JP2003250594A - Method for producing cis-6-dodecene-4-olide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing cis-6-dodecene-4-olide. <P>SOLUTION: This method for producing the cis-6-dodecene-4-olide comprises a process for making lactic acid bacteria act on linoleic acid to form 10- hydroxy-12-octadecenoic acid and another process for making a yeast having β-oxidation activity act aerobically on the octadecenoic acid, wherein the lactic acid bacteria have such an ability as to change the double bond at the 9-position of the linoleic acid into a single bond by introducing hydrogen atom into the carbon atom at the 9-position of the linoleic acid and hydroxy group into the carbon atom at the 10-position. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to cis-6- which can be applied as a raw material for fragrances, food additives and the like from linoleic acid (also known as cis-9-cis-12-octadecadienoic acid).
A method for producing dodecene-4-olide, more specifically,
Lactic acid bacteria act on linoleic acid to give 10-hydroxy-1
Cis-6-dodecene-comprising a step of producing 2-octadecenoic acid and a step of allowing yeast to act on the octadecenoic acid
It relates to a method for producing 4-olid. It also relates to a method for producing 10-hydroxy-12-octadecenoic acid.

[0002]

2. Description of the Related Art Cis-6-dodecene-4-olide is a substance having a milk-like or flower-like aroma and is known as one of the important aroma components of dairy products such as butter. There are great expectations for its application to the world. Such cis-6-dodecene-4-olide has hitherto been obtained by a chemical synthesis method, a fermentation method, or an extraction from a natural product containing the substance. However, it reflects the recent consumer preference for food. Therefore, it is desired to supply a product obtained by fermentation or extraction of a natural product rather than a chemically synthesized product. As a natural product,
Since it is contained in dairy products such as peach fruits, milk and butter in an extremely small amount, it is very difficult to isolate from them and industrially utilize them.

Therefore, a method for producing cis-6-dodecene-4-olide by applying a fermentation method using a microorganism has attracted attention, and various methods have been proposed as follows. For example, Sporoboromyces odorus ( S
cis-6 from linoleic acid by porobolomyces odorus )
-Example of obtaining dodecene-4-olide ("Flavour of Foods
and Beverages Chemistry and Technology '' Academic
Press (1978), 145-167; and "Journal of Agricultur
al and Food Chemistry ”44, 1218-1223 (1996)), hydrolyzed soybean oil is a type of mold, penicillium.
Example of conversion by Rock Follity ( Penicillium roqueforti ) ("Biotechnology Letters" 14, 275-280 (19
92)) is known.

Further, in the method of obtaining cis-6-dodecene-4-olide from linoleic acid, linoleic acid is converted into peroxide by photooxidation and further reduced by 10-hydroxy-.
A method of obtaining 12-octadecenoic acid and β-oxidizing this with yeast to obtain cis-6-dodecene-4-olide (Japanese Patent No. 2969855 and Japanese Patent Laid-Open No. 3-187387),
A method (European patent application EP 578 388) for converting linoleic acid into 10-hydroxy-12-octadecenoic acid by Pseudomonas aeruginosa and obtaining β-oxidation of this with yeast to obtain cis-6-dodecene-4-olide is also proposed. Has been done. Also, "Bioscien
ce Biotechnology, and Biochemistry "(59 (8), 1571-
1572 (1995)), linoleic acid was microbiologically cis-6-
An example of conversion to dodecene-4-olide is shown.

However, in the above-mentioned production method, the concentration of cis-6-dodecene-4-olide produced is low, or the raw materials and microorganisms used are poor for humans to eat, so cis-6-dodecene In the case where -4-olide is produced and added to food, the culture solution (fermentation solution) containing the product cannot be added to the food as it is, and therefore a complicated purification operation is indispensable prior to food addition. There was a problem. Further, such a refining operation has a drawback that it directly leads to a high cost of the product and cannot be said to be an industrially advantageous production method.

Therefore, in the fermentative production of cis-6-dodecene-4-olide as a food additive, it is preferable to use a bacterial species which has been applied to various foods since ancient times and has high safety to human bodies, such as lactic acid bacteria and yeast. Is considered to be optimal. The following are known as conventional techniques relating to such lactic acid bacteria and yeast.

Lactobacillus acidophilus AKU 1137, a type of lactic acid bacterium, produces linoleic acid as a raw material for cis-6-dodecene-4-olide, 10-hydroxy-12.
− Conversion to octadecenoic acid has been reported (“Agricultural Chemistry Society Annual Meeting Abstracts” 2000, 74 Extra edition,
p143; “Applied and Environmental Microbiology” 6
7,1246-1252 (2001)). However, since the above-mentioned strain also has an activity of dehydrating 10-hydroxy-12-octadecenoic acid to convert it to conjugated linoleic acid, 10-hydroxy-
It has a drawback that the productivity of 12-octadecenoic acid cannot be increased. Lactobacillus plantarum ( Lact
obacillus plantarum ) has also been reported to convert linoleic acid to 10-hydroxy-12-octadecenoic acid ("Biochemical and Biophysical Research").
Communications "226, 391-395 (1996)). Although details such as the conversion rate in this reported example are unknown, 200 mg of 10-hydroxy-12-octadecenoic acid was obtained from 2 g of linoleic acid.

Further, conversion of oleic acid into 10-hydroxystearic acid by lactic acid bacteria (see "Journal of Applied").
Microbiology "88, 286-292 (2000)) and 10-hydroxystearic acid of oleic acid and 13-hydroxy-9-octadecenoic acid of linoleic acid by Streptococcus bovis (" FEMS Microbiology Letters 169, 272-282 (199
8)) has also been reported.

