DE1815845C3 - Process for the biochemical separation of 1-menthol - Google Patents

Description

wherein R is an alkyl group or an alk .-: nyl group with 1 to 2! Carbon atoms means of djl-Mc'thol. optionally in a mixture with it Isomers, at about 20 to 45 C with a carboxylic ester hydrolase treated by inoculating a nutrient medium with Penicillum frequentans or spinulo-ramigenum. Gliocladium roseum. Trichoderma v> ride or koningi, Geotrichum candidum, Aspergillus flavus var. asper or japonicus. Pullularia pullulans. Fusarium roseum or graminearum, Absidia elauca var. paradoxa or hyalospora. Cunninghamella elegans. Rhizopus peka or nigricans, Actinomucor repens, Mucor hiemalis or griseocyar.us. Gibberella fujikuroi or sanbiinetii. Streptomyces griseus or Bacillus subtilis var. niger or mesentericus or pumilus has been formed. and subsequently separating the optically active 1-menthol in the usual way from Jem Reaktiunseemisch.

The invention relates to a method for the biochemical separation of 1-menthol.

Due to the stereochemical structure of menthol, there can be four stereoisomers; H. Menthol, isomentho !, neomenthol and neoisomenthol. All of these stereoisomers are optical Isomers (counter-clockwise d-form and levorotatory I-form), so that there are a total of eight optical isomers are. Of these isomers, I-menthol is one of the main components of natural or peppermint Mint oil and shows the strongest refreshing effect, so it is widely used in perfumes and medicines is used.

I-menthoi can be made by a number of methods. For example, crystals can be Isolate from I-menthol from natural mint oil by cooling. Synthetic can! -Menthol from I-menthone, which is contained in natural mint oil, can be obtained by reduction with metallic sodium. On an industrial scale, 1-menthol is turned off d-Citronellal, which is a component of citronella oil represents, obtained by cyclization and hydrogenation. However, these are natural raw materials (1-menthone and d-citronellal), which are optically active, are only available in very limited quantities, so that it is cheap, 1-menthol from easily accessible and cheap., chemical raw materials suitable for industry to manufacture.

When I-menthol is produced from optically inactive raw materials, there is an important problem in separating only the optically active 1-menthol from a mixture of the dl-menthol isomers, since the simultaneous formation of all dl-isomers (dl-menthol, dl-isomenthol, dl-neo-menthol and dl-neoisomenthol) is inevitable. V, ele attempts have already been made to separate i-menthol. _{; r} -, taken. However, so far not effective _processes suitable for industry have been found, and for this reason dl-menthol is currently used in Meier, grooves without complete iWuktre.nheu ieiu "i of the hydrogenation ν on Thvmol and a J,. ^ ^ poor GIc . * -

., ^ ight setting oe> obtained mixture u, r Al isomers to form a mixture of the following composition dl-Men.h "'ei" a' 0%. dl-isomenthol era V dl-Neomen.lu-i et * j 10% and dl-Neo.som, -., - thol'spurenmer ^ e. vveiteihin the behavior varies

this isomerΛ _{! 1:} «citci: scope depending on the reac: ,,!. e.Hangungen. so that the combination of P = luks-Juücrst is variable.

Next «\ enV-κη / ur production of dl-menthol consists in making a mixture of

dl-menthol isomers. animal components mainly dl-menthoi, about 70%). dl-nomenthol u: = d are dl-neomenthol, through cyclization and hydrogenation by dl-Citrondlal. The dl-menthol is made from this severed.

The previously known methods for optical separation of the dl-menthol obtained thereby order over in the optical isolation of this connection their phthalic acid half esters. 1-menthoxyacetic acid eggs and dl-camphoric acid ester. These procedures are very

awkward and expensive and too complicated to be used on an industrial scale, and they are therefore only used as a laboratory procedure.

It has now been found that optically active

i-menthol can be isolated by a process that involves the biochemical separation of certain Carboxylic acid esters of dl-menthol, optionally mixed with its isomers (dl-menthol. Dl-isomenthol. dl-neomenthol and dl-neoisomenthol)

stands. . .

The object of the invention is thus a method for the biochemical separation of I-menthol.

