JP4764309B2 - Method for producing methyl ketones - Google Patents

Description

  The present invention relates to a method for producing methyl ketones from natural fats and oils by microbial conversion. More specifically, the present invention relates to a method for producing a high yield of methyl ketones that efficiently converts fats and oils to methyl ketones even when using two or more times the weight of fats and oils in a liquid medium.

  Since methyl ketones are key flavors of blue cheese and are useful as flavoring materials for flavors such as dairy products and soaps, methods for producing natural methyl ketones from oils and fats using microorganisms have been studied. It has been. For example, a method of converting a substrate containing 0.4% lauric acid using a microorganism belonging to Penicillium roqueforti (Non-patent Document 1), a slurry containing up to 32% of blue cheese as a substrate. A method of producing using a microorganism belonging to P. roqueforti (Non-patent Document 2), a substrate containing at most 46% lauric acid, belonging to the genus Penicillium / Citrinum or Eurotium A method of producing using a microorganism belonging to the above (Non-patent Document 3), a microorganism belonging to Aspergillus rubber or Aspergillus repens in a substrate containing about 3.2% coconut oil There is known a method of causing the effect to act (Non-patent Document 4). In addition, as a method for improving the production efficiency of methyl ketones in microbial conversion, a method using a microorganism belonging to a specific genus or species (Patent Document 1), using Aureobasidium pullulans as a microorganism, A method for producing a large amount of methyl ketone that has been cultivated while maintaining a substrate concentration that is not harmful to the growth of microorganisms and that can be converted at a maximum fat concentration of 33% in the medium (Patent Document 2), A method for increasing the substrate concentration without inhibiting the growth of microorganisms (maximum oil content of 43% in Examples) (Patent Document 3) and culturing in the presence of divalent alkaline earth metal ions in the medium There has been proposed a method for converting a substrate into methyl ketones even at a high concentration of up to 10% of fats and oils.

J. et al. gen. Microbiol. 51, p. 289-302 (1968) J. et al. Sci. Food Agric. , 30, p. 197-202 (1979) Phytochemistry, 23 (12) p. 2847-2849, (1984) Phytochemistry, 26 (5), p. 1417-1420 (1987) JP-A-2-265495 JP-A-3-76588 JP-A-3-151886 Japanese Patent Laid-Open No. 4-148688

  In the production of methyl ketones by microbial conversion, by increasing the fat content, the growth of microorganisms is hindered by the antibacterial properties of fats and oils and the movement of nutrients, and the production of methyl ketones is suppressed. For this reason, none of the conventional methods described above have sufficient production efficiency, and a more efficient method for producing methyl ketones has been demanded. Accordingly, an object of the present invention is to provide a method for efficiently converting fats and oils to methyl ketones without inhibiting the growth of microorganisms even if the medium contains a high concentration of fats and oils.

  The present inventors have made various studies in view of the above problems. As a result, in the culture in the liquid medium in the pre-culture, after forming the mycelium in the form of pellets so that the average particle diameter is 0.5 to 3 mm, the oil is added to the liquid medium. It was found that when cultured, fats and oils are efficiently decomposed and converted to methyl ketone. Furthermore, surprisingly, in this method, it has been found that the conversion proceeds even when oils and fats are added in an amount twice or more by weight with respect to the liquid medium, and the present invention has been completed.

  Thus, in the present invention, a liquid culture medium was inoculated with a solid culture of a fungus capable of producing methyl ketone, and cultured to form pellet-shaped mycelium having an average particle size of 0.5 to 3 mm. Then, the manufacturing method of methyl ketone characterized by adding 2-4 times amount of fats and oils by weight ratio with respect to a liquid culture medium, and also culture | cultivating is provided.

  The present invention also provides the method for producing methyl ketone, wherein the fungus capable of producing methyl ketone is Aspergillus and / or Penicillium.