Further, many attempts have been made so far to obtain lactones similar to cis-6-dodecene-4-olide and intermediates thereof by a fermentation method using a microorganism. For example, there is a method in which lactic acid bacteria are allowed to act on oleic acid to produce 10-hydroxystearic acid, and then yeast is allowed to act to produce 4-dodecanolide.
(JP-A-7-274986). However, as described above, the relationship between the activity of converting oleic acid into 10-hydroxystearic acid and the activity of converting linoleic acid into 10-hydroxy-12-octadecenoic acid was unknown.

Although various conventional techniques related to the present invention have been disclosed, no specific report example regarding conversion of linoleic acid to 10-hydroxy-12-octadecenoic acid by lactic acid bacteria other than the above is found. Furthermore, a method for efficiently producing cis-6-dodecene-4-olide from linoleic acid or fats and oils using both lactic acid bacteria and yeast has not yet been known. Linoleic acid is the most polyunsaturated fatty acid in foods, and is a component contained in soybean oil, sunflower oil, corn oil, safflower, and other vegetable oils. In particular, it is said that it accounts for about 70% of fatty acids in safflower oil. Accordingly, linoleic acid, which is easily available and highly safe for the human body, is used as a raw material, and cis-6-dodecene-4-olid is efficiently produced by a fermentation method using microorganisms with high human body safety, such as lactic acid bacteria and yeast. It is earnestly desired to provide a method of manufacturing the same.

[0011]

DISCLOSURE OF THE INVENTION The present invention is directed to the production of cis-6-dodecene-4-olide from linoleic acid as a raw material via the intermediate 10-hydroxy-12-octadecenoic acid. PROBLEM TO BE SOLVED: To provide a method for continuously and efficiently producing cis-6-dodecene-4-olide without performing isolation or purification, and to produce an intermediate 10-hydroxy-12-octadecenoic acid from linoleic acid. In this regard, it is an object of the present invention to provide an efficient production method in which the yield is high and by-products are not generated. Further, it is possible to add cis-6-dodecene-4-olide to a food product in the form of a medium containing the substance without performing operations such as isolation and purification of the produced cis-6-dodecene-4-olide. It is an object to provide a method for producing dodecene-4-olide.

[0012]

The present inventors have found that cis-6
As a result of screening a microorganism that produces 10-hydroxy-12-octadecenoic acid, which is an intermediate for the synthesis of -dodecene-4-olide, a lactic acid bacterium having an extremely high conversion rate without producing a by-product was found, and It was completed. That is, according to the present invention, lactic acid bacteria capable of introducing hydrogen into the 9-position carbon of linoleic acid and a hydroxyl group into the 10-position carbon of linoleic acid to form a double bond at the 9-position into a single bond act on 10- A method for producing cis-6-dodecene-4-olide, which comprises a step of producing hydroxy-12-octadecenoic acid and a step of aerobically acting yeast having β-oxidation ability on the octadecenoic acid. Is. In addition, the present invention, linoleic acid, Lactobacillus
Casei, Lactobacillus bulgaricus, Lactobacillus helvetics, Lactobacillus paracasei,
A method for producing 10-hydroxy-12-octadecenoic acid, which comprises reacting at least one lactic acid bacterium selected from Lactobacillus rhamnosus, Lactococcus lactis, and Streptococcus thermophilus.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. In the present invention, lactic acid bacteria are allowed to act on linoleic acid to produce 10
-Step of producing hydroxy-12-octadecenoic acid (hereinafter referred to as "first step"), then produced 10-
It comprises a step of reacting hydroxy-12-octadecenoic acid with yeast to produce cis-6-dodecene-4-olide (hereinafter referred to as "second step"). In these steps, a mixture of at least one substance selected from foods and food additives and water is used as a medium or dispersion medium (hereinafter, the medium or dispersion medium is generically referred to as "medium").
The first and second steps can be performed in the same medium, but after the first step, 10-hydroxy-12-octadecenoic acid is isolated and purified, and a new medium is added to the second step. You may perform the process of. In addition, depending on the conditions, the lactic acid bacterium and the yeast may be simultaneously added to the medium to perform the first and second steps in one batch. Each step will be specifically described below.

(1) First Step The first step is a step in which lactic acid bacteria are allowed to act on linoleic acid in an appropriate medium to produce 10-hydroxy-12-octadecenoic acid. The linoleic acid, which is a raw material for linoleic acid, is not limited to a highly purified product at a reagent level, and a product liberated from fats and oils can also be used. As the lipid that is a raw material of linoleic acid, linoleic acid salts such as sodium linoleate and potassium linoleate, esters such as methyl linoleate and ethyl linoleate, and edible oil and fat containing linoleic acid as a constituent can be used. Edible oils and fats containing linoleic acid as a constituent are particularly desirable. As edible oils and fats,
The weight of linoleic acid produced when this is hydrolyzed is preferably 1% or more, more preferably 10% or more of the weight of the original fat or oil.

Examples of such edible oils and fats include cottonseed oil, sunflower oil, grape seed oil, corn oil, soybean oil, sesame oil, rice bran oil, safflower oil, rapeseed oil and the like. When the medium originally contains linoleic acid, a linoleic acid salt, an ester of linoleic acid, or an oil or fat containing linoleic acid as a constituent component, these may be used as raw materials for linoleic acid.

When an oil or fat containing linoleic acid as a constituent is used as a raw material, it is necessary to release linoleic acid from the above raw material by hydrolysis. A known method can be used for the hydrolysis. For example, an enzyme having the activity of neutralizing with a suitable base or acid after hydrolysis with an acid or base, or hydrolyzing an ester bond such as lipase,
Alternatively, a substance containing these can be used. In particular, hydrolysis using lipase having activity in the neutral to acidic region can be carried out simultaneously with the conversion reaction of linoleic acid using lactic acid bacterium, so that the reaction time can be shortened. In addition, when an enzyme that does not have activity or deactivates in the neutral or acidic range is used and designed so that the enzyme activity disappears as the pH decreases due to lactic acid fermentation, the control of fat and oil hydrolysis becomes easy.

