JP3479342B2 - Method for producing γ-dodecalactone and liquid composition containing γ-decalactone - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to γ-dodecalactone and
And a method for producing a liquid composition containing γ-decalactone
Concerned. More specifically, when used for alcoholic beverages, foods, flavors, etc., it is said that it has a good balance of aroma.
, Especially when used for what humans ingest
-A liquid containing dodecalactone and γ-decalactone
The present invention relates to a method for producing a liquid composition containing γ-dodecalactone and γ-decalactone, which enables efficient production of the composition.

[0002]

2. Description of the Related Art γ-Dodecalactone and γ-decalactone are perfume compounds having a peach-like aroma, and have been conventionally used as a preparation component of perfume compositions. It can also be used as an intermediate for insects or other attractants or repellents, deodorants, pharmaceuticals and the like. [gamma] -dodecalactone and [ gamma] -decalactone are contained in natural products such as fruits, but since only a trace amount is contained in the natural products, it is difficult to concentrate and separate [gamma] -dodecalactone and [ gamma] -decalactone from the natural product.
γ- dodecalactone and γ- decalactone the structural formula (I) are shown below.

[0003]

[Chemical 1] (In the above formula, when n is 7, it is γ-dodecalactone, and when n is 5, it is γ-decalactone.)

Therefore, by a fermentation method utilizing microorganisms,
Attempts have been made to obtain lactones such as γ-dodecalactone and γ-decalactone. Ricinoleic acid in castor oil is decomposed into γ-hydroxydecanoic acid by the action of a microorganism having β-oxidation ability (Okui et al., J. Biochem.,
54, 1963). Here, γ-hydroxydecanoic acid easily undergoes a dehydration reaction to produce γ-decalactone. Further, in JP-A-59-82090, is hydrolyzed by the action of microorganisms in castor oil, a mixture composed mainly of ricinoleic acid, a microorganism having a β- oxidation ability to ricinoleic acid in this mixture Is further reacted to produce γ-hydroxydecanoic acid, and then this γ-hydroxydecanoic acid is lactonized to give γ-decalactone.

Furthermore, <br/> in Japanese Unexamined Patent Publication No. 3-198787 is that contains the process for producing γ- dodecalactone by the action of microorganisms having a β- oxidation ability in 10-hydroxystearic acid. However, in this document, γ- de de
Note that it is possible to obtain caralactone and γ-decalactone at the same time.
Not listed. Further, 10-hydroxy stearic acid is obtained Rukoto by artificially synthesized from oleic acid
It is only described by microorganisms such as lactic acid bacteria,
Obtaining 0-hydroxystearic acid is specifically not serial <br/> mounting.

[0006]

Definitive in Japanese of the above-mentioned The object of the invention is to be Solved
Sea urchin, 10 the steps of the γ- Dodekara <br/> transfected emissions of substrates such hydroxystearic acid chemically synthesized, the fermentation step of culturing the following microorganism, be mixed γ- dodecalactone and There is a problem that it is not preferable as a production system of γ-decalactone. In addition, γ-dodecalactone and γ-decalactone are used as flavors in the case of adding to foods such as humans or animals ingested by animals including livestock, in consideration of safety as foods, the chemical synthesis process is highly advanced. quality control of is required, trough the system is complicated
I have a problem . On the other hand, in the process of producing an alcoholic beverage, meal is produced as a by-product, but the meal was only used for animal feed and the like, and its effective use has been achieved.
The current situation is that there is no such thing .

[0007]

Therefore, the present inventor has proposed a means for effectively utilizing these lees and the like, and γ-dodecalactone.
And means for obtaining a substrate of γ- decalactone by attaching technically <br/> relevant, and completed the present invention. In the present invention, an organic substance such as meal or yeast is hydrolyzed or treated with lipase .
Oleic acid and palmitole released by
The free acid containing in acid, in Rukoto reacted with specific microorganisms, gamma - a substrate dodecalactone and γ- decalactone can be obtained easily. Or reverse this order,
A specific microorganism may be allowed to act on the organic matter, and then hydrolysis or lipase treatment may be performed to obtain a substrate for γ-dodecalactone and γ-decalactone.