  The present invention also provides that the microorganism belonging to the genus Aspergillus is Aspergillus oryzae, Aspergillus tamari, Aspergillus parasites, Aspergillus awamori, Asp. Aspergillus kawachii, Aspergillus niger (Asp. Niger), or Aspergillus pheonicis (Asp. Pheonicis), microorganisms belonging to the genus Penicillium (Penicillium rockforti (Pen. Roqueorium)・ Camemberti, Penicillium casei (Pen.ca) ei) or Penicillium Janshineramu (Pen. janthinellum) production method of the methyl ketone is there is provided a.

  The present invention also provides the above-mentioned method for producing methyl ketone, wherein lipase is further added at the time of adding fat to the liquid medium.

  The present invention also provides the method for producing methyl ketone, wherein the fat is glyceride containing lauric acid, capric acid and caprylic acid as main constituent fatty acids.

Furthermore, the present invention provides the method for producing methyl ketone, wherein the inoculation of the solid culture is 10 ³ to 10 ⁵ conidia / ml with respect to the liquid medium.

  According to the production method of the present invention, a reaction for converting fats and oils to methyl ketones efficiently proceeds even in a medium having a fat content of more than 66%. Therefore, an efficient production method for methyl ketones can be provided. As a result, the yield per batch production is increased, and the production cost can be reduced.

Hereinafter, the present invention will be described in more detail.
The fungi that can be used in the present invention may be any microorganism as long as they can produce methyl ketones. Examples include fungi belonging to the genus Aspergillus and Penicillium. can do. Of these, microorganisms belonging to the genus Aspergillus are Aspergillus oryzae, Aspergillus tamari, Aspergillus parasites, Aspergillus awamori, Asor. Mention may be made of Aspergillus kawachii, Aspergillus niger or Aspergillus pheonicis. Moreover, Penicillium rockforti (Pen.roqueforti), Penicillium camanberti (Pen.casei) or Penicillium jancineram (Pen.janthin) as microorganisms which belong to the genus Penicillium (Penicillium). Can be mentioned. Further, examples of these type cultures include Aspergillus oryzae IAM2152, and Penicillium roqueforti IFO 4622.

  As the fats and oils that can be used as starting materials for methyl ketone in the present invention, fatty acids alone or those containing fats or other esters can be used, but in reality, fatty acid esters of glycerin are preferred because of their availability. . Specific oils that can be used include soybean oil, sesame oil, corn oil, rapeseed oil, rice bran oil, cottonseed oil, castor oil, peanut oil, olive oil, palm oil, palm kernel oil, palm oil, safflower oil, and wheat germ Oil, coconut oil, sunflower oil, camellia oil, cocoa butter, sardine oil, salmon oil, mackerel oil, shark oil, tuna oil, whale oil, dolphin oil, squid oil, saury oil, garlic oil, arabe oil, beef tallow, chicken There may be mentioned animal and vegetable oils and fats such as oil, pork fat and butter. When the produced methyl ketones are used as a fragrance, the fat is preferably a glyceride containing lauric acid, decanoic acid, and octanoic acid as main constituent fatty acids, and among them, coconut oil and palm kernel oil are particularly preferable. Can be mentioned.

  It is preferable to prepare a solid culture as a preliminary step for culturing fungi in a liquid medium. By inoculating the solid culture into the liquid medium, it becomes easy to control the amount of conidia inoculated into the liquid medium and make the dispersion uniform. Solid cultures have a wide specific surface area such as cereals, potatoes, beans, bread crumbs, etc., and sterilize them by heat-dissolving raw materials as they are, or by immersing them in water as necessary to absorb water, and also for starch gelatinization The fungus is inoculated as a culture medium and cultured at about 20 ° C. to about 40 ° C. for about 3 days to about 5 days. In the solid culture obtained in this way, the mycelium spreads throughout the interior of the tissue, producing a large number of conidia, and the whole is easy to loosen, so the number of conidia inoculated into the liquid medium is controlled and the distribution is uniform. Can be very easily performed.

  Subsequently, the fungi are cultured in a liquid medium. The composition of the liquid medium is cultured in a medium moderately containing a carbon source, a nitrogen source, an inorganic substance, micronutrients and the like while adjusting the temperature, pH and the like under aerobic conditions.