Lactic acid bacterium The lactic acid bacterium used in the present invention contains 10-hydroxy-12-octadecenoic acid in which hydrogen is introduced into the 9th carbon of linoleic acid and a hydroxyl group is introduced into the 10th carbon to replace the double bond at the 9th position with a single bond. Must have activity to generate. Further generated 10-
A strain that does not react with hydroxy-12-octadecenoic acid such as dehydration or hydration is preferable. In addition, it is a strain that humans have ingested into the body in some form from the past,
That is, those who have experience in eating are desirable. Examples of such lactic acid bacteria include, for example, Lactobacillus delbrue
ckii subsp. delbrueckii ), Lactobacillus bulgaricus, Lactobacillus del
brueckii subsp. lactis), Lactobacillus helveticus, Lactobacillus paracasei, lactic acid bacteria of the genus Lactobacillus, such as Lactobacillus rhamnosus, lactic acid bacteria Lactococcus such as Lactococcus lactis,
And lactic acid bacteria of the genus Streptococcus represented by Streptococcus thermophilus.

Lactic acid bacteria selected from the following strains are particularly preferable. That is, Lactobacillus casei IAM 104
5, Lactobacillus casei L casei 01, Lactobacillus casei IFO 15883, Lactobacillus casei L-1
4, Lactobacillus casei JCM 8129, Lactobacillus bulgaricus IAM 1120, Lactobacillus helvetics IFO 15019, Lactobacillus paracasei IFO 353
3, Lactobacillus paracasei IFO 3953, Lactobacillus paracasei IFO 14709, Lactobacillus paracasei JCM 1109, Lactobacillus paracasei JCM 111
1, Lactobacillus paracasei JCM 1133, Lactobacillus paracasei JCM 1161, Lactobacillus paracasei JCM 1181, Lactobacillus paracasei JCM 155
6, Lactobacillus paracasei JCM 2769, Lactobacillus paracasei JCM 2770,

Lactobacillus paracasei JCM 8130,
Lactobacillus paracasei JCM 8132, Lactobacillus paracasei JCM 8133, Lactobacillus rhamnosus IFO 3532, Lactobacillus rhamnosus IFO 3863,
Lactobacillus rhamnosus IFO 14710, Lactobacillus rhamnosus JCM 1136, Lactobacillus rhamnosus JCM 1165, Lactobacillus rhamnosus JCM 1553,
Lactobacillus rhamnosus JCM 1561, Lactobacillus rhamnosus JCM 1563, Lactobacillus rhamnosus JCM 2771, Lactobacillus rhamnosus JCM 2772,
Lactobacillus rhamnosus JCM 8134, Lactococcus lactis subsp. Cremoris
H-61), Lactococcus cremoris IFO 3427, and Streptococcus thermophilus IAM 10064.

The above strain names, IFO, JCM, and IAM, indicate that they are strains derived from the Fermentation Research Institute, RIKEN Microorganism Preservation Facility, and Foundation for Applied Microbiology Research Promotion, respectively. .

As a result of examining the biological classification of these lactic acid bacteria and the efficiency of converting linoleic acid into 10-hydroxy-12-octadecenoic acid, lactobacillus casei, lactobacillus paracasei and lactobacillus rhamnosus were all found to contain linoleic acid. 10-hydroxy-1
It was found that there are many strains with high conversion efficiency to 2-octadecenoic acid, and they are a closely related group. Other genera,
Seed lactic acid bacteria include 10-hydroxy-12 of linoleic acid.
-A system having and a system having no conversion activity to octadecenoic acid, and a system converting linoleic acid to 13-hydroxy-9-octadecenoic acid were mixed. Thus, for lactic acid bacteria except for three species of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus, it is necessary to actually examine for each strain whether or not to convert linoleic acid into 10-hydroxy-12-octadecenoic acid. is there.

These lactic acid bacteria can be separated from dairy products, animals and plants. It can also be purchased from a known dispensing organization, a company or a group that manufactures and sells starter cultures of yogurt and cheese. Note that these lactic acid bacteria may have both the ability to convert oleic acid into 10-hydroxystearic acid and the ability to convert linoleic acid into 13-hydroxy-9-octadecenoic acid. Further, depending on the purpose, these strains and other microorganisms such as lactic acid bacteria, bifidobacteria, natto bacteria, and yeasts that do not have the activity of producing 10-hydroxy-12-octadecenoic acid from linoleic acid can be mixed and cultured. .

The lactic acid bacterium is usually pre-cultured in an appropriate medium to prepare a culture solution called starter culture, and a fixed amount of this starter culture is added to the production medium. Alternatively, frozen or freeze-dried starter culture can be purchased and used. The amount to be added must be varied depending on the type of medium used, substrate concentration, and economic efficiency and cannot be specified unconditionally, but usually 10 ³ to 10 ¹² cfu (colony forming unit) of lactic acid bacteria per 1 g of medium, preferably 10 ⁶ to 10 Add to ¹² cfu.

The starter culture of linoleic acid and lactic acid bacterium may be added to the medium either before. For example, linoleic acid may be added to the medium in advance and then sterilized, and after cooling, starter culture may be added, or linoleic acid and starter culture may be added simultaneously. Alternatively, linoleic acid may be added after the starter culture has been added to grow the lactic acid bacteria. Further, linoleic acid may be added several times during the culture.

Culture condition In the present invention, the medium used must be one in which lactic acid bacteria can survive. The medium may have a composition such as a buffered salt solution, but a composition capable of growing lactic acid bacteria by further adding a nutrient source is desirable. Examples of such products include dairy products and processed products thereof, fruits and processed products thereof, vegetables and processed products thereof,
Examples include grains and processed products thereof, meat and processed products thereof, seafood and processed products thereof, yeast hydrolyzate solution, molasses solution, sugar solution, organic acid solution, organic acid salt solution and mixtures thereof. be able to. If necessary, inorganic salts, vitamins, nucleic acids, enzymes and the like may be added.

