Method for producing and purifying γ-dodecalactone

Abstract

This invention provides a method for efficiently producing γ-dodecalactone shown in formula (1) below, using a two-step method employing different microorganisms, and a method for purifying the obtained product. [Solution] A composition containing γ-dodecalactone, shown in formula (1) below, is obtained by first culturing lactic acid bacteria obtained from camellia petals using saponified camellia oil as a raw material, and then treating the resulting primary culture with yeast obtained from camellia petals. Subsequently, the obtained product is purified by adsorption onto a solid phase to obtain a composition containing γ-dodecalactone with less malodorous odor. [Formula 1] JPEG2026062594000007.jpg2194

Description

[Technical Field]

[0001] The present invention relates to a method for producing highly purified, odorless γ-dodecalactone without using chemically synthesized starting materials. More specifically, the present invention relates to a method for producing γ-dodecalactone by fermentation using lactic acid bacteria and yeast derived from camellia petals, with saponified camellia oil as the raw material. [Background technology]

[0002] Lactones are cyclic esters produced by the dehydration condensation of a hydroxyl group (-OH) and a carboxyl group (-COOH) within the same molecule. They are named according to the number of carbon atoms in the heterocycle, using Greek letters such as α (2 carbon atoms), β (3 carbon atoms), γ (4 carbon atoms), and δ (5 carbon atoms), along with the name of the carboxylic acid used as a precursor. Structurally, γ and δ lactones are stable and are found in fruits and dairy products, respectively, where they are known to be the aroma components. Because lactones have a pleasant scent to humans, they are used industrially as fragrances in foods, beverages, and perfumes.

[0003] For example, γ-dodecalactone is found in natural products such as fruits and has a peach-like aroma, so it has been conventionally used as a compounding agent in fragrance compositions. Furthermore, γ-dodecalactone is known to be usable as an attractant or repellent for insects, a deodorant, and an intermediate for pharmaceuticals, etc. (Patent Document 1).

[0004] Furthermore, meadowfoam-δ-lactone, contained in meadowfoam oil obtained from the seeds of meadowfoam, an annual plant native to America, is known to protect the hair cuticle when incorporated into shampoos and conditioners (Non-Patent Literature 1). Figure 3 shows the infrared absorption spectrum (hereinafter sometimes referred to as "IR spectrum") of commercially available meadowlactone VE.

[0005] Furthermore, γ-docosalactone, a cyclic ester derived from erucic acid, is known to rehydrophobize the surface of hair by binding with heat from hair dryers and curling irons, thereby imparting smoothness, slipperiness, shine, and luster to the hair surface, as well as having an excellent effect in improving firmness and elasticity (Patent Document 2). For this reason, γ-docosalactone is widely used mainly in shampoos, conditioners, treatments, hair packs, hair color treatments, and also in leave-in treatments.

[0006] As described above, lactones have various effects and are used in a variety of products. However, because lactones are present in only trace amounts in plants and other natural products, it is difficult to concentrate and isolate lactones alone. For this reason, semi-synthetic methods using chemically synthesized raw materials and synthetic methods using microbial fermentation have been attempted. For example, it is known that ricinoleic acid in castor oil is broken down into γ-hydroxydecanoic acid by the action of microorganisms with β-oxidizing ability (Non-Patent Literature 2), and that γ-hydroxydecanoic acid produces γ-decalactone through a dehydration reaction.

[0007] Furthermore, it is known that by hydrolyzing castor oil with microorganisms, a mixture mainly composed of ricinoleic acid can be produced, and by further reacting the ricinoleic acid in this mixture with microorganisms having β-oxidizing ability, γ-hydroxydecanoic acid can be produced. Subsequently, by esterifying this γ-hydroxydecanoic acid, the cyclic ester γ-decalactone can be obtained (Patent Document 3).

[0008] Furthermore, a method for producing γ-dodecalactone by reacting 10-hydroxystearic acid with microorganisms having β-oxidizing ability is also known (Patent Document 4, hereinafter referred to as "Prior Art 1"). Moreover, a method for obtaining γ-dodecalactone by reacting oleic acid with lactic acid bacteria to obtain a fermentation product containing 10-hydroxystearic acid, and then reacting this fermentation product with yeast is also known (Patent Document 5, hereinafter referred to as "Prior Art 2"). [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Meadowfoam-δ-lactone ingredient explanation, safety, and role | Hair dye and color treatment comprehensive site Recolor (recolor.jp) [Non-Patent Document 2] Okui et al., J. Biochem., 54, 1963. [Patent Documents]

[0010] [Patent Document 1] Special Publication No. 2015-515979 [Patent Document 2] Japanese Patent Publication No. 2017-025004 [Patent Document 3] Japanese Patent Application Publication No. 59-82090 [Patent Document 4] Japanese Patent Application Publication No. 3-198787 [Patent Document 5] Japanese Patent Application Publication No. 07-274986 [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] As described above, various methods have been attempted to produce lactones, but several problems remain to be solved. For example, because γ-dodecalactone contains an asymmetric carbon, there is a problem in that yields cannot be obtained through chemical synthesis (Japanese Patent Publication No. 05-024912). On the one hand, the prior art 1 using microorganisms is an excellent invention in that γ-dodecalactone can be obtained by fermentation using 10-hydroxystearic acid obtained by artificially chemically synthesizing based on oleic acid as a raw material. Also, the prior art 2 is an excellent invention in that γ-dodecalactone can be obtained by making the entire process a fermentation process using microorganisms.

[0012] However, in the production methods of the prior arts 1 and 2, since nutrients for microorganism culture such as yeast extract and peptone are added to the medium, when the culture is obtained, there are problems that the peculiar off-odor derived from these nutrients is strong and the oleic acid used as a raw material remains. These problems can be solved by making the pH of the obtained culture alkaline and performing hexane extraction.