  As the carbon source used in the liquid medium, a carbon source usually used for culturing filamentous fungi such as glucose, sucrose, glycerin, and dextrin is used. Nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium tartrate, sodium nitrate, asparagine, sodium glutamate, yeast extract, peptone, corn steep liquor, etc., and inorganic and trace nutrients such as magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate Calcium chloride, sodium chloride, manganese sulfate, zinc sulfate, copper sulfate, ferric chloride, ammonium molybdate, potassium iodide, boric acid, various vitamins (calcium pantothenate, inositol, pyridoxine, etc.) are used. Moreover, corn steep liquor, whey, nonfat dry milk, dry yeast, sake lees, etc. can also be added as what also served as a carbon source, a nitrogen source, and an inorganic nutrient source. Further, a bactericidal substance such as chloramphenicol may be contained in the medium in order to avoid contamination by bacteria during the culture.

  In addition, it is preferable to add about 0.1% to about 10% of the whole liquid medium in advance to the liquid medium, the same fats and oils used as starting materials for methyl ketones. This makes it possible to efficiently induce enzyme activity for fungi to convert fats and oils to methyl ketone.

Liquid culture is performed as follows. First, after mixing and sterilizing the above-mentioned liquid medium raw materials, an aqueous suspension of the solid culture obtained above is inoculated and cultured. In the present invention, it is important to culture so as to form a pellet-like mycelium. This makes it possible to efficiently convert all of a large amount of oil and fat into methyl ketones even when a large amount of oil and fat is added later. When mycelium grows in the form of pulp, the transfer of oxygen and nutrients is hindered due to the viscosity of the medium, causing a bias in oxygen and nutrients at the site of the medium. The conversion efficiency of fats and oils into methyl ketones decreases. The culture method and conditions may be any method as long as the mycelium grows in a pellet form. As one method for growing the mycelium in a pellet form, a method of controlling the inoculation of the solid culture so that the number of conidia is 10 ³ to 10 ⁵ / ml with respect to the liquid medium can be exemplified. In culture in a liquid medium, it is known that the growth form of the mycelium changes depending on the number of initial bacteria. However, when the initial conidia number is 10 ³ to 10 ⁵ / ml, the mycelium is in a pellet form. It is observed to grow. On the other hand, when the initial number of conidia is larger than 10 ⁵ / ml, the mycelium grows in a pulp state, and when the initial number of conidia is smaller than 10 ³ / ml, the mycelium is a pulp. It is observed that the mycelium grows on the wall of the culture tank, and none of them forms a pellet.

  As culture conditions, the temperature is about 15 ° C. to about 50 ° C., preferably about 20 ° C. to about 40 ° C., and conditions such as aeration, agitation, and shaking are adopted as necessary, and the culture is performed for about 4 hours to about 48 hours. . Moreover, it is also possible to make it react, adjusting pH in the range most suitable for the growth of microorganisms as needed.

  Subsequently, the oil and fat more than twice as much by weight ratio are added with respect to a liquid culture, and oil and fat are converted into methyl ketone. The time when the oil and fat is added is preferably a time when a pellet-like mycelium having an average particle diameter of 0.5 to 3 mm is formed. When the mycelium pellets are within the range of the average particle size, it is recognized that the fats and oils can be stably converted into methyl ketones even if the fats and oils are added in an amount twice or more by weight with respect to the medium. . On the other hand, if the pellet exceeds 3 mm, the contact between the hypha in the pellet and the oil and fat becomes worse, and the efficiency is lowered.