A surfactant is added to the above oils and fats and the above medium.
Antioxidants such as tocopherol and 2,6-di-t-butyl-4-methylphenol (BHT) can be added. It is also possible to add enzymes such as protease and lactase. Further, it is possible to add an edible oil / fat or an organic solvent such as ethanol, which has a low content of linoleic acid such as butterfat or coconut oil and is not suitable as a source of linoleic acid, for the purpose of dilution or the like. Further, it is also effective to emulsify the mixture of oil and fat and the medium by using an appropriate method.

The reaction conditions for reacting lactic acid bacteria with linoleic acid may be general lactic acid bacteria culture conditions, and the temperature is in the range of 25 ° C to 45 ° C, preferably 35 ° C to 42 ° C.
The reaction is allowed to stand or stirred. Anaerobic conditions are desirable for the reaction. During the reaction, it is also possible to add a suitable alkali or acid solution so that the pH is changed or is not changed. The reaction time can be appropriately set depending on the medium, the substrate concentration, the amount of lactic acid bacteria added, and the desired yield of 10-hydroxy-12-octadecenoic acid, but it is usually 78 to 240 hours.

(2) Second step The second step is a step of aerobically acting 10-hydroxy-12-octadecenoic acid with yeast to produce cis-6-dodecene-4-olide. The yeast used in the second step of the fermentation mother is 10-hydroxy-12-
It must have the activity of β-oxidizing octadecenoic acid to convert it to 4-hydroxy-6-dodecenoic acid. Further, those having a low activity of assimilating 4-hydroxy-6-dodecenoic acid and cis-6-dodecene-4-olide obtained in the first step are desirable. Further, like the above-mentioned lactic acid bacterium, it is desirable that the strain has been ingested by the human body in some form from the past, that is, a strain that has experienced eating. Examples of this are Candida colicloza, Candida
The yeasts of the genus Candida such as Uchiris, the genus Kliberomyces such as Kleiberomyces lactis, the genus Saccharomyces such as Saccharomyces cerevisiae, or the yeast of the genus Yarrowia such as Yarrowia lipolytica can be exemplified.

[0029] In particular, Candida colicolosa IAF7 (Christian Hansen), Candida utilis IFO386, Kliberomyces lactis IFO433, Saccharomyces cerevisiae IAM4125, baker's yeast (Saccharomyces cerevisiae IFO2044), baker's yeast ("Nisshin Seifun Co., Ltd." Nisshin Super Camellia (trade name);
(Sold by several companies including S. Co., Ltd.), yeast for sake brewing (Saccharomyces cerevisiae IFO2376), Yarrowia repolitica IFO1658, Yarrowia repolitica IFO16
59, Yarrowia lipolytica IFO10073 and the like can be preferably used. These yeasts can be isolated from dairy products, fermented foods, animals and plants. It can also be purchased from a publicly known dispensing organization. In particular, yeast cakes and dry yeasts that are generally commercially available for bread making can be preferably used from the viewpoints of easy availability, storage, and safety.

The yeast used is one that has been precultured in an appropriate medium. At this time, yeast cells can be collected, concentrated, and added by a centrifugation operation or the like, if necessary. When a commercially available baker's yeast such as yeast cake or dry yeast is used, it can be used as it is or after only a short time of reconstitution culture, which is preferable.

From the viewpoint of efficiently producing cis-6-dodecene-4-olide, a suitable combination of the lactic acid bacterium used in the first step and the yeast used in the second step is shown in Table 1 below. It is as follows.

[0032]

[Table 1]

Culturing conditions After the completion of the first step, yeast can be added to start the second step simply by changing the conditions as described below. However, a sterilization step or 10 steps can be performed between the first step and the second step. -Hydroxy-12
-A step of concentrating octadecenoic acid may be included. Further, for the purpose of facilitating the supply of oxygen into the medium, the medium may be dehydrated so that the medium may be changed into a cheese-like semi-solid. Substances that add yeast nutrients or components that change the pH of the medium between the first step and the second step or in the middle of the second step, and promote β-oxidation such as ricinoleic acid and castor oil A defoaming agent may be added. Also,
Fermentation broth flavor of improvement and microbial protease and lipase for the purpose of growth promotion of, amylase, and enzymes, such as lactase, Penicillium lock Folli tea and Penicillium Kamanbe Faculty (Penicillium camemberti)
A fungal culture or spores such as may be added.

The amount of yeast added cannot be unconditionally specified because it needs to be increased or decreased depending on the type of medium used, substrate concentration, and economic efficiency, but usually 10 ³ to 10 ¹⁰ cf per 1 g of medium.
u (colony forming unit), preferably 10 ⁶ to 10
Add to ⁸ cfu. 10-hydroxy-1
Conditions for the reaction of yeast with 2-octadecenoic acid are as follows:
It may be in accordance with general yeast aerobic culture conditions. That is, the reaction temperature is 20 ° C. to 40 ° C., preferably 25 ° C. to 35 ° C., and shaking culture or aeration culture is performed. The reaction time depends on the medium and substrate concentration, aeration conditions, the amount of yeast added,
It can be appropriately set depending on the desired yield of cis-6-dodecene-4-olide, but it is usually 20 to 120 hours.

In the above production method, 4-hydroxy-6- which is a precursor of cis-6-dodecene-4-olide is rare.
Dodecenoic acid may accumulate. In this case, lactonization may be promoted by a known method. For example, an acid such as lactic acid or citric acid may be added or heated to promote lactonization. Conversely, increase the pH of the medium by 4
-Hydroxy-6-dodecenoic acid may be produced, purified by a method such as ion exchange, and then lactonized.