[0013] However, when trying to blend γ-dodecalactone into cosmetics, it is necessary to consider the quality and safety as cosmetic raw materials. That is, since two processes with different natures, namely the chemical synthesis process of 10-hydroxystearic acid and the subsequent fermentation process of converting 10-hydroxystearic acid into γ-dodecalactone by microorganisms, are required, the manufacturing system becomes complicated. Also, it is not easy to perform quality control in each of these processes with different natures.

[0014] In addition, using hexane to extract γ-dodecalactone from the culture obtained in the above fermentation process has risks in operation for workers because the flash point of hexane is as low as -22°C and the boiling point is 69°C, and there are also problems of chronic toxicity to humans. To avoid such risks, using ethanol or other highly polar solvents for extraction will extract not only the target substance γ-dodecalactone but also substances causing the above-mentioned off-odor, and there is a problem that it is not suitable as a blending agent for cosmetics. Currently, due to the increasing awareness of beauty and other factors, the demand for γ-dodecalactone as a cosmetic raw material is rapidly growing in addition to food flavors. Therefore, there has been a strong social demand for a manufacturing method of γ-dodecalactone that has good production efficiency, the final purified product has no off-odor, and is highly safe for workers.

Means for Solving the Problems

[0015] Under such circumstances, the inventors of the present invention have conducted intensive research and completed a manufacturing method of γ-dodecalactone that has high production efficiency, the final purified product has no off-odor, and does not use non-polar solvents such as hexane, so it is highly safe for workers.

[0016] The first aspect of the present invention is a method for producing γ-dodecalactone represented by the following formula (1) using a composition containing a saponified product of natural materials and a plurality of microorganisms, and the method for producing γ-dodecalactone includes a first step and a second step; the first step is a primary culture step using the composition and lactic acid bacteria derived from camellia petals; the second step is a secondary culture step using only the culture obtained in the first step and yeast derived from camellia petals. Using the lactic acid bacteria in the first step is preferable in that a final culture with a high content of γ-dodecalactone can be obtained.

[0017]

Chem.

[0018] The composition is preferably obtained by mixing a saponified product obtained from camellia oil as a material and a surfactant selected from the group consisting of Tween 80, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan oleate, and polyglyceryl-10 oleate. Further, the mixing ratio of the saponified product of camellia oil and the surfactant in the composition is preferably 5:1 to 15:1 (weight ratio) in a buffer solution because it does not suppress the growth of lactic acid bacteria.

[0019] The lactic acid bacteria derived from camellia petals is preferably at least one bacterium selected from the group consisting of NITE P-03292 and NITE P-03920, and the yeast derived from camellia petals is preferably NITE P-01807. The ratio of the weight (g) of the saponified product, the amount (number) of lactic acid bacteria, and the amount (number) of yeast is (1) 1:2 x 10 9 ~2 x 10 10 :3.2 x 10 8 ~1 x 10 11 , or (2) 1:2 x 10 7 ~2 x 10 10 :1 x 10 9 ~1 x 10 11 It is preferable that this is the case. Furthermore, it is preferable that the amount of yeast added in the second step is 1 to 10% by weight of the primary culture obtained in the first step, and 10 to 1,000 times (w / w) the amount of lactic acid bacteria added in the first step. Here, it is preferable that the culture in the second step uses only the primary culture obtained in the first step and the camellia petal-derived yeast mentioned above, as this can suppress the generation of off-odors.

[0020] A second aspect of the present invention is a method for purifying γ-dodecalactone, wherein a secondary culture obtained by the above-described method for producing γ-dodecalactone is purified using a solid phase having hydrophobic interactions and an elution solvent that is a lower alcohol. Here, the solid phase is preferably one selected from the group consisting of styrene-divinylbenzene synthetic resins and alkyl-modified silica, and the elution solvent is preferably one selected from the group consisting of ethanol, acetone, and propanol. [Effects of the Invention]

[0021] The present invention provides a method for producing γ-dodecalactone, which uses only the primary culture obtained in the first step and the camellia petal-derived yeast in the second step. The production method of the present invention is efficient because it does not require the addition of nutrients that require sterilization in the second step. The present invention also provides a production method that is safer for workers because hexane is not used when purifying the secondary culture obtained in the second step, while also providing a final purified product with a small amount of impurities and no off-odor. [Brief explanation of the drawing]

[0022] [Figure 1] Figure 1 shows the IR spectral data of commercially available γ-dodecalactone. [Figure 2] Figure 2 shows the IR spectral data of a hexane extract of lactone produced by the method of the present invention. [Figure 3] Figure 3 shows the IR spectral data of a commercially available meadowlactone. [Figure 4] Figure 4 shows the IR spectral data of commercially available oleic acid.

[0023] [Figure 5] Figure 5 shows the IR spectral data of commercially available δ-dodecalactone. [Figure 6] Figure 6 shows the ion chromatogram of the secondary product prepared by the method of the present invention. In the figure, numbers 1 to 7 represent the following compounds: 1 phenylethyl alcohol, 2 γ-nonanolactone, 3 γ-decalactone, 4 γ-undecalactone, 5 γ-dodecalactone, 6 4-hydroxy-6-dodecenoic acid lactone, and 7 γ-tetradecalactone. [Figure 7] Figure 7 shows the ion chromatogram of the final purified product produced by the method of the present invention. The numbers 1 to 7 in the figure are the same as in Figure 6. [Modes for carrying out the invention]

[0024] An embodiment of the present invention will be described below. In the following description, the same or equivalent elements will be denoted by the same reference numerals, and redundant descriptions will be omitted.

[0025] The present invention provides a biological method for producing γ-dodecalactone, comprising first and second steps, using a composition containing a saponified product obtained by processing natural materials as a raw material and employing two different types of microorganisms.

[0026] The saponified product contained in the above composition refers to an oil obtained from a plant of the Camellia genus, which is a natural material, which is saponified using an alkaline solution such as sodium hydroxide or potassium hydroxide according to a conventional method. The type of Camellia genus plant used is not particularly limited, as long as it yields an oil containing oleic acid.