That is, according to the present invention, a decomposition step of hydrolyzing or lipase-treating an organic substance containing a lipid to obtain a free fatty acid (free acid), and the obtained free acid is mixed with the carbon-carbon dicarbonate. Lactobacillus (Lac ) is the first microorganism having the ability to hydroxylate the carbons involved in heavy bonds.
a hydroxylation step of allowing a lactic acid bacterium belonging to the genus Tobacillus) to act to obtain a substrate of γ-dodecalactone and γ-decalactone, and β-
As a second microorganism having an oxidizing ability, Saccharomyces (Sa
and a β-oxidation step of obtaining a solution containing γ-dodecalactone and γ-decalactone by allowing a microorganism belonging to the genus ccharomyces) to act, and a method for producing a liquid composition containing γ-dodecalactone and γ-decalactone. The organic matter containing the lipid in the decomposition step is characterized by using an organic matter that liberates a free acid containing oleic acid and palmitoleic acid from at least a part thereof by the hydrolysis or the lipase treatment. -A method for producing a liquid composition containing dodecalactone and γ-decalactone is provided . In the present invention, it is preferable to use a yeast or an alcoholic beverage, a soy sauce, vinegar, or a solid by-product obtained when fermenting a dairy product as the organic substance containing the lipid in the decomposition step . Also, in the present invention, the decomposition step, in the hydroxylation step and said β- oxidation step it is preferable to use the same reaction vessel.

[0009]

Further, according to the present invention, silage produced by lactic acid fermentation of cereals or the like is hydrolyzed or treated with lipase to obtain a substrate for γ-dodecalactone and γ-decalactone from at least a part of the silage. The process and the obtained substrate are Saccharomyces as a second microorganism having β-oxidation ability.
s) a β-oxidation step of allowing a microorganism belonging to the genus to act to obtain a solution containing γ-dodecalactone and γ-decalactone, and a liquid containing γ-dodecalactone and γ-decalactone. A method of making the composition is provided . Also, in the present invention, in the decomposition step and the β- oxidation step, it is preferable to use the same reaction vessel.

[0011] In the present invention, the β- said obtained in the oxidation step γ- dodecalactone and γ- Dekaraku
It is preferred to add the solution containing ethanol to the solution containing tonnes and then distill .

[0012] Also, according to the present invention, in any of the above
Contains the described γ-dodecalactone and γ-decalactone
Using the liquid composition obtained by the method for producing a liquid composition
Te, .gamma.-dodecalactone and .gamma. decalactone manufacturing method characterized by and this <br/> obtaining .gamma. dodecalactone and .gamma. decalactone is provided.

[0013]

According to the present invention, "organic material containing lipids"
As a raw material, the hydroxylation step may be performed after the decomposition step, or the decomposition step may be performed after the hydroxylation step. Here, lipids are roughly classified into simple lipids, which are esters of fatty acids and alcohols, and complex lipids, in which the ester further contains phosphorus or nitrogen such as phosphoric acid. In the present invention, the organic matter-containing lipid contains an ester of oleic acid and palmitoleic acid with an alcohol. In the decomposition step, the lipid is decomposed into fatty acids, especially oleic acid and palmitoleic acid, and alcohol. In the hydroxylation step, fatty acids, especially oleic acid and palmitoleic acid, are hydroxylated. Fatty acids liberated during the hydroxylation process after the decomposition process
Hydroxylate (free acid) . On the other hand, when the decomposition step is performed after the hydroxylation step, the fatty acid portion of the lipid is hydroxylated, and then the hydroxylated fatty acid is released. Further, in the present invention , silage and the like produced by lactic acid fermentation of grains and the like is used as a raw material, and a decomposition step and a β-oxidation step are performed to obtain γ-dodecalactone and
And a liquid composition containing γ-decalactone can be obtained.

[0014] In the present invention, "organic matter containing lipid" is a raw material. This organic substance is preferably a solid by-product obtained when yeast or an alcoholic beverage, soy sauce, vinegar, or a dairy product is obtained by fermentation, and more preferably lees which is a by-product for producing an alcoholic beverage. When the raw material is such yeast or solid by-product, in the culture of microorganisms in the decomposition step and the β-oxidation step, only by adding them to water, a medium is formed, and adjustment of other nutrients may be unnecessary. Many are preferred. In addition, the raw material is
It is preferably derived from natural products, especially plants or dairy products.

Yeast refers to a group of lower eukaryotes that generally have a relatively long unicellular trophozoite generation. Many yeasts ferment, but some do not. The yeast used as a raw material is not fermented by yeast but decomposes or hydroxylates the lipid contained in the yeast, and is not particularly limited. However, yeast having no pathogenicity is preferable.
Further, although the concept overlaps with that of lees, the yeast after fermentation such as alcohol fermentation and amino acid fermentation may be reused, or the fermentation ability may already be lowered by these fermentations. For example, as yeast, the second microorganism used in the β-oxidation step or already used can be preferably used.