(3) cis-6-dodecene-4 produced
-Olido cis-6-dodecene-4 produced through the above steps
-Oride can be used as a food, a raw material of a food, a food additive or a raw material of a food additive as it is contained in a medium. Further, unnecessary solids can be removed by filtration or centrifugation, or cis-6-dodecene-4-olide can be concentrated or purified by a method such as distillation, solvent extraction, or fractionation by column.

A medium containing the produced cis-6-dodecene-4-olide, or cis-6 obtained by purifying the medium.
-Dodecene-4-olide may be prepared as a mixture with known food and drink or flavor composition, for example, lipase degradation product of dairy product or fresh cream, fruit juice, recoveries obtained by concentrating fruit juice, liqueurs and the like. it can.

Further, limonene, myrcene, α-pinene, β-pinene, α-terpinene, γ-terpinene, which are generally used for imparting a flavor to fruits, coffee and the like,
Hydrocarbons such as caryophyllene; hexanol, heptanol, octanol, nonanol, decanol, ci
s-3-hexenol, cis-6-nonenol, ci
s-3,6-nonadienol, citronellol, geraniol, nerol, linalool, α-terpineol,
Alcohols such as γ-terpineol, terpinen-4-ol, menthol, thymol, eugenol; hexanal, cis-3-hexenal, trans-2
Aldehydes such as hexenal, heptanal, octanal, nonanal, decanal, undecanal, 10-undecenal, 2,6-nonadienal, citral, citronellal, vanillin, ethyl vanillin, etc.
And acetals thereof; carvone, pulegone, menthone, camphor, nootkatone, acetoin, p-methylacetophenone, p-methoxyacetophenone, ketones such as raspberry ketone, anisylacetone, zingerone, acetylfuran, and ketals thereof; ethyl. Acetate, propyl acetate, butyl acetate, isoamyl acetate, hexyl acetate, heptyl acetate, geranyl acetate, linalyl acetate, ethyl propionate, isoamyl propionate, hexyl propionate, geranyl propionate, citronellyl propionate, ethyl Butyrate,
Esters such as isoamyl butyrate, geranyl butyrate, butyl pentanoate, methyl butyrate, methyl anthranilate; fatty acids such as acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid;
Maltol, ethylmaltol, cyclotene, dimethylhydroxyfuranone, γ-hexanolactone, γ-heptanolactone, γ-octanolactone, γ-nonanolactone, γ-decanolactone, cis-7-deceno-γ-lactone, γ-unde Canolactone, γ-dodecanolactone, δ-heptanolactone, δ-octanolactone, δ
-Nonanolactone, delta-decanolactone, 2-deceno-
A preparation may be prepared by appropriately mixing flavor components such as δ-lactone, δ-undecanolactone, δ-dodecanolactone, and δ-tridecanolactone according to the desired flavor.
Further, it may be dried to obtain a powder form of the preparation. At this time, an excipient such as sugar or dextrin may be added.

Example to which a medium containing cis-6-dodecene-4-olide produced according to the present invention, or cis-6-dodecene-4-olide obtained by purifying the medium and a preparation containing the same are added Examples thereof include dairy products such as milk, butter, cheese, yogurt and processed products of dairy products, dairy drinks and ice creams, and foods and drinks such as milk chocolate and lactoice. Also, dairy substitutes such as coffee whitener, margarine or fat spread, fruit juice beverages, fruit liquors, carbonated beverages, favorite beverages such as coffee, green tea, black tea, oolong tea, frozen desserts, Japanese and Western confectionery, jams. , Chewing gums, breads, soups, flavor seasonings, various instant beverages and foods, various snack foods, and the like, and raw materials thereof, food additives such as flavors, and the like.

[0040]

EXAMPLES The present invention will be described in more detail with reference to examples, but the present invention is not limited to these. [Example 1] (Production of 10-hydroxy-12-octadecenoic acid) To a 200 ml Erlenmeyer flask, 100 ml of SMY medium (900 ml of distilled water plus 100 g of skim milk powder and 1 g of yeast hydrolyzate) was added, and 115 Sterilized by holding at 15 ° C for 15 minutes. Lactic acid bacterium culture solution shown in Table 1 in each flask (lactic acid bacterium sterilized in SMY medium for 1 to 3 days) 1 m
After adding l, 0.5 g of linoleic acid (linoleic acid manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in 1 ml of ethanol was added. These were left to stand at 37 ° C. for 7 days to culture lactic acid bacteria. After completion of the culture, 5-tridecanolide as an internal standard substance was added to the culture medium, followed by extraction with diethyl ether. This extract was analyzed by gas chromatography (hereinafter referred to as "GC"), and the concentration of the produced 10-hydroxy-12-octadecenoic acid was measured. The qualitative analysis of 10-hydroxy-12-octadecenoic acid was performed by the retention time in GC. The results are shown in Table 2.

[0041]

[Table 2]

[0042]

[Table 3]

Example 2 (10-hydroxy-12-
Production of octadecenoic acid) 500 g of skim milk powder, 5 g of yeast hydrolyzate, and 4.5 kg of distilled water were added to a 10-liter culture tank, sufficiently dissolved by stirring, and sterilized by holding at 115 ° C for 15 minutes and culturing (medium). ) Got. After cooling this culture solution to 37 ° C., a mixed solution of 25 g of linoleic acid (manufactured by Wako Pure Chemical Industries, Ltd.) and 50 g of ethanol, and a freeze-dried powder of Lactobacillus casei L casei 01 strain as a lactic acid bacterium starter.
1 g (manufactured by Christian Hansen) was added to the culture solution.
Furthermore, nitrogen was passed through a sterile filter to replace oxygen in the system, and then the mixture was stirred at 100 rpm and kept at 37 ° C for 7 hours.
After culturing for a day, 10-hydroxy-12-octadecenoic acid was produced. When a part of this culture solution was collected and analyzed by GC, 2100 ppm of 10-hydroxy-12-octadecenoic acid was formed. Furthermore, 500 of this culture solution
g was extracted with ethyl acetate to give 0.80 g of an oily extract. The extract was fractionated by a chromatogram using a silica gel column to give 0.35 g of 10-hydroxy-12.
-Octadecenoic acid was obtained. NM of such octadecenoic acid
The R spectrum is based on literature (“Applied and Environmental Mi
crobiology "(2001), 67, p1246-1252).

