CA1310925C - Quality improvement of alcoholic liquors - Google Patents

Abstract

The Abstract of Disclosure Ethyl carbamate in alcoholic liquors can be decomposed by bringing a culture broth or processed matter thereof obtained from a microorganism which belongs to the genus Gluconobacter, Flavobacterium, Arthrobacter, Achromobacter, Alcaligenes, Pseudomonas, Klebsiella, Rhodotorula, Rhodosporidium, Trichosporon or Candida, and is capable of decomposing ethyl carbamate, into contact with said alcoholic liquors.

Description

1 09~5 Quality Improvement of Alcoholic Liquors This invention relates to a method of improving quality of alcoholic liquors.
In alcoholic liquors, all brewages such as refined sake, beer, wine, samshu or the like, and spirit such as whisky, brandy, shochu or the like, is contained ethyl carbamate which is considered as a carcinogenic substance. For people who regularly drink alcoholic liquors, content of the carcinogenic substance is desired to be as low as possible.
The reason why ethyl carbamate is produced in alcoholic liauors has not been elucidated, and no method of removing ethyl carbamate from alcoholic liquors has been known.
As mentioned in the foregoing, development of a method of removing ethyl carbamate from alcoholic liquors with a practical advantage has been desired.
For producing alcoholic liquors containing a relatively low content of ethyl carbamate, the present inventors diligently searched for m~croorganisms capable of decomposing the substance and succeeded in obtaining such microorganisms, and they found that, by treating alcoholic liquors with the enzyme to efficiently remove ethyl carbamate, and the study was further carried out to accomplish the present invention.
That is, the present invention relates to (1) a method of improving quality of alcoholic liquors, which comprises treating alcoholic liquors containing ethyl carbamate with an enzyme capable of decomposing ethyl carbamate, and (2) a method of improving quality of alcoholic liquors, which comprises bringing a culture broth or processed matter thereof obtained from a microorganism which belongs to the genus Gluconobacter, Rhodotorula, Rhodosporidium, Trichosporon, Candida, Flavobacterium, Arthrobacter, Achromobacter, Alcaliaenes, Pseudmonas or Klebsiella and is capable of decomposing ethyl carbamate, into contact with alcoholic liquors containing ethyl carbamate.

1310~25 As the enzyme of decomposing ethyl carbamate, any one can be employed so long as it decomposes ethyl carbamate to produce one or more of ammonia, carbon dioxide gas and ethanol, and there is no particular limitation with respect to the general properties of the enzyme, such as optimal pH, pH
stability, optimal temperature, ~ ~rcn~ stability, substrate specificity, etc.
In the present invention, there is no particular limitation with respect to microorganisms capable of decoposing ethyl carbamate, if only they have, in the culture broth or its processed matter, an activity of decomposing ethyl carbamate as mentioned above. Examples of strains of such microorganisms include bacteria belonging to e.g. the genus Gluconobacter, Flavobacterium, Arthrobacter, Achromobacter, Alcaliqenes, Pseudomonas or Klebsiella or yeasts belonging to e.g. Rhodotorula, Rhodoporidium, Trichosporon, Candida, and, as representable ones are mentioned Gluconobacter albidus, Flavobacterium meninaosepticum, Arthrobacter ramosus, Achromobacter xerosis, Alcaliaenes faecalis, Pseudomonas oleovorans or Klebsiella ~neumoniae; Rhodotorula alutinis, Rhodotorula minuta var.
texensis, Rhodotorula rubra, Rhodosporidium toruloides, Trichosporon cutaneum, Candida parapsilosis, Candida intermedia, Candida quilliermondii, Candida krusoides, Candida catenulata. There is no limitation with respect to these strains, and, even those newly isolated from soil, food-stuff, animals, plants, etc., can be employed in the method of this invention, so long as they are capable of decomposing ethyl carbamate. In addition, variants obtained by artificially from the strains by W irradiation or by treatment with mutagens, and other microbial cells obtained by recombination of the artificially separated gene fragments necessary for expression of the said decomposition activity can be employed the method of this invention.