The process is characterized in that one uses a formic acid ester or the ester of a carboxylic acid the general formula

RCOOH

wori.i R is an alkyl group or an aikenyl group

with 1 to 21 carbon atoms means from dl-menthol. possibly in a mixture with its isomers, at about 20 to 45 C with a carboxylic acid ester hydrolase treated by inoculating a nutrient medium with Penicillum frequentans or spinuloramigenum, Gliocladium roseum, Trichoderma viride or koningi, Geotrichun candidum, Aspergillus fiavus var. asper or japonicus, Pullularia pullulans, Fusarium roseum or graminearum, Absidia gläuca var. paradoxa or hyalospora, Cunninghamella

elegans, Rhizopus peka or nigricans, Actinomucor repens, Mucor hiemalis or griseocyanus, Gibberella fujikuroi or sanbiinetii, Streptomyces griseus or Bacillus subtilis var. niger or mesentericus or pumilus has been formed, and then the

separating optically active 1-menthol from the reaction mixture in a customary manner.

In the course of extensive investigations into the asymmetric hydrolysis of esters of the

tli-menthol isomers with organic carboxylic acids, for example dI-meth \! ester, dl-neoisomenthyiester and dl-isone ethyl ester by means of the carboxylic acid ester hydrolase. those of some types of microorganisms, v. ic bacteria and yeasts or fungi. it has now been found that the dl-menthyl ester lind dl-isoment! i>! ester to! -menthol or 1-isomenthol l: \ droiysed, but the d-forms are not attacked become and the dl-Neomenthvlester and the C -neoisometh ester in no case by the carbon !. acid ester hydrolase are attacked. The present \ experienced isi compared to dt.ii previous \ erj, mren feasible on an industrial scale.

The selection and sifting of the microorganisms. nc for the production of the carboxylic acid ester hydrolase i. \ mchhar, was determined in practice by determining the activity for the asymmetric hydrolysis <ies di-Monthviesters (hydrolysis \ obtained by angle of rotation This enzyme of carboxylic acid ester hydase. are shown in the table. The investigation of the asymmetric hydrolysis for dl-menth lucetate was carried out as follows: The selected microorganisms were stirred for 2 days at a temperature of 27 ° C. in 100 g of a glucose, phosphate, ie Dipotassium hydrogen phosphate and a medium containing peptone (17 g glucose, dipotassium hydrogen phosphate, pepion, ig yeast> traki. 15 g glucose, Ig agar and

ίο 1000 g water) for yeast and mushrooms, and in one Standard medium (10 g peptone, 4 g meat extract. 1 g glucose and 2 g agar! Cultured in bacteria. Then, dl-menthyl acetate became the culture medium in the concentrations given in the table

'5 added, stirring (Schütteini at a Temperature of 27 C for 2 days was continued. The Medium was subjected to steam distillation and the distillate was analyzed by gas chromatography and the hydrolysis ratio (1-menthol; ■: 100 dl-menthyl acetate -! -menthol) from the tip surfaces the gas chromatography calculated.

table

organism

concentration hydrolysis an dl-Mtnthyl- ratio ^ acetate (Weight percent) 27.4 -> 14.8 j 12.0 1 60.2 7th 57.2 1 80.6 2 68.8 ] 55.2 1 24.0 1 46.2 2 19.0 2 18.8 1 98.4 1 98.6 T 76.4 2 69.2 1 40.0 I. 26.8 2 25.8 1 15.5 1 42.5 1 15. ^ 1 10.0 1 97.5 5 20.4 1 31.3 2

Peniciilum frequontans (AHU 8328)

Penicillum sp ... ulo-ntmigenum (AHLJ 8071)

Gliocladium roseum (A HU 9C'j9)

Trichoderma virido (AH ¹ J 9484)

Trichoderma koningi (AHU 91- ''.)

Trichoderma viride (AHU 9485)

Geotrichum Candidum (AHU 9108)

Aspergillus flavus var. Asper (AHU 7051) ..

Aspergilius japomcus (AHU 7086).

Pullularia pullulans (AHU 9187)

Fusarium roseum (AHU 9056)

Fusarium gramineariim (AHU 9054)

Absidia glauca var. Paradoxa (AHU 9004).