[0016] As a raw material, it is possible to use the lees which are solid by-products removed in the intermediate step of producing an alcoholic beverage before fermentation or after fermentation. The “alcoholic beverage” means a concept including whiskey, beer, brandy, wine, sake, shochu, spirits, liqueurs, hybrids and the like. Here, the term " spirits " means a concept including , for example, gin, rum, vodka, tequila, aquabit, arach and the like.

For example, in the production of whiskey, barley is germinated to malt, the malt is dried, the root is removed from the malt, the saccharified, and then fermented before alcoholic fermentation. In this saccharification step, malt or a mixture to which malt, corn or the like is further added, is saccharified by an enzyme contained in malt to obtain a syrup-like saccharified solution. From this saccharified solution, the solid matter-removed solid matter is further fermented, and the removed solid matter is lees. Since the roots removed from barley are also rich in nitrogen source, they can be used as raw materials as solids, like the solids removed from the saccharified solution.

In the production of beer, cereals containing malt are saccharified to obtain a saccharified solution rich in monosaccharides or disaccharides.
Solids are removed from the saccharified liquid, and the saccharified liquid is fermented with yeast. The lees are removed from the saccharified liquid before alcoholic fermentation. Further, after fermentation, solid matters mainly composed of yeast are removed from the liquid components, and the liquid components are further filtered to remove the solid substances. These solid substances are lees. These solids are dried to have a water content of, for example, about 5%, and then reused as feed or the like.

On the other hand, in the production of sake, after the moromi produced from rice is fermented with yeast, it is separated into a liquid portion and a solid portion by a filtration press or the like. This solid portion is lees.
Yeast is contained in the lees, and koji, rice, etc. may remain. In the production of sake, it is not necessary to remove the solid matter before these steps because alcohol fermentation is carried out simultaneously with saccharification with koji. In the production of fruit wine,
Since the saccharification process is unnecessary, the solid matter before alcohol fermentation such as grape skin and seeds is lees.

In the production of soy sauce, moromi mash produced from soybeans is fermented, and the moromi mash is squeezed to separate it into soy sauce and lees. This meal can be used. In addition, lees, which is a by-product when producing vinegar, particularly brewed vinegar, can be used. Brewed vinegar includes grain vinegar, fruit vinegar and the like. The lees from which mirin is produced can be used. Also,
Depending on the brewing method and the like, after fermentation and the like, squeezing or the like is performed to separate the liquid portion and the solid matter which is the lees, and then the liquid portion is further precipitated and the like to be produced. The precipitated or filtered product can also be used as a raw material. Further, milk products such as milk, yogurt, butter and the like can be used.

The “ alcoholic beverage ” means a product obtained by alcoholic fermentation of grains, fruits and the like as raw materials.
Here, the lipid-containing organic matter may be cereals, fruits, beans and the like, and it is preferable that these are meals. " Cereals " include beer, wheat as a raw material for whiskey or shochu, corn as a raw material for whiskey, rice as a raw material for sake, potatoes as a raw material for shochu, buckwheat, sugar cane and the like. Means a concept . The grains are not limited to those used as raw materials for alcoholic beverages. " Fruit " is a general term that includes grapes, apples, cherries, etc.
Means just in case . " Beans " includes, for example, soybean
Means a concept .

[0022] In cracking step of the present invention, the organic substance containing lipids, pressurized water Kaimata is by lipase treatment may be free fatty acids from lipids, also the lipid hydroxylated via hydroxylation step pressurized water Kaimata may be lipase treatment. Since yeast or these solid by-products contain lipids, oleic acid and palmitoleic acid are liberated by hydrolysis or lipase treatment in the decomposition step. Needless to say, the decomposition of lipids releases not only oleic acid and palmitoleic acid but also other fatty acids.

“ Lipase ” is also called glycerol ester hydrolase and means an enzyme that hydrolyzes glycerol ester to release fatty acid. Thus, the lipase treatment is included in the hydrolysis, the decomposition process, in order to clarify that may be lipase treatment, "pressurized water solution
Or it is described as in lipase treatment ". The “ glycerol ester ” means a lipid whose alcohol is glycerin.