.. ~ ~ 3 ~ 13~09~5 Specific examples of strains producinq enzymes capable of decomposing ethyl carbamate are mentioned below:
Gluconobacter albidus IFO 3250 Rhodotorula qlutinis IFO 1501 Rhodotorula minuta var. texensis IFO 1541 Rhodotorula rubra IFO 0910 Rhodosporidium toruloides IFO 10032 Trichosporon cutaneum IFO 1198 Trichosporon cutaneum IFO 1200 . 10 Candida parapsilosis IFO 0708 Candida parapsilosis ATCC 7336 Candida intermedia IFO 0761 Candida quilliermondii IFO 0838 Candida krusoides ATCC 7345 Candida krusoides ATCC 10755 Candida catenulata IFO 0745 Flavobacterium meninqosepticum IFO 12535 Arthrobacter ramosus IFO 12958 ~chromobacter xerosis IFO 12668 20 . Alcaliaenes faecalis IFO 13111 Pseudomonas oleovorans IFO 13583 Klebsiella pneumoniae IFO 13703 The above IFO numbers mean the accession numbers at Institute for Fermentation, Osaka (IFO) located at 17-85, . 25 Juso-honmachi 2-chome, Yodogawa-ku, Osaka, and these strains ~r~ /<no~
are *n~ones listed, on "List of Cultures, 7th edition, 1984 ' and 8th edition 1988" published by the said Institute. And, the ATCC numbers mean the accession numbers at American Type Culture Collection (ATCC) located at 12301 Parklawn Drive Rockville, Maryland 20852, and these strains are known ones listed on "ATCC Catalogue of FUNGI/YEASTS, 16th edition, 1984"-Enzymes capable of decomposing ethyl carbamate are produced from these strains continuously or intermittently by conventional standing culture, aeration-spinner culture or ......

_ 4 - ~1 Oq~5 solid culture, among others. The culture media used are those of conventional composition wherein the microorganisms used can grow. The carbon sources are appropriately selected from carbohydrates, oils and fats, fatty acids, organic acids or alcohols which can be utilized, and are used independently or in combination. The nitrogen sources include organic nitrogen sources such as peptone, soybean flour, cotton seed flour, corn steep liquor, yeast extract, meat extract, malt extract, whey, urea, etc., and inorganic nirogen sources such as ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphate, etc., which are used independently or in appropriate combination upon necessity. It is desirable that, in addition to the carbon sources and nitrogen sources, essential growth factors or growth promoters such as minerals, amino acids and vitamins are added to the media. To induce the production of the enzyme capable of decomposing ethyl carbamate, ethyl carbamate or its analogues or related compounds thereof, for example, carbamyl phosphate, biuret, etc., or an amide compound such as acetamide, butylamide, acrylamide, lactamide, or the like may, depending on cases, further be added. For th~ control of pH and foam during the cultivation, it is effective to supplement appropriately a caustic alkali solution, a sodium carbonate solution, or calcium salts, or to add an antifoaming agent.
Temperature of cultivation is selected from the ranqe suitable for growth of the microorganisms used, being usually 15C to 55C, preferably 25C to 45C. Cultivation is continued for a time sufficient for growth of the microorganisms used and for production of the enzyme capable of decomposing ethyl carbamate, usually for 5 to 120 hours.
Thus, the culture broth of microorganisms capable of decomposing ethyl carbamate referred to in the present invention can be obtained.
The processed matter of the culture broth referred to in the pFesent invention means an authentic specimen containing , , .