Example 3 (cis-6-dodecene-4-
Generation of Oride) In a 500 ml Erlenmeyer flask, TYG medium (distilled water 100
0 g tryptone (Difco) 10 g, yeast hydrolyzate 5
g, glucose 10 g, salt 5 g added) 100
ml was added for sterilization. Then, 0.1 g of linoleic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 1 ml of ethanol in each flask, and a starter of lactic acid bacteria shown in Table 4 (at 37 ° C. using TYG medium sterilized with lactic acid bacteria). (Cultured for 48 hours) 1 ml was added. This was statically cultured at 37 ° C. for 4 days. Then, 1 g of dried baker's yeast (Dry yeast “Super Camellia (trade name)” sold by Nisshin Seifun Co., Ltd.) was added and shaken at a speed of 120 rpm at a temperature of 30 ° C. for 4
Cultured aerobically for 8 hours. After completion of the culture, 5-tridecanolide as an internal standard substance was added to the culture medium, followed by extraction with diethyl ether. The extract was analyzed by GC to measure the concentration of the produced cis-6-dodecene-4-olide. Depending on the combination of lactic acid bacteria and baker's yeast shown in Table 4, several tens of ppm of cis-6-dodecene-4 was converted from linoleic acid.
-Oride formed. The results are shown in Table 3.

[0045]

[Table 4]

Example 4 (cis-6-dodecene-4-
Generation of Oride) 100 ml of 10% in each 500 ml Erlenmeyer flask
(w / w) non-fat dry milk solution was added, and the mixture was kept at 115 ° C for 15 minutes for sterilization. Then, a solution of 0.5 g of linoleic acid (manufactured by Wako Pure Chemical Industries, Ltd.) in 1 ml of ethanol was added to each flask, and Lactobacillus.
0.01 g of freeze-dried powder of L. casei strain 01 (manufactured by Christian Hansen) was added. 12 at 37 ℃
After culturing for 0 hour, 10-hydroxy-12-octadecenoic acid was produced. Y sterilized with the yeast shown in Table 5
2 ml of culture medium aerobically cultivated in M medium (1,000 ml of distilled water to which 10 g of malt extract, 4 g of glucose, and 4 g of yeast hydrolyzate were added) was added, and the shaking speed was 120 rpm and the temperature was 30 ° C. Cultured aerobically for 24 to 120 hours. When the obtained culture solution was analyzed by GC,
As shown in Table 4, cis-6-dodecene-4-olide was produced in an amount of 50 to 280 ppm in all cases.

[0047]

[Table 5]

Example 5 (cis-6-dodecene-4-
Generation of Olede) Skim milk powder (500 g) and distilled water (4.5 kg) were added to a 10-liter culture tank, and the mixture was sufficiently stirred to dissolve at 15 ° C at 15 ° C.
It was held for a minute and sterilized to obtain a culture solution (medium). This is 37
After cooling to ℃, 25 g of linoleic acid (Wako Pure Chemical Industries, Ltd.) dissolved in 50 g of ethanol and Lactobacillus casei L casei 0 as a lactic acid bacterium starter
1 g of freeze-dried powder (manufactured by Christian Hansen)
Was added. After nitrogen was passed through a sterile filter to replace oxygen in the system, the mixture was cultured at 37 ° C. for 120 hours while stirring at 100 rpm. Then, add 30
The temperature was maintained at 0 ° C., 50 g of dry baker's yeast (“Super Camellia (trade name)” described above) was added, and the mixture was cultured for 72 hours while stirring at 350 rpm while blowing air through a sterile filter at 1.0 vvm. Cis-6 produced after the culture was completed
-The concentration of dodecene-4-olide was measured by GC. This culture contained 890 ppm of cis-6-dodecene-4-olide.

Example 6 1 kg of the culture solution containing cis-6-dodecene-4-olide of Example 5 was extracted with 500 ml of ethyl acetate. This extract was concentrated and fractionated by silica gel column chromatogram. The fraction containing cis-6-dodecene-4-olide was concentrated to give 250 mg.
An oily substance was obtained. This is appropriately diluted with a solvent and analyzed by gas chromatography-mass spectrometry (GC-MS),
It was confirmed from the retention time and the mass spectrum that the main component was cis-6-dodecene-4-olide.

Example 7 (cis-6-dodecene-4-
Formation of Oride) 1 g of corn germ oil was dispersed and emulsified in 100 ml of 10% non-fat dry milk aqueous solution. This was added to a 500 ml Erlenmeyer flask and sterilized by holding at 115 ° C. for 15 minutes to obtain a medium (culture solution). After cooling the culture solution to 37 ° C., lipase (“Lipase A Amano (trade name)” manufactured by Amano Pharmaceutical Co., Ltd.) 10
mg (dissolved in an appropriate amount of distilled water and sterilized by filtration) and lactic acid bacterium starter (Lactobacillus casei IAM 1045 sterilized 10% non-fat dry milk aqueous solution for 48 hours at 37 ° C) added 1 ml did. This was cultured at 37 ° C. for 96 hours. Then, 1 g of dried baker's yeast (the above-mentioned “Super Camellia (trade name)”) was added and aerobically cultured at a shaking speed of 120 rpm and a temperature of 30 ° C. for 48 hours. When the obtained culture solution was analyzed by GC, it was 129 pp
It contained m of cis-6-dodecene-4-olide.