The hydrolysis can be carried out in an aqueous solution in the presence of a catalyst at room temperature, under heating or under reflux. As the catalyst, for example, hydrochloric acid, nitric acid such as sulfuric acid; can be used organic bases like; organic acids; sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia and inorganic bases. However, when the reaction vessel is made of metal,
It is preferable to use a base because it reacts with an acid. Further, in consideration of safety for the human body, it is preferable to use an inorganic base. As a production system, it is preferable to adjust the pH and the like so that the reaction proceeds at room temperature.

In the present invention, since "lipid-containing organic matter" is used as a raw material, in the step of culturing the microorganism in the lipase treatment of the decomposition step, the hydroxylation step and the β-oxidation step,
In general, no further conditioning of the medium is necessary. That is, in these steps, an inorganic nitrogen source such as ammonium sulfate, ammonium nitrate, or diammonium hydrogen phosphate; N
a, K, Mg, Ca, Zn, Fe, and other sulfates, nitrates, chlorides, carbonates, phosphates, and other inorganic metal salts; or yeast nutrients such as yeast extract, polypeptone, gravy, and malt extract Good, but usually not necessary. Also,
Since all of these microbial culture steps are carried out at around pH 7, a buffer solution may be used, but culture can be performed without using a buffer solution.

[0026] hydroxylated step of the present invention, Ri I <br/> oleic acid and palmitoleic acid (II) lactate bacteria described above, be converted to a substrate for γ- dodecalactone and γ- decalactone, or lipid Hydroxylates organic matter containing. Silage is believed to be obtained when hydroxylating lipid containing organics. As mentioned above
In addition, " silage " means a product produced by lactic acid fermentation of cereals or the like under anaerobic conditions, and is used as a feed for livestock. Examples of the raw material for lactic acid fermentation include green picked corn, rice grass, clover, alfalfa, sweet potato vine, astragalus, and weeds, and green picked corn is preferably used. Further, it goes without saying that these plants are also included in the “lipid-containing organic matter”.
Furthermore, in the intermediate step of producing an alcoholic beverage or the like, silage can also be obtained by lactic acid fermentation using the lees, which are solid by-products removed before or after fermentation. For example, malt meal, which is a by-product of beer and whiskey production, can be preferably used.

As the substrate of γ-dodecalactone and γ-decalactone, as shown below, 10-hydroxystearic acid and 10-hydroxypalmitic acid (I
II), but not limited thereto. Moreover, this reaction is not always performed in one step. Genus Lactobacillus
When a lactic acid bacterium belonging to the group is cultured in a medium containing oleic acid and palmitoleic acid, the lactic acid bacterium consumes oleic acid and palmitoleic acid, and γ-dodecalactone and γ-
It produces a substrate for decalactone. This lactic acid bacterium can be cultured in, for example, a buffer solution containing oleic acid and palmitoleic acid and a surfactant.

[0028]

[Chemical 2] (In the above formula , n is 7 or 5.)

[0029] As the microorganism used in this hydroxylation step is Lactobacillus (Lactobacil
Lactic acid bacteria belonging to the genus Lus) are used . This lactic acid bacterium has the ability to hydroxylate the carbon relating to the carbon-carbon double bond, and here, "has the ability to hydroxylate the carbon relating to the carbon-carbon double bond."
"Has the ability to hydroxylate one carbon of the carbon-carbon double bond in compound (V) shown below to give compound (VI)."

[0030]

[Chemical 3]

(In the above formula , R ¹ and R ² are the same or different and each represents a hydrocarbon group.) "The first microbiota having the ability to hydroxylate a carbon atom related to a carbon-carbon double bond <" (lactic acid bacterium described above) " hydroxylates oleic acid and palmitoleic acid (II) to give 10-hydroxystearic acid and 10-hydroxypalmitic acid (II).
It is preferred to have the ability to produce I).

The above-mentioned lactic acid bacterium used in the present invention is a lactic acid bacterium belonging to the genus Lactobacillus.
Lactobacillus belonging to the genus Illus) is Lactobacillus
llusbrevis, Lactobacillusd
elbruechii, Lactobacillusp
lantarum, Lactobacillussan
francisco, Lactobacillus bu
lgaricus, or Lactobacillusc
It is preferably asei . Lactic acid bacteria of this is, for example, are commercially available from Nippon Siebel Heguna companies, etc.. Breast <br/> acid bacteria, for example, culture 1ml per 1 × 10 ⁶ cells to 1
× ^{10 10} pieces Ru is contained.