~~ ~ 5 ~ 1310925 an enzyme system relevant to decomposition of ethyl carbamate, e.g. viable cells, entrapped cells, mashed cells, crude or refined enzyme, insoluble enzyme, etc., prepared from the culture broth obtained by such a method as described aibove.
Examples of the said processed ma~ter include (1) viable cells collected form the culture by ~e~ ~ ~en, sedimentation, aggregation, filtration through porous membrane, poly membrane, ceramic membrane, etc., (2) dried cells of the said viable cells as they are, or those prepared by freeze-drying, spray drying, acetone drying, etc. after killing the cells by heating, chemical treatment, etc.; (3) crude or purified enzyme preparations with higher specific activity than the viable cells, by solubilizing the cells by freeze-thawing, grinding, ultrasonication, osmotic shock, cell-wall-digestion, treating with a surfactant, etc., separately or in combination, and then purifying by means of an appropriate combination of conventional techniques for purification of enzymes, such as protamine treatment, salting out, treatment with an organic solvent, isoelectric precipitation, electrophoresis, ion-exchange chromatography, gel fil-tration, affinity chromatography, crystallization, etc., and (4) entrapped cells in natural polymers such as agar, carrageenan, etc., or in synthetic polymers such as polyacrylamide, urethane resin, etc., or immobilized preparation obtained by binding to a carrier such as activated charcoal, ceramic, dextran, agarose and its related substances, porous glass, etc. When this immobilized enzyme preparation is produced, some other proteins or sugars, an SH group-protecting agent, a chelating agent, an anti-oxidant, an analogue of the substrate, etc. may optionally be added for stabilization of the enzyme.
In the present invention, the culture broth of microorganisms capable of decomposing ethyl carbamate or its processed matter obtained by such a method as mentioned above is brought in-to contact with alcoholic lliquors to decompose and remove ethyl carbamate in the alcoholic liquors.
The alcoholic liquors to be treated with the method in the present invention are those containing ethyl carbamate, including brewages wine such as refined sake, beer, wine, samushu, etc., spirit such as whisky, brandy, shochu (Japanese spirit), etc., and the intermediate products thereof.
When these alcoholic liquors are to be.treated with the enzyme capable of decomposing ethyl carbamate, it is practically advantageous that the enzyme cpable of decomposing ethyl carbamate is added at a level of 0.0001 unit to 100 unit, especially 0.01 unit to 1 unit relative to 1 ml of alcoholic liquors.
The activity of decomposing ethyl carbamate in the culture broth or its processed matter was determined colorimetrically by the nitroprusside method on ammonia produced by the reaction at 30C for 30 minutes of the mixture of a volume of a suspension in sterilized deionized water of the cells collected by centrifugation of the culture which had been diluted appropriately, with an equal volume of 0.2M
citrate buffer (pH 5.5) containing ethyl carbamate. The amount of enzyme to produce one micromole of ammonia per unit time (one minute) is expressed in 1 unit (lU).
e~ ~er~a~(r~
~ uu~ue of the treatment of alcoholic liquors is - 25- usually 10C to 80C, preferably 10C to 60C. The pH is 2 to 7, desirably 3 to 6. The treatment is continued for a time sufficient for allowing ethyl carbamate in alcoholic liquors to disappear, usually for 20 minutes to 200 days, preferably 5 hours to 120 days.
The enzyme which decomposed ethyl carbamate in alcoholic liquors treatrad by the method of this invention can be removed from the alcoholic liquors usually by means of e.g.
filtration. In this case, a filter aid or a settling agent, activated charcoal, etc. may optionally be added, then the mixture is subjected to filtration by the aid of a filter ~ ~ 7 ~ 1310925 cloth. Further, for example, precision filtration or ultrafiltration is an effective means.
According to the present invention, ethyl carbamate in alcoholic liquors can be easily decomposed, thus providing alcoholic liquors of qood quality, the content of ethyl carbamate being low, for example not higher than 100 ppb in the case of refined sake, not higher than 150 ppb in the case of whisky or not higher than 30 ppb in the case of wine. The method of this invention is, therefore, a remarkably useful one from the practical viewpoint.
srief Explanation of The Drawings Figs. l and 2 are graphs illustrating the relations between pH and the enzyme activity regards to the enzyme obtained by Example 5 hereinafter. Figs~. 3 and 4 are graphs illustratig the relations between temperature and enzyme activity regards to the said enzyme.
In the following working example, the present invention is illustrated in more concrete manner. These examples are no more than examples and do not limit the scope of the present invention at all.
Incidentally, the amount of ethyl carbamate contained in alcoholic liquors was determined by means of gas chromatography in accordance with the method of Frank L. Joe, Jr. et al. [disclosed on Journal of the Association of Official Analytical Chemists, J. AOAC, 60, 509 (1977)].

Example 1 Trichosporon cutaneum IFO 1198 grown on a commercially available YM agar medium (manufactured by Difco Co., Ltd.) was inoculated into a 200 ml-Erlenmeyex flask containing 25 ml of a sterilized seed medium (pH 5.5) consisting of glucose (0.25%), potassium monophosphate (0.5 %), ammonium sulfate (0.5%), yeast extract (0.2%), table salt (0.01%), calcium chloride (dihydrate) (0.2%), magnesium sulfate (heptahydrate) (0.05%) and n-butylamide (0.25%), which was incubated at 28OC

- 8 - 1 3 1 0 q25 for 24 hours under shaking. The resulting seed culture (25 ml) was transferred into a 200 ml-Erlenmeyer flask containing a sterilized medium of the same composition as described above, followed by incubating at 28C for 3 days under shaking. The activity of the culture broth of decomposing ethyl carbamate was determined as 0.005 U/ml enzyme potency.
This culture broth was subjected to centrifugation to collect cells. These cells were added to a commercially available refined sake (alcohol 17~, ethyl carbamate 148 ppb, pH 4.3) so that the concentration might be 0.01 U/ml or 0.05 U/ml, which was kept at 28OC to cause decomposition of ethyl carbamate in refined sake; the result is shown in Table 1.