[0027] Examples of the above-mentioned Camellia species include Camellia japonica, Camellia rusticana Honda, Camellia lutchuensis, and Camellia japonica L. var. macrocarpa. Among these, the camellias growing in the Goto Islands and Oshima Island in Japan are preferred in terms of the quality of the oil obtained. The saponified product contained in the above composition may be obtained by saponifying oil from the seeds of the above-mentioned Camellia species according to the method described above, or a soap or other commercially available product made from camellia oil, such as Camellia Soap manufactured by Goto Camellia Co., Ltd., may be used.

[0028] First, let's explain the lactic acid bacteria derived from camellia petals used in the first step. The lactic acid bacteria derived from camellia petals used here can be obtained as follows. Separate only the petals from the camellia flowers collected from the camellia tree and place them in a sterilized test tube containing a sterilized separation medium. Examples of the separation medium include M17 broth, MRS broth, etc. However, since it can suppress the growth of bacteria other than lactic acid bacteria, it is preferable to use MRS broth containing ammonium acetate and sodium citrate. Adding sorbic acid to adjust the pH to about 5.7 can further suppress the growth of bacteria other than lactic acid bacteria.

[0029] Next, perform enrichment culture using the large test tube containing the above separation medium and camellia petals. For enrichment culture, for example, put the above suspension stock solution into a pre-sterilized and cooled MRS medium or GYP medium (hereinafter collectively referred to as "enrichment medium"), and statically culture at about 30 °C for about 24 hours to obtain a culture stock solution. Dilute the obtained culture stock solution with sterilized water to a desired concentration, for example, about 10 times, and repeat this operation to prepare dilution solutions up to 10 5 times dilution. Then, put each of these dilution solutions into a petri dish. After the dilution solution solidifies, pour sterilized agar water over it to cover the surface of the solidified dilution solution. Culture these petri dishes in an incubator at about 36 °C until colonies are clearly formed. Here, using an agar medium containing ammonium acetate and sodium citrate can suppress the growth of bacteria other than lactic acid bacteria. Pick up the colonies formed on the above agar medium and transfer them to a petri dish containing another new agar medium for culture, and confirm the characteristics of the emerged colonies as follows. When no bubbles are generated when hydrogen peroxide is dropped onto the colonies emerged as described above, and the bacteria taken from this colony show Gram-positive, the bacteria forming the colony can be determined as lactic acid bacteria.

[0030] The lactic acid bacteria used in the first step are not limited to those derived from camellia petals, as long as they can produce lactones, but it is preferable that they belong to the genera Lactobacillus, Leuconostoc, or Pediococcus. Among these, it is preferable that they belong to the genus Leuconostoc, and even more preferable that they be Leuconostoc mesenteroides. An example of such lactic acid bacteria is NITE accession number P-03920. Using such lactic acid bacteria makes it possible to obtain a primary culture that can produce more of the target lactone when yeast is used in the second step, as described later. Furthermore, while a single species of lactic acid bacteria may be used, multiple species of lactic acid bacteria may be mixed as appropriate.

[0031] The primary culture in the first step is a step of converting at least the oleates contained in the above-described composition to 10-hydroxystearic acid, and this conversion is carried out by the lactic acid bacteria described above. The lactic acid bacteria consume the oleates contained in the culture medium used here during cultivation and produce 10-hydroxystearic acid. Furthermore, this production reaction is not necessarily carried out in a single step. Note that the composition used for the primary culture may also contain compounds other than oleates, such as linoleic acid, stearic acid, or palmitic acid, so their metabolites may also be produced during the primary culture. The IR spectrum of oleic acid is shown in Figure 4.

[0032] Primary culture can be carried out according to conventional methods and is not particularly limited as long as the conditions are suitable for the lactic acid bacteria used. For example, primary culture of lactic acid bacteria can be cultured in a buffer containing oleic acid, oleates, and / or surfactants. Examples of such buffers include phosphate buffer (pH 6.0-8.0), HEPES buffer, and Tris buffer. Examples of surfactants include nonionic surfactants, particularly Tween® 20, Tween 40, Tween 60, and Tween 80. Adding such surfactants has the effect of promoting the formation of thick-walled spores of yeast-like fungi (e.g., fungi belonging to the genus Candida), uniform dispersion in the culture medium, promotion of fatty acid supply, and promotion of nutrient uptake, thereby improving the yield of the target product, γ-dodecalactone.

[0033] Adding a surfactant to the liquid culture medium described above promotes the dispersion of lactic acid bacteria and oleates in the medium; however, using oleates allows for a reduction in the amount of surfactant added. Furthermore, in the purification process of the secondary culture described later, oleic acid can be removed by adsorption onto the solid phase, thus obtaining a good composition that does not contain oleic acid. For this reason, it is preferable to use a composition containing oleates rather than oleic acid as a raw material for production. As the oleate used here, sodium oleate is preferred, for example.

[0034] The ratio (by weight) of the saponified product, surfactant, and lactic acid bacteria (inoculation amount) in the composition used for the primary culture is preferably 1-10:0.1-1:0.05-1. Needless to say, to avoid contamination by bacteria other than lactic acid bacteria, the culture medium used for the primary culture should be sterilized before adding the lactic acid bacteria. Adding the saponified camellia oil mentioned above slightly increases the pH, but the pH of the culture medium used for the primary culture is preferably about 6-7. Note that there is no need to add nutrients during the primary culture.

[0035] The primary culture is generally carried out under anaerobic conditions. The culture temperature is preferably around 20-40°C, and more preferably 25-35°C. The culture period can be set appropriately according to the composition of the culture medium and the temperature conditions, and is often 24-96 hours. The primary culture may be carried out by static culture, or by gently stirring the culture medium to raise the settling lactic acid bacteria. This primary culture can be completed by sterilization, for example, by autoclaving. Note that even if the primary culture obtained in the primary culture is used as a raw material for the secondary culture, which is the second step, without sterilization, it will not have any particular effect on the completion of the target product.

[0036] In the second step, yeast derived from camellia petals is added to the primary culture obtained in the first step and cultured to produce the target product, γ-dodecalactone, represented by the following formula (I). The IR spectrum of commercially available γ-dodecalactone is shown in Figure 1.