As described above, the aqueous solution after the decomposition step is directly used as the medium for the hydroxylation step and the β-oxidation step, and it is not necessary to adjust the medium. The culture solution may contain a surfactant such as food grade silicone. Thus, oleic acid 及 liquid medium is an aqueous solution
And palmitoleic acid can be easily dispersed.
However, the culture can be performed even in a medium containing no surfactant. The culturing method is performed according to the culturing method of general microorganisms, but the static culturing method is usually advantageous in a liquid medium. Although not necessary, a buffer solution such as a phosphate buffer solution may be used, and the pH is preferably 6-8.

As the culture conditions, it is generally advantageous to culture under anaerobic conditions. Culture temperature is about 20-40 ° C
Is preferable, and 28-37 degreeC is more preferable. The culturing period is appropriately set depending on the composition of the medium and the temperature conditions, and is, for example, 12 to 120 hours. This hydroxylation step can be terminated, for example, by sterilization.
Further, without sterilization, the culture medium of the β-oxidation step can be started by adding the microorganism used in the β-oxidation step to the medium of the hydroxylation step. In either method, the hydroxylation step and the β-oxidation step can be continuously performed in the same container. Alternatively, after the hydroxylation step, the γ-dodecalactone and γ-decalactone substrates may be extracted and the substrate added to fresh medium to initiate the β-oxidation step.

In the β-oxidation step of the present invention, substrates of γ-dodecalactone and γ-decalactone such as 10-hydroxystearic acid and 10-hydroxypalmitic acid are used as microorganisms having β-oxidation ability in Saccharomyces.
It is converted into γ-dodecalactone and γ-decalactone by a microorganism belonging to the genus (Saccharomyces) . In the above, as a microorganism having a β- oxidizing ability
Belongs to the genus Saccharomyces
Microorganism is 1 0-hydroxystearic acid and 10
-Hydroxypalmitic acid (III) is decomposed into γ-hydroxydodecanoic acid and γ-hydroxydecanoic acid (IV), and γ-hydroxydodecanoic acid and γ-hydroxydecanoic acid (IV) are easily lactonized to give γ- Convert to dodecalactone and γ-decalactone (I).

The β- As the microorganism having an oxidizing ability, may be mentioned <br/> Ru yeast belonging to the sub <br/> Kkaromisesu (Saccharomyces) genus. Commercial bread yeast belonging to the support Kkaromisesu genus, Saccharomycescer
eviciae, Saccharomycescars
bergensis, Saccharomycesch
evalieri etc. can be used . Also, the Chuetsu Yeast Co., Ltd., Oriental Yeast Co., Ltd., Lallemand Inc.,
Pressed yeast or dried yeast sold by Littorale or the like can be used.

For the β-oxidation step, an aerobic culture method using a liquid medium is advantageous. As a medium for the β-oxidation step, a buffer solution having a pH of 4 to 8 may be used, though it is not necessary. The culturing method is performed according to the culturing method of general microorganisms, but the aerobic culturing method using a liquid medium is usually advantageous. It is generally advantageous to culture under aerobic conditions. When the culture scale becomes large, for example, aeration and stirring is preferable. The culture temperature is preferably about 20 to 35 ° C, more preferably 25 to 32 ° C. The culturing period is appropriately set according to the composition of the medium and the temperature conditions, but for example, 2
~ 72 hours. Culture under shaking or stirring.

If necessary, a surfactant such as food grade silicone may be added to the culture solution to disperse the γ-dodecalactone and the γ-decalactone substrate in a liquid medium which is an aqueous solution. Each of the γ-dodecalactone and the γ-decalactone, for example, is contained in the culture solution after the culture in the β-oxidation step in an amount of 0.1 to 15 ppm, and
It is often contained at 10 ppm. Further, the concentrations of γ-dodecalactone and γ-decalactone can be concentrated by distillation.

In order to isolate and collect γ-dodecalactone and γ-decalactone from the culture solution, a usual method for isolating a substance from a culture of a microorganism is applied. Since the culture medium and the cells contain the target substance, the cells are separated by centrifugation or filtration, and then the active substance is extracted from the filtrate and the cells. That is, the difference in solubility and solubility in a suitable solvent,
Separation, collection, and purification are carried out by means used for the production of general compounds utilizing the difference in adsorption affinity for various adsorbents, the difference in partition between two liquid phases, and the like. γ-
Since dodecalactone and γ-decalactone are soluble in an organic solvent and have a relatively high boiling point, they can be extracted with a polar or nonpolar organic solvent and the organic solvent can be distilled off. These methods can be used alone, or can be combined and repeated in any order, if desired.

