa, Pseudomonas, Brevibacterium, Aerobacter, grasp a very complicated process sequence, see above Agrobacterium, Achromo'mcter, Lactobacillus, Strep- that they are not used on an industrial scale tocoecus. Clostridium, Flavobacterium, Mycobacte-50 can and therefore only in the laboratory and Arthrobacter has been formed. procedure can be used. The 1-menthol can be found in perfumes and medicinal products. A process for the biochemical isolation of agents can be used. 1-menthol from dl-menthol or a mixture of According to the stereochemical configuration of dl-menthol isomers containing dl-menthol, became Menthol can be present in four stereoisomers, namely already proposed. The procedure persists borrowed menthol, ismenthol, neomenthol and neoiso- in that a formic acid, alkane and alkenemol, respectively. Each of these stereoisomers has optical carboxylic acid esters having 2 to 22 carbon atoms of Isomers (dextrorotatory [d] and levorotatory II] dl-menthol or a mixture of this ester with a Forms), so that a total of alas! optical isomers formic acid, alkane or alkene carboxylic acid ester are present. Of these isomers, 1-menthol is 60 with 2 to 22 carbon atoms of the other dl-isociners the main components of natural mint oil are kidney treated with a carboxylic acid ester hydrolase and has the strongest accessibility to refreshments, so that it is produced by the action of microorganisms it is widely used in perfumes and drugs of the classes Penicillium, Gliocladium, Trichoderma, is applied. Geotrichum, Aspergillus, Pullularia, Fusarium, Absi- l-Menlhol can be obtained by a number of methods 65 dia, Cunninghamella, Rhisopus, Actinomucor, ChIahergeslellt will. For example, crystals from mydomueor, Mucor, Gibberella, Streptomyoes and I-Menlhol has been made from natural mint oil by cooling Bacillus: the enzymes are to be isolated. ! -Menthol can be synthetically removed from the cell bodies and the culture medium.