[0037] [ka]

[0038] The yeast derived from camellia petals used in the second step described above can be obtained as follows. The petals are separated from camellia flowers collected from a camellia tree and placed in a large, sterile test tube containing a sterile isolation medium. For yeast isolation, for example, koji extract or YMA medium can be used. When using koji extract as an isolation medium, first, place the desired weight of rice koji in a cloth bag, add 4 to 5 times the weight of the rice koji in water, and maintain at a predetermined temperature for a predetermined time, for example, at approximately 50 to 63°C for approximately 4 to 6 hours. Next, take a small amount of liquid from the bag, check the starch reaction using an iodine-potassium iodide solution, wait for the starch reaction to disappear, then lift the bag and squeeze lightly, and boil the extracted liquid. If the extracted liquid is not clear, add egg white, stir well, squeeze lightly, and boil this extracted liquid. After boiling, filter all boiled liquids using, for example, an Advantec membrane filter.

[0039] Next, water is added using a sugar analyzer to adjust the Balling reading to a predetermined value, for example, around 10, to obtain the koji extract. Here, the Balling reading represents the number of grams of sucrose contained in 100 g of sucrose aqueous solution at 17.5°C. Note that the Balling reading is sometimes abbreviated as Ballg. reading. Finally, it is preferable to add an antibiotic, such as chloramphenicol, to the culture medium to prevent bacterial contamination.

[0040] Next, the koji extract (isolated medium) prepared as described above and the camellia petals are placed in a large test tube and cultured at approximately 30°C for 2 to 5 days. After the culture, the koji extract is diluted as appropriate and spread onto an agar plate in a petri dish. For example, the agar plate used here is YMA agar plate (NBRC medium No. 108), which is then cultured at a predetermined temperature for a predetermined period, for example, approximately 3 days at approximately 30°C, to allow colonies to appear. From the resulting colonies, those with different morphologies are selected and spread onto a new agar plate to grow, thereby obtaining yeast as single colonies. The yeast thus isolated can then be used in the second step of secondary culture.

[0041] In the second step described above, it is preferable to use yeast belonging to the genera Saccharomyces, Pichia, Hansenula, or Candida as the camellia petal-derived yeast, because it is possible to produce a large amount of the target lactone from the primary culture. The target lactone here is γ-dodecalactone, represented by formula (I) above.

[0042] In the secondary culture performed in the second step described above, it is preferable to use yeast in an amount of approximately 100 to 1,000 times the amount of lactic acid bacteria used in the primary culture, and more preferably 300 to 800 times the amount by wet weight. In this secondary culture, yeast extract, polypeptone, and other nitrogen-producing nutrients may be added to the primary culture as appropriate. In that case, it goes without saying that these nutrients should be sterilized before adding the yeast to be used for the secondary culture. It should be noted that lactones will be produced even if the above nutrients are added to the culture medium used for the secondary culture, i.e., the primary culture. On the other hand, when a large amount of yeast is added, the amount of lactone produced will be higher if nutrients are not added. Therefore, the addition of nutrients in the secondary culture should be appropriately determined in relation to the amount of yeast used. It should be noted that if the above nutrients are not added at the start of the secondary culture, sterilization of the primary culture is unnecessary, and the yeast can be added immediately after the end of the primary culture to start the secondary culture, thereby improving production efficiency.

[0043] The secondary culture using the above yeast is preferably carried out under aerobic conditions, unlike the primary culture. The culture temperature is preferably about 20 to 40°C, and more preferably about 25 to 35°C. The culture period can be set appropriately depending on the composition of the culture medium and the temperature conditions, but for example, it is generally about 24 to 72 hours. The culture may be carried out by standing or by slow shaking. This secondary culture can be terminated, for example, by performing a sterilization procedure.

[0044] To recover γ-dodecalactone from the secondary culture obtained as described above, methods for isolating substances from conventional microbial cultures can be applied. For example, after separating the microbial cells from the culture medium by centrifugation or filtration, the target substance, γ-dodecalactone, can be extracted from the supernatant or filtrate. This extraction can be performed by solution partitioning, chromatography utilizing differences in affinity to various solid phases, etc. γ-dodecalactone is practically insoluble in water and has a relatively high boiling point. Therefore, it can also be obtained by solvent extraction followed by evaporation, and the crude product obtained in this way can be further purified using chromatography or other separation methods. These purification methods can be used in appropriate combinations as needed.

[0045] The present invention employs a purification method that does not use hexane, an organic solvent. First, the secondary culture obtained in the secondary culture is adsorbed onto a suitable solid phase. Examples of such solid phases include styrene-divinylbenzene synthetic resins and alkyl-modified silica. Examples of the styrene-divinylbenzene synthetic resins include resins suitable for ion chromatography, such as Diaion HP-20 and Diaion HP-21, which have hydrophobic interactions. Examples of the alkyl-modified silica include octadecyl silica (hereinafter sometimes abbreviated as "ODS") having a C18 alkyl chain. γ-dodecalactone is adsorbed onto this solid phase, and then a polar solvent such as ethanol, acetone, and propanol is used as an eluent to obtain a fraction containing γ-dodecalactone. The solvent of the above fraction is then removed by distillation under reduced pressure to obtain a composition containing γ-dodecalactone without unpleasant odor. [Examples]

[0046] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited thereto.

[0047] (Comparative Example 1) Preparation of lactic acid bacteria and yeast used in the comparative example and production of comparative culture 1 In a 500 mL Erlenmeyer flask, 0.5 g of oleic acid (manufactured by Yoneyama Pharmaceuticals), 50 mg of surfactant (Tween® 80 (manufactured by Yoneyama Pharmaceuticals)), and 200 mL of 0.05 M phosphate buffer (pH 6.8) were added, and the mixture was autoclaved at 121°C for 15 minutes.