Further, in the present invention, γ-dodecalactone and
The culture beauty γ- decalactone was produced, a solution containing ethanol, may then be distilled. Distillation is performed under reduced pressure, if necessary. Examples of the solution containing ethanol include a brewing solution brewed for producing an alcoholic beverage, an aqueous solution containing ethanol, and the like. As the brewing liquid, a fermentation liquid obtained in an intermediate step of manufacturing whiskey, beer, wine, sake, shochu, etc. can be used. In the solution containing ethanol, the content of ethanol is not particularly limited, but may be, for example, 1 to 60% or 2 to 45%. However, it does not prevent the addition of ethanol itself.

The liquid composition thus distilled contains γ-
Each of dodecalactone and γ-decalactone contains, for example, 0.1 to 200 ppm, and often 1 to 100 ppm. Further, a composition containing [gamma] -dodecalactone and [ gamma] -decalactone at a higher concentration may be added to this liquid composition to adjust the concentration thereof appropriately.

[0042]

【Example】

Reference Example Beer was subjected to bottom fermentation, the fermentation liquor was removed, and the solid matter precipitated during beer fermentation was separated. The solid matter was dried to obtain dried yeast, which was a by-product of beer. This dried yeast was used as a raw material in Examples 1, 2 and 4, and the composition of this dried yeast is as follows.

Example 1 In a 500 ml Sakaguchi flask, 1 g and 1 of the above-mentioned dried yeast was added.
10 ml of a specified aqueous sodium hydroxide solution was added, and the mixture was stirred at room temperature for hydrolysis. Dilute hydrochloric acid was added to adjust the pH to 6, then water was added to bring the aqueous solution to 50 ml. This solution was kept at 120 ° C. for 20 minutes for sterilization.
Next, dry lactic acid bacteria powder (Lactoba
Cillus brevis) (Christian Hansen L-62) was inoculated with 1 × 10 ⁸ Lactobacillus brevis and kept at 30 ° C. for 12 hours to obtain γ-dodecalactone.
And γ-decalactone substrates were produced.

This aqueous solution was kept at 120 ° C. for 20 minutes for sterilization. Then, a small amount of pressed baker's yeast made by Chuetsu Yeast Co., Saccharomyces cereviciae (0.5 g) and food additive silicone was added. While shaking at 180 rpm, the culture was carried out with aeration and stirring at 30 ° C. for 24 hours. Next, the concentrations of γ-dodecalactone and γ-decalactone in this culture solution were measured. A mixed solution of ether and pentene (1: 1) was added to a part of this culture solution to extract an oil layer. Then, by addition of sodium hydroxide solution, and the like oleic acid and palmitoleic acid unreacted dissolved in the aqueous phase was extracted an oil layer. The extracted oil layer was analyzed by gas chromatography. The results are shown in Table 1 below.

Further, as the concentration of ethanol 8% by addition of further concentration of 95% ethanol aqueous solution to the culture solution (5 ml), and distilled at 40 to 60 ° C., .gamma. dodecalactone 及
An aqueous ethanol solution having a concentration of 10 to 30 ppm of γ-decalactone was obtained. The ethanol concentration of this ethanol aqueous solution was 10 to 25%. Example 1 was repeated changing only the amount of dry yeast as the raw material. Table 1 shows the contents of γ-dodecalactone and γ-decalactone in the culture solution, which are the results of the above gas chromatography analysis.

[0046]

[Table 1]

Example 2 Instead of being hydrolyzed with an aqueous sodium hydroxide solution, it was hydrolyzed with lipase. In a 500 ml Sakaguchi flask, 1 g of the above-mentioned dried yeast was added to 50 ml of a pH 7 phosphate buffer solution.
And suspended at 120 ° C. for 20 minutes for sterilization. Next, a lipase (Amano Pharmaceutical Co., Lipase AY) was added to the mixture.
1% was added and the mixture was kept at 30 ° C. for 12 hours for hydrolysis.
Then, this solution was kept at 120 ° C. for 20 minutes for sterilization.