Absidia hyalospora (AHU 9005)

Cunninghameüa elegans (AHU 9445)

Rhizopus peka (AHU 6543)

Rhizopus nigricans (AHU 6529)

Actinomucor repens (AHU 6026)

Mucor hiemalis (AHU 6005)

Mucor griseocyanus (AHU 6044)

Gibberella fujikuroi (AHU 9078)

Gibbereila sanbiinetii (AHU 6264)

Streptomyces griseus (IAM 0084)

Bacillus subtilis var. Niger (IFO 3108)

Bacillus mesentericus (AHU 1033)

Bacillus pumilus (AHU 1386)

AHU = Faculty of Agriculture at Hokkaido University, Sapporo, Japan IAM = Institute for Applied Microbiology, University of Tokyo, Japan IFO = Institute for Fermentation, Osaka, Japan

The classification of fungi was made according to Ainsworth & Bisby's Dictionary of the Fungi, and that of bacteria according to Bergey's Manual of Determinative Bacteriology, 7th edition.

Formic acid and organic carboxylic acids of the formula RCOOH, in which R is an alkyl or alkenyl group having 1 to 21 carbon atoms, are used as the organic carboxylic acids for the production of the most dl-menthol. 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. Formic acid, acetic acid, propionic acid, and myristic acid are the best of these carboxylic acids. Use according to the invention suitable, and in particular

Acetic acid is preferred because it is the lowest Costs and a high level of risk HyJrolysis ratio results. '

In the [(imitation of the foundation, the hydrolysis of the l-menthol and l-isomenthol solvents can not only be carried out Medium in contact with the growing micro-organisms or the intact cells thereof, but also by mixing with one Culture medium free of living cells or with / cell-free extracts of those described above Microorganisms.

In the Durchführune this procedure, the applied Reaktionstemperalur between 20 and 45 "C. is preferably from 25 to 30 C, _ir d so that the inactivated-Mennig the enzymes ν held by heat denaturation at the minimum. The reaction time amounts to about 24 to 48 hours at 25 to 30 ° C., but becomes shorter at higher temperatures. The amount of the enzyme used in raw form (see Example 4) is about 0.1 to 0.5 °, '", based on the reaction mixture.

The amount of organic Carb "iisäur <: esier of Gemis' ^ s of dl-Mentholisomeren is preferably in the range of about 1 to 10 °, ₀ the reaction mixture at higher concentrations, the llydrolysegeschwindigkeit may decrease However, even at higher concentrations the reaction rate.. can be increased by using a larger amount of the enzymes.

In general, a proportion of 1 to 10 ⁿ / _{0 of} the ester to 0.1 to 0.5% of the crude enzyme solution or the corresponding culture liquid can be used as the economical amount of the ester.

Of the above dl-menthol isomers, dl-neoisomenthol can be converted into other menthol isomers by thermal isomerization with a hydrogenation catalyst can be converted, so that even a mixture which is free from dl-neoisomenthol, easily can be used as a starting material for ester production.

The separation of the I-menthol from the reaction mixture can easily be done in the usual way by fractional distillation.

Can continue to l-menthol (melting point 42 to 43 ° C. Boiling point 216.5 C ^Ü) and 1-isomenthol (melting point 82.5 ° C, boiling point 218.6 ° C) slightly (by Rektifizierimg, derivatization cyanoacetic acid ester, or Monochloressigsäureester Monophthalsäureester ), Recrystallization or chromatography. The following examples serve to illustrate preferred embodiments of the present invention.

β e 1 s ρ 1 e 1 J

The asymmetric hydrolysis of dl-menthyl acetate by Absidia hyalospora was carried out in the following manner accomplished:

20 kg of the glucose-peptone-dipotassium hydrogen phosphate medium described above were introduced into a 30-1-fermenter after heat sterilization (120 ⁰ C, 15 minutes). 500 g of a Absidia hyalospora starting culture (cultivated in the same medium at 27 ° C within 2 days with shaking) were added to the fermentation vessel as an inoculation medium and the mixture was 24 hours at 27 ° C, an air supply of 5 l / min, and a J * üJ ^ir 8speed of 200 rev / min. cultivated. ² OOg dl-menthylacelate were added to the culture fluid

added and this mixture at .7 C under cow
held at 300 rpm for 24 hours
was the Fermcnticrlösung with steam each.
lierl and the HeMiHa! tram with toluene, etc.
finally concentrated, being ISUg des kon / enin
th oil were obtained.