[0048] Lactic acid bacteria (Leuconostoc mesenteroides, accession number NITE P-03290 (manufactured by Goto Tsubaki Co., Ltd.)) obtained from camellia petals were inoculated into MRS medium in two platinum loops and pre-cultured for 48 hours in a 30°C incubator. The resulting pre-culture solution was centrifuged at 5969xg at room temperature for 5 minutes, and the supernatant was discarded by decantation. The resulting wet bacterial cells amounted to 0.1g (approximately 1x10⁻⁶) 9 The bacteria (cells / g) were inoculated into 1 mL of phosphate buffer and then kept in an incubator at 30°C for 72 hours. Afterward, the buffer was sterilized by autoclaving at 121°C for 15 minutes. The lactic acid bacteria and yeast used in the following comparative examples are the same as those used in this comparative example.

[0049] To the above buffer solution, 0.6 g of yeast extract (manufactured by Oriental Yeast Co., Ltd.) and 1.2 g of polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.) were added. Then, 0.3 g of frozen yeast (Saccharomyces cereviciae, accession number NITE P-01807, manufactured by Goto Tsubaki Co., Ltd., approximately 1.6 x 10) obtained from camellia petals was added. 8 The culture was obtained by adding (particles / g) to the above buffer solution and culturing at 30°C for 48 hours with shaking at 100 rpm to obtain the culture of Comparative Example 1 (hereinafter referred to as "Comparative Example 1").

[0050] (Comparative Example 2) Production of Comparative Culture 2 The culture for Comparative Example 2 was obtained in the same manner as for Comparative Example 1, except that the oleic acid was replaced with saponified camellia oil (GT-TB100, manufactured by Goto Tsubaki Co., Ltd., mainly containing sodium oleate) and the amount of Tween 80 was changed from 0.1 g to 0.05 g.

[0051] (Comparative Example 3) Production of Comparative Culture 3 The culture for Comparative Example 3 (Comparative Example 3) was obtained in the same manner as in Comparative Example 2, except that the wet weight of the lactic acid bacteria used in the primary culture was changed from 0.1 g to 0.01 g.

[0052] (Example 1) Preparation of lactic acid bacteria and yeast used in the example (1) Preculture In a 500 mL Erlenmeyer flask, 200 mL of pH 6.8 phosphate buffer was added to contain 0.5 g of saponified camellia oil, mainly sodium oleate, and 50 mg of Tween 80. This buffer was maintained at 121°C for 15 minutes to sterilize. Next, 0.1 g (approximately 1 x 10⁻¹⁶) of wet cells of lactic acid bacteria (Leuconostoc mesenteroides, accession number NITE P-03290) (Goto Camellia Co., Ltd.) obtained from camellia petals was added. 9 The solution was inoculated with (1.6 x 10¹ / g) and kept at 30°C for 72 hours. This buffer solution was sterilized by keeping it at 121°C for 15 minutes. Then, 0.3g (approximately 1.6 x 10¹) of frozen yeast (Saccharomyces cerevisiae, accession number NITE P-01807) (Goto Tsubaki Co.) obtained from camellia petals was added. 8 The lactic acid bacteria (particles / g) were added to the buffer solution and cultured at 30°C for 48 hours while shaking at 100 rpm. The lactic acid bacteria and yeast used in the following examples are the same as those used in this example.

[0053] (2) Production of culture 1 by this culture 200 mL of pH 6.8 phosphate buffer was added to a 500 mL Erlenmeyer flask to contain 0.5 g of camellia oil saponified mainly of sodium oleate and 50 mg of Tween 80. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 0.01 g of wet cells of lactic acid bacteria (Leuconostoc mesenteroides) obtained from camellia petals (Goto Tsubaki Co., Ltd.) were inoculated into the buffer and kept at 30°C for 72 hours. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 0.3 g of frozen yeast (Saccharomyces cerevisiae, Goto Tsubaki Co., Ltd., the same number as in Comparative Example 1) obtained from camellia petals was added to the buffer and cultured at 30°C for 48 hours with shaking at 100 rpm to obtain Culture 1.

[0054] (Example 2) Production of Culture 2 0.5 g of saponified camellia oil, mainly sodium oleate, and 200 mL of pH 6.8 phosphate buffer were added to a 500 mL Erlenmeyer flask to a concentration of 50 mg of Tween 80. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 0.1 g of wet cells of lactic acid bacteria (Leuconostoc mesenteroides) (Goto Tsubaki Co.) obtained from camellia petals were inoculated and kept at 30°C for 72 hours. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 3 g of frozen yeast (Saccharomyces cerevisiae) (Goto Tsubaki Co.) obtained from camellia petals was added to the buffer and cultured at 30°C for 48 hours with shaking at 100 rpm to obtain culture 2.

[0055] (Example 3) Production of Culture 3 0.5 g of saponified camellia oil, mainly sodium oleate, and 200 mL of pH 6.8 phosphate buffer were added to a 500 mL Erlenmeyer flask to a concentration of 50 mg of Tween 80. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 0.01 g of wet cells of lactic acid bacteria (Leuconostoc mesenteroides, Goto Tsubaki Co.) obtained from camellia petals were inoculated and kept at 30°C for 72 hours. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 1 g of frozen yeast (Saccharomyces cerevisiae, Goto Tsubaki Co.) obtained from camellia petals was added to the buffer and cultured at 30°C for 48 hours with shaking at 100 rpm to obtain culture 3.

[0056] (Example 4) Production of Culture 4 0.5 g of saponified camellia oil, mainly sodium oleate, and 200 mL of pH 6.8 phosphate buffer were added to a 500 mL Erlenmeyer flask to a concentration of 50 mg of Tween 80. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 0.01 g of wet cells of lactic acid bacteria (Leuconostoc mesenteroides, Goto Tsubaki Co.) obtained from camellia petals were inoculated and kept at 30°C for 72 hours. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 3 g of frozen yeast (Saccharomyces cerevisiae, Goto Tsubaki Co.) obtained from camellia petals was added to the buffer and cultured at 30°C for 48 hours with shaking at 100 rpm to obtain culture 4.