Hereinafter, dry lactic acid bacteria powder (Lactobacillus bre
vis) hydroxylation process and Saccharomyces cere
The β-oxidation step by viciae was performed under the same experimental conditions as in Example 1 including the reaction scale. The oil layer was extracted from this culture solution and analyzed by gas chromatography in the same manner as in Example 1. Further, the culture solution was distilled in the same manner as in Example 1 to obtain an aqueous ethanol solution having a concentration of γ-dodecalactone and γ-decalactone of 10 to 30 ppm.
The ethanol concentration of this ethanol aqueous solution is 10 to 25
%Met. Table 2 shows the contents of γ-dodecalactone and γ-decalactone in the culture solution, which are the results of the above gas chromatography analysis.

[0049]

[Table 2]

The real施例3 Chuetsu yeast manufactured by the press like baker's yeast, Saccharomyces cere
viciae 100g and 1N sodium hydroxide aqueous solution 1
1 liter was added, and the mixture was stirred at room temperature for hydrolysis. Dilute hydrochloric acid was added to adjust the pH to 6, and then water was added to make the aqueous solution 5 liters. This solution was kept at 120 ° C. for 20 minutes for sterilization. Then, dry lactic acid bacteria powder (L
actobacillus brevis) (Christian Hansen L-6
2) Add 10 g, hold at 30 ° C. for 12 hours, and
Substrates for dodecalactone and γ-decalactone were produced.

This aqueous solution was kept at 120 ° C. for 20 minutes for sterilization. Put this culture solution in a sterilized jar fermenter with a capacity of 10 liters, and press-type baker's yeast Saccharomyces cereviciae (50 g) manufactured by Chuetsu Yeast Co., Ltd.
And a small amount of food additive silicone was added. 180r
While shaking at pm, the cells were cultured with aeration and stirring at 30 ° C. for 48 hours. The oil layer was extracted by the above method and analyzed by gas chromatography. The results are shown in Table 2 below.

Further, a 95% aqueous ethanol solution was added to the culture solution to adjust the alcohol concentration before distillation to 8% by weight, and the mixture was distilled at 40 to 60 ° C. to obtain γ-dodecalactone and γ.
An aqueous ethanol solution with a decalactone concentration of 20-100 ppm was obtained. The ethanol concentration of this ethanol aqueous solution was 10 to 25%.

Example 4 Hydrolysis was carried out under the same reaction conditions as in Example 3 except that 100 g of the dry yeast was used instead of the pressed baker's yeast.
Then, the hydroxylation step with dry lactic acid bacteria powder (Lactobacillus brevis) and the β-oxidation step with Saccharomyces cereviciae were performed under the same experimental conditions as in Example 3 including the reaction scale. The oil layer was extracted from this culture solution and analyzed by gas chromatography in the same manner as in Example 1. Further, the culture solution was distilled in the same manner as in Example 1 to obtain γ
-The concentration of dodecalactone and γ-decalactone is 10 to 10
A 30 ppm aqueous ethanol solution was obtained. The ethanol concentration of this ethanol aqueous solution was 10 to 25%.

In the β-oxidation step, instead of culturing at 30 ° C. for 48 hours, culturing with aeration and stirring was carried out at 30 ° C. for the following time.
The other experimental conditions were the same. after the β-oxidation step,
Tables 2 and 3 show the contents of γ-dodecalactone and γ-decalactone in the culture solution before the distillation step.

[0055]

[Table 3]

Example 5 In a process for producing whiskey, 1/1000 weight of dry lactic acid bacterium powder (Lactobacillus brevis) was added to malt meal (water content 63%) obtained by saccharifying malt to remove the saccharified solution, and the mixture was placed in a closed container. Degas thoroughly with, then 2 at 30 ° C.
On day, it was cultured to produce silage. This silage 5
20 ml of 1N aqueous sodium hydroxide solution was added to g, and the mixture was stirred at room temperature for hydrolysis. After neutralizing with hydrochloric acid,
Phosphate buffer was added to make a 50 ml aqueous solution. This aqueous solution was kept at 120 ° C. for 20 minutes for sterilization.

Next, a small amount of pressed baker's yeast manufactured by Chuetsu Yeast Co., Saccharomyces cereviciae (0.5 g) and silicone for food addition was added. While shaking at 180 rpm, the culture was carried out with aeration and stirring at 30 ° C. for 24 hours. The oil layer was extracted from this culture solution and analyzed by gas chromatography in the same manner as in Example 1. The concentrations of γ-decalactone and γ-dodecalactone were both 5 ppm.
Furthermore, an ethanol aqueous solution with a concentration of 95% was added to this culture solution to make the alcohol concentration before distillation 8% by weight,
Distillation was performed at 0 ° C. to obtain an ethanol aqueous solution having a concentration of γ-dodecalactone and γ-decalactone of 20 to 100 ppm. The ethanol concentration of this aqueous ethanol solution is 1
It was 0 to 25%.