  Moreover, at the time of adding fats and oils, conversion of fats and oils to methyl ketone can be performed more efficiently by adding lipase. In the conversion of fats and oils to methyl ketones, the fats and oils are first decomposed into fatty acids and glycerin by lipase activity, and the fatty acids are β-oxidized, and subsequently decarboxylated to be converted into methyl ketones. Therefore, by adding lipase at this time, the decomposition of fats and oils into fatty acids can be promoted, and the conversion efficiency from fats and oils to methyl ketones can be increased. The lipase that can be used is not particularly limited. For example, Aspergillus genus, Rhizomucor genus, Rhizopus genus, Penicillium genus, Candida genus, Pichia genus, Chromobacterium genus, Alkagenes genus, Stress tomyces Lipase collected from various microorganisms such as genus, Actinomadura, and Bacillus, lipase obtained from porcine pancreas, goat, lamb, oral lipase collected from calf oral secretory gland, etc. it can. Commercially available products include lipase A, lipase L, lipase M, lipase AP, lipase AY, lipase P, lipase AK, lipase CES, lipase M-AP, lipase D, lipase N, lipase CT, lipase R (above, Amano Enzyme), lipase MY (manufactured by Meisei Sangyo Co., Ltd.), lipase P (manufactured by Nagase ChemteX Corporation), porcine pancreatic lipase (manufactured by Sigma Aldrich Japan), lecitase, paratase, paradase M Examples include lipase “Saiken” (manufactured by Yakult Honsha Co., Ltd.), and lipase (manufactured by Tanabe Seiyaku Co., Ltd.). These lipases can be used singly or in combination. The amount of lipase used varies depending on the titer and the like and cannot be generally specified, but is usually 0.1 to 10000 u / g, preferably 0.5 to 2000 u / g, more preferably 2 to 2 on the basis of the weight of the fat raw material. A range of 500 u / g can be exemplified.

  As the culture conditions in the conversion of fats and oils to methyl ketones, the temperature is about 15 ° C. to about 50 ° C., preferably about 25 ° C. to about 40 ° C., and conditions such as aeration, agitation, and shaking are adopted as necessary. Moreover, it is also possible to make it react, adjusting pH in the range most suitable for the growth of microorganisms as needed. Examples of the reaction time include a method of culturing for about 20 hours to about 100 hours, preferably about 40 hours to about 80 hours, but it is preferable to appropriately select the method while measuring the amount of methyl ketones produced. .

  Recovery of methyl ketones from the conversion reaction completion liquid can be performed by a conventional method. For example, in the method of the present invention, since the amount of methyl ketones obtained is large, an oil layer rich in methyl ketones can be obtained as an oil phase in the upper layer of the culture solution. Therefore, by removing the bacterial cells from the conversion reaction completion liquid, further collecting the oil phase by a separation means such as centrifugation, by collecting a fraction containing methyl ketones by atmospheric pressure or vacuum distillation method, Methyl ketones can be purified. It is also possible to obtain methyl ketones from the oil phase collected by centrifugation by column separation operations such as liquid chromatography and silica gel column chromatography. Methyl ketones are also contained in the separated bacterial cells and the aqueous layer during centrifugation, but these are extracted with an organic solvent such as hexane, ethyl acetate, petroleum ether, chloroform, and the solvent is recovered. It can be recovered by collecting a fraction containing methyl ketones by reduced pressure or reduced pressure distillation. In addition, methyl ketones can be recovered from the separated cells or the aqueous layer during centrifugation by steam distillation. It is also possible to perform solvent extraction or steam distillation from the beginning of purification without performing separation operations such as separation of bacterial cells and centrifugation of the culture solution. What method should be selected may be determined in consideration of the recovery rate of methyl ketones, workability, production cost, and the like.

The methyl ketones thus obtained include flavors of foods such as blue cheese, mayonnaise, wine, whiskey, liquor, miso, soy sauce and bread, shampoos, cosmetics such as cosmetics, and fragrances for pharmaceuticals. It can be used as a raw material for various blended fragrances.
Hereinafter, the present invention will be described more specifically with reference to examples and comparative examples.