[0057] (Example 5) Production of Culture 5 0.5 g of saponified camellia oil, mainly sodium oleate, and 200 mL of pH 6.8 phosphate buffer were added to a 500 mL Erlenmeyer flask to a concentration of 50 mg of Tween 80. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 0.01 g of wet cells of lactic acid bacteria (Leuconostoc mesenteroides, Goto Tsubaki Co.) obtained from camellia petals were inoculated and kept at 30°C for 72 hours. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 6 g of frozen yeast (Saccharomyces cerevisiae, Goto Tsubaki Co.) obtained from camellia petals was added to the buffer and cultured at 30°C for 48 hours with shaking at 100 rpm to obtain culture 5.

[0058] (Example 6) Production of Culture 6 0.5 g of saponified camellia oil, mainly sodium oleate, and 200 mL of pH 6.8 phosphate buffer were added to a 500 mL Erlenmeyer flask to a concentration of 50 mg of Tween 80. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 0.1 g of wet cells of lactic acid bacteria (Leuconostoc mesenteroides, Goto Tsubaki Co.) obtained from camellia petals were inoculated and kept at 30°C for 72 hours. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 6 g of frozen yeast (Saccharomyces cerevisiae, Goto Tsubaki Co.) obtained from camellia petals was added to the buffer and cultured at 30°C for 48 hours with shaking at 100 rpm to obtain culture 6.

[0059] (Example 7) Production of Culture 7 0.5 g of saponified camellia oil, mainly sodium oleate, and 200 mL of pH 6.8 phosphate buffer were added to a 500 mL Erlenmeyer flask to a concentration of 50 mg of Tween 80. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 0.01 g of wet cells of lactic acid bacteria (Leuconostoc mesenteroides, Goto Tsubaki Co.) obtained from camellia petals were inoculated and kept at 30°C for 72 hours. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 10 g of frozen yeast (Saccharomyces cerevisiae, Goto Tsubaki Co.) obtained from camellia petals was added to the buffer and cultured at 30°C for 48 hours with shaking at 100 rpm to obtain culture 7.

[0060] (Example 8) Production of Culture 8 0.5 g of saponified camellia oil, mainly sodium oleate, and 200 mL of pH 6.8 phosphate buffer were added to a 500 mL Erlenmeyer flask to a concentration of 50 mg of Tween 80. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 0.1 g of wet cells of lactic acid bacteria (Leuconostoc mesenteroides, Goto Tsubaki Co.) obtained from camellia petals were inoculated and kept at 30°C for 72 hours. This buffer was kept at 121°C for 15 minutes to sterilize it. Next, 10 g of frozen yeast (Saccharomyces cerevisiae, Goto Tsubaki Co.) obtained from camellia petals was added to the buffer and cultured at 30°C for 48 hours with shaking at 100 rpm to obtain culture 8.

[0061] (Example 9) Measurement of γ-dodecalactone concentration in culture medium The concentration of γ-dodecalactone in the cultures obtained in Comparative Examples 1-3 and Examples 1-8 was measured. 30 mL of hexane was added to the culture medium and stirred. Liquid-liquid extraction was performed using a separatory funnel, and the hexane layer was transferred to a separate container. The same procedure was repeated two more times, and the resulting hexane layers were combined. Hexane was removed by distillation under reduced pressure using a water bath at 40°C for 5-10 minutes. The residue was dissolved in 6 mL of hexane, 0.3 mL of this solution was taken, and 0.7 mL of hexane was added to prepare the sample solution for gas chromatography. For the γ-dodecalactone standard, hexane solutions of γ-dodecalactone (manufactured by Tokyo Chemical Industry Co., Ltd.) with concentrations of 0.1%, 0.05%, 0.025%, and 0.0125% were prepared. 300 μL of each solution was taken into a tube, and 700 μL of hexane was added to each to prepare the samples for calibration curve preparation.

[0062] <Device> A GC-2010plus (manufactured by Shimadzu Corporation) was used for gas chromatography, and the procedure was performed according to the manual under the following conditions. <Gas chromatography conditions> Column: DB-1 (J&W), 15m x 0.25 (id), 0.1 μm (ft) Column temperature: 40°C (3 min), 4°C / min to 170°C, 25°C / min to 325°C (7 min) Injection temperature: 370℃ Detector temperature: 345℃ Injection volume: 1.0μL

[0063] [Table 1]

[0064] As shown in Comparative Examples 1-3 of Table 1, conventional patented methods for producing lactones involve adding nutrients such as yeast extract and polypeptone necessary for yeast growth during secondary culture. In contrast, the method of the present invention was able to produce at least the same amount of lactone even when yeast was added and cultured without adding nutrients during secondary culture. The amount of lactone produced in this case depended on the amount of yeast added. Furthermore, it was confirmed that when the amount of yeast used in the primary culture was 50 times or more by weight compared to the amount of lactic acid bacteria used, the amount of lactone produced was greater than when no nutrients were added. In particular, when the ratio of saponified product (g), amount of lactic acid bacteria (cells), and amount of yeast (cells) was 1:2 x 10⁻⁶ 9 ~2 x 10 10 :3.2 x 10 8 ~1 x 10 11 , or 1:2 x 10 7 ~2 x 10 10 :1 x 10 9 ~1 x 10 11 At that time, lactone production was increasing.

[0065] (Example 10) Analysis of components contained in the culture Of the above cultures, GCL-1 (culture 1 cultured using the lactic acid bacteria of the present invention), GCL-2 (culture 2 cultured using the lactic acid bacteria 2 of the present invention), and KGC (control culture using a related lactic acid bacteria provided by the Food Analysis Center) were used as measurement samples, and gas chromatography-mass spectrometry was requested from the Tokyo Metropolitan Industrial Technology Center.