Table 1 Change of concentration of ethyl carbamate in refined sake Decomposition Days elapsed activity (U/ml) 0 2 4 6 10 .
0.01 148 86 44 25 16 0.05 148 24 0 0 0 . .
(unit : ppb) Example 2 The strains listed in following Table 2 grown on commercially available YM agar media (manufactured by Difco Co., Ltd.) were incubated in the same manner as in Example 1.
The activities of these cultures of decomposing ethyl carbamate were determined, and the results are shown in Table 2.

- 9 - ~ 3 1 0925 Table 2 Activities o culture broth for decomposition of ethyl carbamate 5 Strain (U/ml) Trichosporon cutaneum IFO 1200 , 0.004 Rhodotorula qlutinis IFO 1501 0.001 Rhodotorula minuta var. texensis IFO 1541 0.001 Rhodotorula rubra IFO 0910 O. 002 Gluconobacter albidus IFO 3250 0.004 Rhodosporidium toruloides IFO 10032 0.001 Candida Parapsilosis IFO 0708 0.006 Candida parapsilosis ATCC 7336 0.005 Candida intermedia IFO 0761 0.003 Candida auilliermondii IFO 0838 0.002 Candida krusoides ATCC 7345 0.006 Candida krusoides ATCC 10755 0.006 Candida catenulata IFO O 745 0.006 - The culture broth of each strain in Table 2 was subjected to centrifugation to collect cells, which were added to the same commercially available refined sake as in Example 1 so that the activity might be 0.02 U/ml. The respective mixtures were kept at 28OC for 7 days while stirring occasionally. The concentrations of ethyl carbamate in refined sake were as follows.

t 3 t 0925 Table 3 Concentratlon of ethyl carbamate after treatment tppb) . _ Gluconobacter albldus IFO 3250 94 Rhodotorula rubra IF0 0910 66 Rhodotorula ~lutlnls IFO 1501 82 Rhodotorula mlnuta var. texensls IFO 1541 48 RhodosPorldlum toruloldes IFO 10032 22 TrlchosPoron cutaneum IFO 1200 15 Candlda ~araPsllosls IFO 0708 0 Candlda ~araPsllosls ATCC 7336 12 Candlda lntermedla IFO 0761 22 Candlda aullllermondll IFO 0838 38 Candlda krusoldes ATCC 7345 0 Candlda krusoldes ATCC 10755 0 Candlda catenulata IFO 0745 3 . . .
ExamPle 3 The stralns llsted ln the followlng Table 4 grown on a nutrltlon agar medlum were transferred lnto a 200 ml-Erlenmeyer flask contalnlng 25 ml of a sterlllzed seed medlum (pH 4.5) conslstlng of glucose (0.25%), potasslum monophosphate (0.03%), polypeptone (0.25%), table salt (0.5%) and formylamlde (0.1%), whlch were cultlvated at 30C for 48 hours. The resultlng seed culture (25 ml) was transferred lnto a lQ-Erlenmeyer flask contalnlng 250 ml of a sterlllzed medlum of the same composltlon as descrlbed above, followed by lncubatlng at 30C for 3 days under shaklng. The actlvlty of culture broth of decomposlng ethyl carbamate were determlned, and the-results are shown ln Table 4.

Table 4 Activities of culture broth for decomposltlon of ethyl carbamate _ _ _ _ Straln (U/ml) Flavobacterlum menlnqose~tlcum IFO 125350.002 Arthrobacter ramosus IFO 12958 0.001 Achromobacter xerQsis IFO 12668 0.001 Alcallqenes faecalls IFO 13111 0.001 :
(2) Pseudomonas oleovorans IFO 13583 grown on a nutrltlon agar medlum was lnoculated lnto a 200 ml-Erlenmeyer flask contalnlng 25 ml of a sterllized seed medium (pH 4.5) conslstlng of dlpotasslum phosphate (0.1%), magneslum sulfate (heptahydrate) (0.02%), calclum carbonate (0.1%), ferrous sulfate ~0.01%), sodlum molybdate (0.0005%), table salt (0.02%) and ethyl carbamate (0.5%), whlch was lncubated at 30C for 72 hours under shaklng.
The resultir.g seed culture (25 ml) was transferred lnto a l~-Erlenmeyer flask containing 250 ml of a sterlllzed medlum havlng the same composltlon as descrlbed above, followed by lncubatlon at 30C for 3 days under shaklng. The actlvlty of the culture broth of decomposlng ethyl carbamate was determlned as 0.005 U/ml.