Examples 6 to 8 Instead of Lactobacillus brevis,
Lactobacillus plantarum (Example 6), Lactobacillus bulgaric
us (Example 7), Lactobacillus cas
ei (Example 8 ) using the same conditions as in Example 1,
γ-Dodecalactone and γ-decalactone were produced.
In each case, it was confirmed by gas chromatography that 0.1 to 15 ppm of γ-dodecalactone and γ-decalactone were produced in the culture solution.

[0059]

INDUSTRIAL APPLICABILITY In the present invention, γ-dodecalactone and γ
-A liquid composition containing decalactone and having an excellent balance of aroma can be easily obtained by culturing microorganisms. In addition, since an organic substance containing a lipid is used as a starting material, a liquid containing γ-dodecalactone and γ-decalactone without adjusting the medium when culturing a microorganism in a hydroxylation step, a β-oxidation step, etc. A composition can be obtained. Moreover, since only natural products can be used as starting materials, the liquid composition of γ-dodecalactone and γ-decalactone can be safely used for foods and other human ingested products. Furthermore, since the hydroxylation step and the β-oxidation step are both culturing of microorganisms, it becomes easy to continuously operate in the same container.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI (C12P 17/04 C12R 1:85 C12R 1: 245) 1: 865 (C12P 17/04 C12R 1:25) (C12P 17/04 (C12R 1:85) (C12P 17/04 C12R 1: 865) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12P 17/00-17/18 BIOSIS / WPI (DIALOG)

Claims (7)

(57) [Claims] 1. A decomposition step of hydrolyzing or lipase-treating an organic substance containing a lipid to obtain a free fatty acid (free acid), and a carbon relating to the carbon-carbon double bond is added to the obtained free acid. LA as the first microorganism having the ability to hydroxylate
Belongs to the genus Lactobacillus
Lactic acid bacteria by the action that the hydroxylation step to obtain a substrate for γ- dodecalactone and γ- decalactone, the substrate obtained, a second microorganism having the β- oxidizing ability
Then the genus Saccharomyces
A β-oxidation step of obtaining a solution containing γ-dodecalactone and γ-decalactone by acting a microorganism belonging to
A method for producing a liquid composition containing γ-dodecalactone and γ-decalactone, which comprises, as the organic substance containing the lipid in the decomposition step, at least part of oleic acid by the hydrolysis or lipase treatment. And a method for producing a liquid composition containing γ-dodecalactone and γ-decalactone, which comprises using an organic substance that liberates a free acid containing palmitoleic acid. As wherein organic material containing said lipid in said decomposition step, gamma according to claim 1 which comprises using a solid by-product when obtained by fermenting yeast or alcoholic beverage, soy sauce, vinegar or milk products -A method for producing a liquid composition containing dodecalactone and γ-decalactone. In wherein the degradation step, the hydroxylation step and the β- oxidation step, containing γ- dodecalactone and γ- decalactone according to claim 1 or 2, characterized by using the same reaction vessel A method for producing a liquid composition. 4. A decomposing step of hydrolyzing or lipase-treating silage produced by lactic acid fermentation of cereals or the like to obtain a substrate for γ-dodecalactone and γ-decalactone from at least a part of the silage. the substrate, and a second microorganism having the β- oxidizing ability
Then the genus Saccharomyces
And a β-oxidation step of obtaining a solution containing γ-dodecalactone and γ-decalactone by causing a microorganism belonging to the group γ-dodecalactone and a liquid composition containing γ-decalactone to be obtained. Production method. 5. The decomposition step and the β-oxidation step,
The same reaction container is used, The manufacturing method of the liquid composition containing (gamma) -dodecalactone and (gamma) -decalactone of Claim 4 characterized by the above-mentioned. 6. A solution containing the γ-dodecalactone and γ-decalactone obtained in the β-oxidation step,
A solution containing ethanol, then according to any one of claims 1 to 5, characterized in that distillation γ-
A method for producing a liquid composition containing dodecalactone and γ-decalactone. 7. Using the claim 1 or the .gamma.-dodecalactone and .gamma. decalactone liquid composition obtained by the method for producing a liquid composition containing a description of 6, .gamma.-dodecalactone and γ A method for producing γ-dodecalactone and γ-decalactone, which comprises obtaining decalactone.