[0066] 500 μL of each sample was collected in a vial (20 mL capacity), and the volatile components generated were measured. The measurement conditions were as follows. <Device> Pre-treatment device: AOC-6000 (manufactured by Shimadzu Corporation) Gas chromatograph-mass spectrometer: GCMS-TQ8050 (manufactured by Shimadzu Corporation) <Pretreatment device conditions> Volatile component collection method: SPME Arrow (DVB / PDMS, φ 1,1 mm) Sample heating temperature (time): 60°C (10 minutes) Sample extraction time: 10 minutes

[0067] <Gas chromatograph conditions> Carrier gas: Helium Column pressure: 83.5 kPa (constant pressure) Column: InertCap Pure-WAX (Inner diameter 0.25 mm, length 30 m, film thickness 0.25 μm) Split ratio: 5:1 Column oven temperature: Initial temperature 50°C, held for 5 minutes, heating rate 10°C / min Final temperature 250℃ held for 10 minutes

[0068] <Mass spectrometry conditions> Ionization method: Electron ionization method Interface concentration: 230℃ Ion source temperature: 230℃ Scan range: m / z 30-500 A GC-2010plus (manufactured by Shimadzu Corporation) was used for gas chromatography, and the procedure was performed according to the manual under the following conditions.

[0069] <Gas chromatography conditions> Column: DB-1 (J&W), 15m x 0.25 (id), 0.1 μm (ft) Column temperature: 40°C (3 min), 4°C / min to 170°C, 25°C / min to 325°C (7 min) Injection temperature: 370℃ Detector temperature: 345℃ Injection volume: 1.0μL

[0070] The results of gas chromatography-mass spectrometry are shown in Figures 6 and 7. Furthermore, the infrared absorption spectra of γ-dodecalactone, lactones obtained from meadowfoam oil, oleic acid, δ-dodecalactone, and lactones obtained as hexane extracts from camellia oil, all commercially available reagents, were measured under the following conditions. The results are shown in Figures 1-5. This analysis demonstrated that the constituent fatty acids of meadowfoam oil are similar to eicosenoic acid (C20:1) and oleic acid (C18:1).

[0071] <Sample Preparation> The test sample was dissolved in ethanol (99.5%), 500 μL was taken into a vial (20 mL), and the volatile components generated were measured. <Device> Pre-treatment device: AOC-6000 (manufactured by Shimadzu Corporation) Gas chromatograph-mass spectrometer: GC-MS-TQ8050 (manufactured by Shimadzu Corporation)

[0072] <Pretreatment conditions> Volatile component collection method: SPME Arrow (DVB / PDMS, φ1.1mm) Sample heating temperature and time: 60°C (10 minutes) Sample extraction temperature: 10 minutes <Gas chromatograph conditions> Carrier gas: Helium Column pressure: 83.5 kPa, Column: InertCap Pure-WAX (inner diameter 0.25 mm, length 30 m, film thickness 0.25 μm) Split ratio: 5:1 Column oven temperature: Initial temperature 50°C, held for 5 minutes; heating rate 10°C / min; final temperature 250°C, held for 10 minutes.

[0073] <Mass spectrometry conditions> Ionization method: Electron ionization method Interface temperature: 230℃ Ion current temperature: 230℃ Scan range: m / z 30-500

[0074] (Comparative Example 4) Preparation of comparative purified product 1 by purification of comparative product 1 Comparative example culture 1 was centrifuged (5960xg, 5 minutes), and the supernatant was filtered. 30 mL of hexane was added to 185 g of the resulting filtrate, and liquid-liquid extraction was performed to obtain the hexane layer. The same procedure was repeated two more times, the hexane layer was collected, and the solvent was removed by distillation under reduced pressure using a water bath at 40°C to obtain comparative purified product 1.

[0075] (Comparative Example 5) Preparation of comparative purified product 2 by purification of comparative product 2 Comparative culture 2 was centrifuged (5960xg, 5 minutes), and the supernatant was filtered. 30 mL of hexane was added to 185 g of the resulting filtrate, and liquid-liquid extraction was performed to obtain the hexane layer. The same procedure was repeated two more times, and the hexane layer was collected. The solvent was removed by distillation under reduced pressure using a water bath at 40°C to obtain comparative purified product 2.

[0076] (Comparative Example 6) Preparation of comparative purified product 3 by purification of comparative product 2 The culture of Comparative Example 2 was centrifuged (5960xg, 5 minutes), and the supernatant was filtered. Water was removed from 185 g of the obtained supernatant under reduced pressure. 30 mL of ethanol was added to the residue, and the dissolved portion was collected by decantation. This procedure was repeated two more times, and the ethanol was removed from the collected ethanol solution by heating it in a 40°C water bath under reduced pressure to obtain Comparative Purified Product 3.

[0077] (Example 10) Preparation of purified product 1 by purification of culture 1 A 185g culture 1 obtained in Example 2 was applied to an HP-20 column (manufactured by Mitsubishi Chemical Corporation, inner diameter 10mm, h=300mm) that had been washed with ethanol and equilibrated with water. The column was washed with 50mL of water, and then the adsorbed material was eluted with 50mL of ethanol. The solvent of the eluate was removed by distillation by heating in a water bath at 40°C under reduced pressure to obtain purified product 1.

[0078] (Example 11) Preparation of purified product 5 by purification of culture 5 The 185 g of culture 5 obtained in Example 5 was subjected to a water bath at 40°C under reduced pressure to remove the water. 30 mL of ethanol was added to the residue, the mixture was thoroughly stirred, filtered, and the solvent was removed from the resulting filtrate by a water bath at 40°C under reduced pressure to obtain purified product 5.

[0079] (Example 12) Preparation of purified product 6 by purification of culture 5 The 185 g culture 5 obtained in Example 5 was applied to an HP-20 column (10 mm inner diameter, 300 mm height) that had been washed with ethanol and equilibrated with water. The column was washed with 50 mL of water, and then eluted with 50 mL of 95% ethanol. The solvent of the eluate was removed by distillation under reduced pressure in a water bath at 40°C to obtain purified product 6.