From the results of the gaschromatischenι muI
the content of I-menthol was 40., "botm- (inert solid carrier: kieselguhr, stationary_Hu ^ iu ^
Silicone grease 20 ° ' _n , column dimensions: _ m 0.3,
T-temperature 155 C. Carrier gas: helium, pressure
Entry: 1 kg / cm'-).

By fractional distillation ues üls v.iir
75 g I-menthol, boiling point 98'QK) mm Hg:;

-50 (c 3 in ethanol) and 95 g d-Menthv! Av
Boiling point KW C; 10 mm Hg: [λ] J, · - -SO C (<:
in ethanol).

Example ^?

The asymmetric hydrolysis \ on dl-Menthoh> omeren by Trichoderma mde was conducted in the following manner:

20 kg of the above-described glucose-di-lium hydrogenphosphate-peplon medium "were poured into a 30 l flask fermentation vessel C and an air supply of 5 l / min, and cultivated with stirring at 300 rev / min., Whereupon 200 g of the acetates of the dl-menthol isomers containing dl-menthol (dl-neomenthol 5.8%, dl-menthol 63 , 8 ⁰ Z ₀ , dl-isomenthol 30.4 ° / ₀ ), were added and the solution was kept at 28 ° C. with stirring at 300 rpm for 24 hours extracted toluene to give 173 g of the concentrated oil. Based on the results of the gasckronn'ographischen analysis under the same conditions as in example 1, the content of dl-Neomentholacetat to 5 ° / ₀ to d-menthyl acetate and d-lsomenthylacetat to 48.5% of I-menthol to 28.8 ° / ₀ and I-Iso menthol to 17.4 ° / _o determined.

100 g of the oil obtained were subjected to chromalography using aluminum oxide (1 kg) fed. After development with η-hexane, elution was carried out with 2 liters of η-hexane, Wherein 50 g of a mixture of dl-neomenthol acetate, d-menthyl acetate and d-isomenthyl acetate were obtained became. Then a further elution was carried out with 2 l of ethyl acetate and 45 g of a mixture obtained from I-menthol (62.3%) and I-isomenthol (37.7%).

This mixture finally obtained was esterified with monochloroacetic acid and recrystallized from methanol and the pure 1-menthylmonochloroacetic acid ester [a] V = -77.5 ° (in ethanol), melting point 38 ° C., was separated off.

By hydrolysis of this ester with an aqueous solution of caustic soda, about 23 g of I-menthol M? = -50 ° (in ethanol).

. .
Example 3

The separation of the carboxylic acid ester hydrolase from the parent organisms and the asymmetric hydrolysis of dl-menthyl acetate using the carboxylic acid ester hydrolase was prepared in the following manner accomplished:

7 8

Trichoderma SP 9484 was in 1 kg of the above fermentation solution, the distillate was extra-

listed glucose dipotassium hydrogen phosphate hiert and dried with magnesium sulfate, whereby

Peoton medium 3 days with shaking were obtained 1.8 g of oil after concentration at 27 ⁰ C. on

cultivated. To 900 g of a low temperature based on the results of the gas chromatographic

removed filtrate of the culture medium was 5 Analysis of the content of the I-Menthol 40.2 ° / ₀

Ammonium sulfate determined up to a saturation of 70 ¹ V ₀ (inert solid carrier: kieselguhr, stationary

admitted. This solution was left to stand for 12 hours - liquid: silicone grease 2O% - column dimensions:

left, whereupon the crude enzymes through high 3 m 0.3 cm, temperature: 180 ⁰ C, carrier gas: helium,

speed centrifuge separation from the lo- Pressure at the inlet: 1 kg / cm ² ).

solution were separated off and freeze-dried, with io By applying the above mass to 30 g

3 g of crude enzyme was obtained. an aluminum that can be used for chromatography