[Example 8] cis-6 obtained in Example 5
5 parts by weight of a culture solution containing dodecene-4-olide
It was added to 000 parts by weight of milk (commercial product). As a result of a sensory evaluation by a flavor list, the milk to which the culture solution was added had an enhanced natural milk fat feeling.

[Example 9] cis-6 obtained in Example 5
5 parts by weight of a culture solution containing dodecene-4-olide
It was added to 000 parts by weight of a lactic acid bacterium beverage (commercially available product). As a result of a sensory evaluation by a flavor list, the lactic acid bacteria beverage to which the culture solution was added was given a thick taste with a cheese-like or butter-like flavor.

Example 10 The cis-obtained in Example 5
1 part by weight of a culture solution containing 6-dodecene-4-olide was mixed with 4 parts by weight of a milk flavor (flavor) having the composition shown in Table 5. This mixture was kept at -5 ° C for 24 hours, and then insoluble matter was removed using filter paper to obtain a new fragrance.
The resulting fragrance was subjected to a sensory evaluation of a flavor list,
Table 5 containing no cis-6-dodecene-4-olide
When compared with the milk flavor of the composition described, it was a flavor with a natural milk feel.

[0054]

[Table 6]

[0055]

(1) Using linoleic acid as a starting material, 10-
In the method of the present invention for producing the target substance cis-6-dodecene-4-olide by a fermentation method via hydroxy-12-octadecenoic acid, 10-hydroxy-12-octadecenoic acid is produced at a high concentration to produce a by-product. Lactic acid bacteria that do not generate are used. Therefore, it is not necessary to perform a complicated operation such as isolation and purification of octadecenoic acid from the lactic acid bacterium culture solution prior to the step of allowing the yeast to act on the octadecenoic acid, and yeast can be directly added to the lactic acid bacterium culture solution.
6-dodecene-4-olide can be produced continuously and efficiently.

(2) The method of the present invention for producing 10-hydroxy-12-octadecenoic acid using linoleic acid as a starting material produces 10-hydroxy-12-octadecenoic acid in a high concentration and does not generate a by-product. Since lactic acid bacteria are used, 10-hydroxy-12-octadecenoic acid can be efficiently produced, and isolation or purification of the substance is not necessary.

(3) Since the method of the present invention is a fermentation method using highly safe lactic acid bacteria and yeast, cis-
It is not necessary to perform a complicated operation such as isolating and purifying 6-dodecene-4-olide, and it is possible to directly add to a food or the like in the form of a medium containing lactic acid bacteria or yeast. Furthermore, according to the prior art, cis-6-dodecene-4-
A rich milk-like or butter-like flavor can be imparted to foods, which is not inferior to the refined products of Orido.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ identification code FI theme code (reference) (C12P 7/64 C12R 1:72 C12R 1:72) 1: 865 (C12P 7/64 C12R 1: 645 C12R 1: 865) 1:01 (C12P 7/64 C12R 1:24 C12R 1: 645) 1: 245 (C12P 7/64 C12R 1: 225 C12R 1:01) 1:46 (C12P 7/64 C12R 1:85 C12R 1:24) (C12P 7/64 C12R 1: 245) (C12P 17/04 C12R 1: 245) (C12P 17/04 C12R 1: 225) (C12P 17/04 C12R 1:01) (C12P 17/04 C12R 1:46) (C12P 17/04 C12R 1:72) (C12P 17/04 C12R 1: 645) (C12P 17/04 C12R 1: 865) (C12P 17/04 C12R 1:85) F term (reference) 4B001 AC99 BC01 EC01 4B035 LC01 LG07 LK02 4B064 AE45 CA02 CA06 CB14 CB17 CC03 CD07 4H059 BA36 BB15 BB22 BB44 DA09

Claims (16)