[0080] (Example 13) Preparation of purified product 7 by purification of culture 5 185 g of culture 5 obtained in Example 5 was applied to an ODS column (manufactured by Mitsubishi Chemical Corporation, Φ10 mm, h=300 mm) that had been washed with ethanol and equilibrated with water. The column was washed with 50 mL of water, and then eluted with 50 mL of 95% ethanol-5% water. The solvent of the eluate was removed by distillation under reduced pressure in a water bath at 40°C to obtain purified product 7. The refined product described above was evaluated for its odor, solubility in camellia oil, and residual oleic acid. The evaluation criteria are shown in Table 2.

[0081] [Table 2]

[0082] [Table 3]

[0083] As shown in Table 3, in Comparative Examples 4 and 5, using a camellia oil saponified product mainly composed of sodium oleate as the raw material for oleic acid tended to result in less oleic acid being present in the hexane layer of hexane extraction. As a method that does not use hexane, in Comparative Example 6, the water of the secondary culture was removed by distillation, the residue was dissolved in ethanol, and the ethanol was removed by distillation under reduced pressure. The resulting solution was undesirable, as it extracted many impurities other than lactones, had a strong culture medium odor, and contained a large amount of oleic acid.

[0084] On the other hand, as shown in Example 10, when using HP-20, a solid phase utilizing hydrophobic interactions, the culture obtained in Comparative Example 2, in which nutrients were added (Comparative Example 2), passed through the column without adsorbing the culture medium components or the raw material oleic acid. Therefore, when the adsorbed substances were eluted with ethanol, a composition containing γ-dodecalactone with a weak culture medium odor could be obtained (see Figure 6). Furthermore, as shown in Example 11, when using a method that does not use nutrients during secondary culture, a composition with a weak culture medium odor could be obtained even with ethanol elution, but it contained oleic acid. Finally, as shown in Examples 12 and 13, by treating the culture medium obtained without using nutrients during secondary culture with a solid phase having hydrophobic interactions and eluting with ethanol, a good γ-dodecalactone-containing composition with almost no culture medium odor and almost no oleic acid could be obtained without using hexane (see Figure 7).

[0085] In this invention, γ-dodecalactone can be produced by culturing lactic acid bacteria and yeast obtained from camellia petals, using saponified camellia oil as a raw material. Furthermore, since both the primary and secondary cultures are microbial cultures, continuous production in the same container is easy. Moreover, in the secondary culture of this invention, the amount of γ-dodecalactone produced is large because no nutrients are used, and sterilization after the primary culture can be omitted, simplifying the manufacturing process. Furthermore, by purification using solid-phase and ethanol extraction with hydrophobic interactions, it is possible to obtain a composition containing a large amount of γ-dodecalactone with little off-odor and low contamination of oleic acid and hexane. The method for producing γ-dodecalactone of this invention can be used to produce compositions that can be used in hair cosmetics for damage care, etc.

[0086] Conventional manufacturing methods do not use hexane as an extraction solvent; instead, the solid phase described above is used, followed by extraction with a highly polar solvent. Using hexane or other low-polarity solvents prevents the extraction of odor components derived from the culture medium and makes it difficult to extract unused saponified products, but it poses health problems for workers.

[0087] In contrast, the method of the present invention selectively adsorbs γ-dodecalactone onto a solid phase such as Diaion HP-20 or other styrene-divinylbenzene resins or alkyl-modified silica such as ODS, thereby separating it from odor components and unused saponified products contained in the secondary culture. Subsequently, by eluting the adsorbed components with a highly polar solvent, a γ-dodecalactone composition with less off-odor from the culture medium can be obtained. Furthermore, extraction is performed using ethanol or other highly polar solvents with the solid phase described above. As a result, the use of organic solvents that are difficult to handle and harmful to the human body, such as hexane, is avoided, making this a highly safe method for workers. [Industrial applicability]

[0088] The present invention's method for producing γ-dodecalactone is useful in the field of cosmetics.

Claims

1. A method for producing γ-dodecalactone represented by the following formula (I) using a composition containing a saponified substance and a plurality of microorganisms, The method for producing γ-dodecalactone comprises a first step and a second step; The first step is a primary culture step using the composition and lactic acid bacteria derived from camellia petals; The method for producing γ-dodecalactone, wherein the second step is a secondary culture step using only the culture obtained in the first step and yeast derived from camellia petals. 【Chemistry 1】

2. The method for producing γ-dodecalactone according to claim 1, characterized in that the composition is obtained by mixing a saponified product obtained from camellia oil as a material with a surfactant selected from the group consisting of Tween 80, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan oleate, and polyglyceryl-10 oleate.

3. The method for producing γ-dodecalactone according to claim 2, characterized in that the mixing ratio of the saponified product and the surfactant in the composition is 5:1 to 15:1 by weight in the buffer solution.

4. The method for producing γ-dodecalactone according to claim 1, characterized in that the lactic acid bacteria derived from camellia petals is at least one bacterium selected from the group consisting of NITE P-03292 and NITE P-03920.

5. The method for producing γ-dodecalactone according to claim 1, characterized in that the yeast derived from camellia petals is NITE P-01807.

6. The ratio of the weight (g) of the saponified product to the amount (number) of lactic acid bacteria and the amount (number) of yeast is 1:2 x 10 9 ~2 x 10 10 : 3.2 x 10 8 ~1 x 10 11 , or 1:2 x 10 7 ~2 x 10 10 : 1 x 10 9 ~1 x 10 11 A method for producing γ-dodecalactone according to claim 1, characterized in that...

7. The method for producing γ-dodecalactone according to claim 1, characterized in that the amount of yeast added in the second step is 1 to 10% by weight of the culture obtained in the first step, and is 10 to 1,000 times (w / w) the amount of lactic acid bacteria added in the first step.

8. A method for purifying γ-dodecalactone, comprising adsorbing a culture obtained by the manufacturing method described in claim 1 onto a solid phase and eluting it with a polar solvent.

9. The purification method according to claim 8, characterized in that the solid phase is selected from the group consisting of styrene-divinylbenzene synthetic resins and alkyl group-modified silica, and the polar elution solvent is a solvent selected from the group consisting of ethanol, acetone, and propanol.

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