The 3 g of the crude enzyme was dissolved in 800 g of water were oxide by elution with hexane-η, Ig, and 200 g of a / _o strength Polyvinylalkoholemulsion d-menthyl acetate, and dissolved by further elution with 2 °. 20 g of dl-menthol acetate were added and 0.7 g of I-menthol [*] {f = 50 ° in ethanol was obtained for benzene, and the solution was shaken at 27 ° C. for 2 days. After the 15th
Example 6 was steam distilled of the reaction mixture
Distillate extracted with ether, dried and concentrated. The asymmetric hydrolysis of dl-menthyl, 19 g of oil being obtained. On the basis of formiat was carried out in the following way:
the results of the gas chromatographic analysis 2 g of dl-menthyl formate were added to 100 g of the liquid, the content of I-menthol was determined to be 43.3 ° / y, to culture medium of Trichoderma SP 9484 (example 3) (inert solid carrier: kieselguhr, stationary Liquid added, previously by shaking at 27 ° C: silicone grease 20 ° / ₀ , column dimensions: half of 2 days in the above 3 m x 0.3 cm, temperature: 180 ⁰ C, carrier gas: helium, glucose Dipotassium hydrogen phosphate peptone medium pressure at the inlet: 1 kg / cm ² ). had been made. The mass was at 27 ° C

The oil was shaken for 2 days by fractional distillation. After steam distillation

I-menthol was obtained in a yield of 7.3 g. [«]" Of the fermentation solution became the distillate with ether

= - 50 * " (c = 4 in ethanol, boiling point 98 ° C / ev. Extracted and the oil concentrate in one yield

10 mm Hg). obtained from 1.9 g. Based on the results of the

R e i s D i e I 4 gas chromatographic analysis of the concentrated

^H 30 oil was the content of a .. I-menthol 17.45 ° / _0th To

The asymmetric hydrolysis of dl-menthyl acetate further treatment by chromatography

by Bacillus subtilis var. niger was on the following aluminum oxide, as in Example 5, were 300 mg

Way done: I-menthol received, [α]? = -45 °.

Bacü'us subtilis var. Niger was in 1 kg of the general. .

my standard medium at 27 ° C for 2 days under 35 Example 7

Shaking cultivated. 30 g of dl-menthyl acetate were carried out as in Example 6, but

added and the solution ^{shaken at 27 °} C. for 2 days. 2 g dl-menthyl propionate instead of 2 g dl-menthyl

After steam distillation of the fermentation solution, formate according to Example 6 was applied. D. j yield at

the distillate was extracted with ether and concentrated oil from dl-menthyl propionate was

narrowed. The yield was 27 g. Because of the 40 1.8 g and the I-menthol content 49.98%. From the

Results of the gas chromatographic analysis dl-menthyl propionate were determined by chromatographic

the content of l-menthol to 50.3 ° / ₀ was determined. see separation 800 mg I-menthol [*] 5? = -49 °

The concentrated oil was obtained on 350 g of chromium ^ to-.

graphical aluminum oxide applied and thereby B e i s ο i e 1 8

by eluting with n-hexane 14 g of d-menthyl acetate ₄ S ^v

[«]« = -82 ° (c = 3 in ethanol) and by further The asymmetric hydrolysis of dl-menthyl-

Elution with benzene, 12 g I-menthol [x] " = 49 ° myristate by Trichoderma viride was on the following

(c = 5 in ethanol). Way carried out:

Trichoderma viride was supine in 1 kg dci

B is ρ 1 c 1 5 ₅ "listed glucose dipotassium hydrogen phosphate

Asymmetric hydrolysis of dl-menthyl acetate peptone medium at 27 ° C for 2 days with shaking

cultivated by the liquid culture medium of Geötrichuöi. 20 g of dl-menthyl myristate were added

candidium was carried out in the following way: and the solution was shaken at 27 ° C. for a further 3 days

Geotrichum cand'dum was grown in a wheat bran after steam distillation of the fermentation

medium (10 g wheat bran and 90 g water) at 27 ° C 55 solution was the distilled I-menthol with Äthei

Cultured with shaking for 3 days. 2 g dl-menthyl acetate extracted, dried with magnesium sulfate and a ·

were added and the solution was concentrated at 27 ° C. for 2 days, 1.7 g of I-menthol [#] * = -30 "(c = ί

shaken. After the steam distillation of were / receive in 95 ° _cent, ethanol).

Claims (1)

Claim: Process for biochemical separation i-menthol. characterized, that one has a formic acid ester or the ester of a carboxylic acid of the general formula RCOOH