[Claims] 1. A lactic acid bacterium capable of introducing hydrogen at the 9-position carbon of linoleic acid and a hydroxyl group at the 10-position carbon of linoleic acid to form a double bond at the 9-position into a single bond is reacted with 10-
Cis-characterized in that it comprises a step of producing hydroxy-12-octadecenoic acid, and then a step of aerobically acting yeast having β-oxidation ability on the octadecenoic acid.
Process for producing 6-dodecene-4-olide. 2. The lactic acid bacterium is Lactobacillus
us ) genus, Lactococcus ( Lactococcus ) genus or Streptococcus ( Streptococcus ) genus strain selected from at least one strain selected from the cis-6-dodecene-4-olide Production method. 3. The method for producing cis-6-dodecene-4-olide according to claim 1, wherein the lactic acid bacterium is at least one strain selected from the following strain group. [Strains] Lactobacillus cas
ei subsp. casei ), Lactobacillus delbrue
ckii subsp.bulgaricus ) , Lactobacillus helve
ticus), Lactobacillus paracasei (Lactobacillus paracasei
subsp. paracasei), Lactobacillus rhamnosus (Lactobacillus rhamnosu
s ), Lactococcus lactis , and Streptococcus thermophilus ( Streptococcus s).
alivarius subsp. thermophilus ). 4. The method for producing cis-6-dodecene-4-olide according to claim 1, wherein the lactic acid bacterium is at least one strain selected from the following strain group. (Strains) Lactobacillus casei IAM 1045, Lactobacillus casei L casei 01, Lactobacillus casei IFO 15883, Lactobacillus casei L-14, Lactobacillus casei JCM 8129, Lactobacillus bulgaricus IAM 1120, Lactobacillus I19 herbetics Paracasei IFO 3533, Lactobacillus paracasei IFO 3953, Lactobacillus paracasei IFO 14709, Lactobacillus paracasei JCM 1109, Lactobacillus paracasei JCM 1111, Lactobacillus paracasei Jaca 1CM, Lactobacillus paracasei Jaca 1CM, Lactobacillus paracasei JCM 1133 JCM 1556, Lactobacillus paracasei JCM 2769, Lactobacillus paracasei JCM 2770, Lactobacillus paracasei JCM 8130, Lactobacillus・ Paracasei JCM 8132, Lactobacillus paracasei JCM 8133, Lactobacillus rhamnosus IFO 3532, Lactobacillus rhamnosus IFO 3863, Lactobacillus rhamnosus IFO 14710, Lactobacillus rhamnosus JCM 153, Lactobacillus rhamnosus JCM 1165, Lactobacillus rhamnosus JCM 1165, Lactobacillus rhamnosus JCM 1165, JCM 1561, Lactobacillus rhamnosus JCM 1563, Lactobacillus rhamnosus JCM 2771, Lactobacillus rhamnosus JCM 2772, Lactobacillus rhamnosus JCM 8134, Lactococcus cremoris ( Lactococcus lactis subs)
p. cremoris H-6), Lactococcus cremoris IFO 3427, and Streptococcus thermophilus IAM 10064. 5. The yeast is genus Candida, genus Kluyveromyces , saccharomyces Sacch.
The cis-6 according to any one of claims 1 to 4, which is at least one strain selected from the group of strains belonging to the genus aromyces ) or the genus Yarrowia.
-Method for producing dodecene-4-olide. 6. The yeast is at least one strain selected from the following strain group:
The method for producing cis-6-dodecene-4-olide according to any one of 1. (Strain group) Candida utilis , Candida colliculosa , Kluyveromyces lactis
, Saccharomyces cerevicia
e ), Saccharomyces pasto
rianus ), and Yallowia lipolytica . 7. The yeast is at least one strain selected from the following strain group:
The method for producing cis-6-dodecene-4-olide according to any one of 1. (Strains) Candida coliculosa IAF7, Candida utilis IFO386, Kliberomyces lactis IFO433, Saccharomyces cerevisiae IAM4125, Saccharomyces cerevisiae IFO2044, Saccharomyces cerevisiae IFO2376, Yarrowia wyropia ricolia IFO3376 IFO10073. 8. The cis-6-according to any one of claims 1 to 7, characterized in that at least one substance selected from foods, food additives and water is used as the medium or dispersion medium. Method for producing dodecene-4-olide. 9. The food is dairy products or processed products thereof, fruits or processed products thereof, vegetables or processed products thereof, grains or processed products thereof, meat or processed products thereof, seafood or processed products thereof, molasses or solutions thereof. 9. The cis-6 according to claim 8, which is at least one selected from the group consisting of saccharides, sugars or solutions thereof, acids, inorganic salts and yeast hydrolysates.
-Method for producing dodecene-4-olide. 10. The method for producing cis-6-dodecene-4-olide according to claim 8, wherein the food additive is at least one selected from acids, salts of organic acids or inorganic acids, vitamins, and nucleic acids. . 11. Linoleic acid is obtained by hydrolysis of an oil or fat containing linoleic acid salt, linoleic acid ester and linoleic acid as a constituent, or by hydrolysis of a medium or a dispersion medium containing an oil or fat containing linoleic acid as a constituent. The method for producing cis-6-dodecene-4-olide according to any one of claims 1 to 10, which is obtained. 12. An oil or fat containing linoleic acid as a constituent,
The method for producing cis-6-dodecene-4-olide according to claim 11, which is a fat and oil which releases 1% or more of linoleic acid based on the weight of the original fat and oil when hydrolyzed. 13. A flavoring composition comprising cis-6-dodecene-4-olide obtained by the method according to any one of claims 1 to 12. 14. A food or drink containing cis-6-dodecene-4-olide obtained by the method according to any one of claims 1 to 12. 15. A method for producing 10-hydroxy-12-octadecenoic acid, which comprises reacting linoleic acid with at least one lactic acid bacterium selected from the following strain group. [Strains] Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactococcus lactis, and Streptococcus thermophilus. 16. The lactic acid bacterium is at least one strain selected from the following strain group:
5. The method for producing 10-hydroxy-12-octadecenoic acid according to 5. (Strains) Lactobacillus casei IAM 1045, Lactobacillus casei L casei 01, Lactobacillus casei IFO 15883, Lactobacillus casei L-14, Lactobacillus casei JCM 8129, Lactobacillus bulgaricus IAM 1120, Lactobacillus I19 herbetics Paracasei IFO 3533, Lactobacillus paracasei IFO 3953, Lactobacillus paracasei IFO 14709, Lactobacillus paracasei JCM 1109, Lactobacillus paracasei JCM 1111, Lactobacillus paracasei Jaca 1CM, Lactobacillus paracasei Jaca 1CM, Lactobacillus paracasei JCM 1133 JCM 1556, Lactobacillus paracasei JCM 2769, Lactobacillus paracasei JCM 2770, Lactobacillus paracasei JCM 8130, Lactobacillus・ Paracasei JCM 8132, Lactobacillus paracasei JCM 8133, Lactobacillus rhamnosus IFO 3532, Lactobacillus rhamnosus IFO 3863, Lactobacillus rhamnosus IFO 14710, Lactobacillus rhamnosus JCM 153, Lactobacillus rhamnosus JCM 1165, Lactobacillus rhamnosus JCM 1165, Lactobacillus rhamnosus JCM 1165, JCM 1561, Lactobacillus rhamnosus JCM 1563, Lactobacillus rhamnosus JCM 2771, Lactobacillus rhamnosus JCM 2772, Lactobacillus rhamnosus JCM 8134, Lactococcus cremoris ( Lactococcus lactis subs)
p. cremoris H-61), Lactococcus cremoris IFO 3427, and Streptococcus thermophilus IAM 10064.