(3) Klebslella Pneumonlae IFO 13703 grown on a nutrltlon agar medlum was lnoculated lnto a 200 ml-Erlenmeyer flask contalnlng 25 ml of a sterlllzed seed medlum (pH 5.5) conslstlng of ammonlum sulfate (0.2%), monopotasslum phosphate (0.6%), dlpotasslum phosphate (1.4%), magneslum sulfate (heptahydrate) (0.02%), sodium ",~,~

~ 3 t 0925 12 2~205-~511 cltrate (0.1%) and ethyl carbamate (0.5%), whlch was lncubated at 30C for 48 hours under shaklng. The resultlng seed culture (25 ml) was transferred lnto a l~-Erlenmeyer flask contalnlng 250 ml of a sterlllzed medlum havlng the same composltion as described above, followed by lncubation at 30C for 3 days under shaking.
The actlvity of the culture broth of decomposlng ethyl carbamate was determined as 0.001 U/ml.
Each culture broth obtalned by the manner descrlbed above (1), (2) and (3) was sub~ected to centrifugation to collect cells. The collected cells were fully washed with physlological sallne solutlon and then washed wlth dlstill~d water. These cells were added to a test wlne (ethyl carbamate 120 ppb), which was prepared by adding ethyl carbamate to a commerclally avallable whlte wine (alcohol 12%, pH 3.2), so that the concentration mlght be 0.05 U/ml. Thls wlne was kept at 20C for 20 days to decompose ethyl carbamate5 the results are shown ln Table 5.

- 1310q25 12a 24205~811 Table 5 The concentratlon of ethyl carbamate after treatment StralnsResldual concentration (ppb) Flavobacterlum menlnqosePtlcum IF0 12535 18 Arthrobacter ramosus IF0 12958 27 Achromobacter xerosls IF0 12668 25 Alcallqenes faecalis IF0 13111 25 Pseudomonas oleovorans IF0 13583 12 Klebslella Pneumonlae IF0 13703 21 None 120 Example 4 Candlda krusoldes ATCC 7345 was cultlvated ln the same manner as descrlbed ln Example 1 and the obtalned culture broth (1~) was sub~ected to centrlfugatlon to collect cells, followed by washlng wlth physlologlcal sallne solutlon. The - 13 - l 3 ~ 0925 collected wet cells (5 g) was suspended in 15 ml of physiological saline solution, followed by mixing with 15 ml of sterilized sodium algina~e solution (1%) and then was dropwised into calucium chloride solution (1%) to give entrapped cells. The thus obtained entrapped cells, which were filtered and washed with water, were dipped into 30 ml of a test whisky (ethyl carbamate 229 ppb) prepared by adding ethyl carbamate to a commercially available whisky (alcohol 43%, pH 4.8) at the enzyme activity of 0.04 U/ml and kept at 30C for 20 days. ~fter that, the residual amount of ethyl carbate was determined; the result was 81 ppb.

Example 5 Candida krusoides ATCC 7345 was cultivated by the same manner as described in Example l to give 2~ of a culture broth having 0.007 U/ml of the activity of decomposing ethyl carbamate. The cells were collected from the culture broth by centrifugation, washed with 0.05 M phosphate buffer (p~ 7.2) twice, suspended in a solution containing l mM of EDTA 1 mM of dithiothreitol, broken down by treating with a cell mill containing glass beads having 0.1-0.2 mm in diameter and was treated at 4500 rpm for 20 minutes. The treated product was centrifuged and to the supernatant was added ammonium sulfate so that the concentration comes to 90%. The precipitate was collected by centrifugation, dissolved in 0.05 M phosphate buffer (pH 7.0) containing 1 mM 2-mercapto ethanol, adsorbed on a Sephadex-G*200 column (4.5 cm in diameter, 150 cm in length) bufferized with the above mentioned buffer solution, eluted with the buffer solution to collect the activ~ fractions.
\~, This showed the specific activity of 0.0032 ~ protein and - - the activity yield was 43.6%.
The enzymological and chemical properties are as follows:
(1) Substrate specificity *Trademark - 14 - ~310925 This acts on ethyl carbamate, and besides on methyl carbamate and formaldehyde.

Table 6 substrate specifity SubstrateRelative activity (%) Ethyl carbamate 100 Methyl carbamate 90 Butyl carbamate 6 Urea O
Formamide 94 (2) Optimal pH and pH stability As shown in Fig. 1, the optimal pH is 7. Fig. 2 shows residual activities when kept at 37C for 15 minutes. As is clear from Fig. 2, the enzyme is stable at pH 6-8.

(3) Inhibitors As shown in Table 7, the enzymatic action is inhibited by salts of metals such as mercury, iron, copper and zinc.

Table 7 Inhibitors Inhibitors Concentration (mM) Relative activity (~) -HgCl 2 5 mM 49 FeCl3.nH20 5 mM 14 CuSO4~SH20 5 mM 54 ZnSO4-7H20 5 mM 26 None 100 (5) Km value The Km value is 65 mM (pH 4, 0.2 M citrate buffer) This crude enzyme was added to the commercially available refined sake as mentioned in Example 1 80 that the concentration might be 0.05 U/ml, which was kept at 3~C for 5 days to cause decomposition of ethyl carbamate. The concentration of ethyl carbamate was 14 ppb.

lû

Claims (18)

1. A method of improving the quality of an alcoholic liquor, which comprises treating an alcoholic liquor containing ethyl carbamate with an enzyme capable of decomposing ethyl carbamate, thereby reducing the quantity of ethyl carbamate and obtaining the alcoholic liquor having reduced ethyl carbamate content.

2. A method of improving the quality of an alcoholic liquor, which comprises bringing a preparation containing an enzyme capable of decomposing ethyl carbamate produced by a microorganism which belongs to the genus Gluconobacter, Flavobacterium, Arthrobacter, Achromobacter, Alcaligenes, Pseudomonas, Klebsiella, Rhodotorula, Rhodosporidium, Trichosporon or Candida, into contact with an alcoholic liquor containing ethyl carbamate, thereby reducing the quantity of ethyl carbamate and obtaining the alcoholic liquor having reduced ethyl carbamate content.

3. The method according to claim 2, wherein the microorganism is Gluconobacter albidus.

4. The method according to claim 2, wherein the microorganism is Flavobacterium meningosepticum.

- 16a - 24205-811

5. The method according to claim 2, wherein the microorganism is Arthrobacter ramosus.

6. The method according to claim 2, wherein the microorganism is Achromobacter xerosis.

7. The method according to claim 2, wherein the microorganism is Alcaligenes faecalis.

8. The method according to claim 2, wherein the microorganism is Pseudomonas oleovorans.

9. The method according to claim 2, wherein the microorganism is Klebsiella pneumoniae.

10. The method according to claim 2, wherein the microorganism Rhodotorula glutinis, Rhodotorula minuta var.texensis or Rhodotorula rubura.

11. The method according to claim 2, wherein the microorganism is Rhodosporidium toruloides.

12. The method according to claim 2, wherein the microorganism is Trichosporon cutaneum.

13. The method according to claim 2, wherein the microorganism is Candida parapsilosis, Candida intermedia, Candida quilliermondii, Candida krusoides or Candida catenulata.

14. The method according to claim 1 or 2, wherein the alcoholic liquor is one selected from the group consisting of refined sake, beer, wine, samush, whisky, brandy or shochu.

15. The method according to claim 1, wherein the enzyme capable of decomposing ethyl carbamate is added at a level of 0.0001 unit to 100 unit relative to 1 ml of alcoholic liquors.

16. The method according to claim 2, wherein the processed matter is one selected from the group consisting of viable cell, entrapped cell, mashed cell, crude enzyme, refined enzyme or insoluble enzyme.

17. The method according to any one of claims 2 to 13 and 16, wherein. the culture broth and the processed matter contain an enzyme capable of decomposing ethyl carbamate and are used in such an amount that the enzyme is 0.01 to 1 unit relative to 1 ml of the alcoholic liquor.

18. The method according to any one of claims 1 to 13, 15 and 16, wherein the treatment is conducted at a temperature of 10 to 60°C at a pH of 3 to 6.