Example 1
130g of polished rice is soaked in water overnight, drained, sterilized at 120 ° C for 15 minutes, inoculated with 1g of Aspergillus oryzae IAM2152 culture cultured in the same medium, cultured at 30 ° C for 4 days, solid culture Got.
Separately, 129 g of potassium monophosphate, 12 g of sodium hydrogen phosphate · 12H ₂₀ 18 g, 166 g of crystalline glucose, 112 g of yeast extract, 500 g of coconut oil, and 9075 g of water are put into a 50 L jar fermenter, and sterilized by heating at 121 ° C. for 20 minutes. After performing, it cooled to 30 degreeC and was set as the liquid culture medium. To this liquid medium, 25 g of the previously cultured solid culture suspended in 125 g of sterilized water (number of conidia 6 × 10 ⁵ / ml) was added (the number of conidia in the liquid medium at this time was calculated). 9 × 10 ³ pieces / ml). When culturing was performed for 28 hours while aeration was performed at a culture temperature of 30 ° C., a stirring speed of 400 rpm, and 0.5 vvm, the mycelium grew in a pellet form, and the average particle size was about 0.9 mm. Furthermore, 20 kg (66% of the whole medium including fats and oils) and 15 g of lipase A (manufactured by Amano Enzyme Co., Ltd.) and 15 g of coconut oil dissolved here (heated once at 60 ° C. and cooled to 35 ° C.) and cultured The culture was performed while aeration was performed at a temperature of 30 ° C., a stirring speed of 400 rpm, and 0.5 vvm. The relationship between the culture time and the amount of methyl ketones produced is shown in FIG. In addition, the production | generation amount (%) of the methyl ketone in FIG. 1 represents the weight (%) with respect to the weight of the added coconut oil. Analysis of methyl ketones was performed by gas chromatography. The analysis conditions are as follows.
Equipment: Hewlett-Packard 5890
Column: DB-WAX (30 m × 0.25 nm)
I. D = 0.25 μm
Carrier gas: 1 ml / min. (He) Drawer inlet pressure = 14 psi
Column temperature: 100 ° C (1 min.) To 250 ° C (10 ° C / min.)
Injector: 250 ° C, splitless, 1 μl
Detector: FID
Moreover, the general fatty acid composition (%) of coconut oil is as follows.
Caproic acid 0-0.8%
Caprylic acid 5-9%
Capric acid 6-10%
Lauric acid 44-52%
Myristic acid 13-19%
Palmitic acid 8-11%
Stearic acid 1-3%
Oleic acid 5-8%
Linoleic acid trace ~ 2.5%
Microbial conversion produces 2-heptanone from caproic acid, 2-nonanone from caprylic acid, and 2-undecanone from capric acid.
As a result of culturing for 77 hours, 2-heptanone and 2-nonanone, which are important as fragrances, were produced in amounts that were considered to be obtained by converting almost all caproic acid and caprylic acid in the raw palm oil.
After 77 hours from the addition of coconut oil, the culture was terminated, heat sterilized at 75 ° C. for 15 minutes, cooled to 40 ° C., allowed to stand at 40 ° C., and the upper oil part was separated by decantation. Thereafter, 9.8 kg of an oil phase was recovered by centrifugation. This oil phase was subjected to atmospheric distillation to obtain 2.36 kg of a fraction at 150 ° C. to 200 ° C. (Product 1 of the present invention).
The composition ratio of methyl ketones in the product 1 of the present invention was as follows.
2-Heptanone 28.1%
2-nonanone 24.5%
2-Undecanone 36.0%

Example 2
In Example 1, since 2-heptanone and 2-nonanone, which are important as fragrances, were produced up to an amount in which almost all caproic acid and caprylic acid in the raw palm oil were converted, the culture was continued for up to 77 hours. However, if the culture is further continued, the production of 2-undecanone is thought to increase further with time. Therefore, the culture was continued under exactly the same conditions as in Example 1 until 245 hours. The relationship between the culture time and the amount of methyl ketones produced is shown in FIG.
When culturing was continued until 245 hours, 2-undecanone was produced to an amount considered that all capric acid in the raw material palm oil was converted. However, 2-heptanone and 2-nonanone, which are important as fragrances, peaked at about 80 hours and thereafter decreased. This may be due to the utilization of microorganisms or volatilization due to aeration culture, but the cause is not clear.

Comparative Example 1 (Example in which the number of inoculated bacteria is large and a pulp-like mycelium is formed)
In Example 1, instead of adding 25 g of the solid culture suspended in 125 g of sterile water to the liquid medium, 1000 g of the solid culture suspended in 5000 g of sterile water was added to the liquid medium. The number of conidia at this time was 2.4 × 10 ⁵ / ml. Subsequently, the cells were cultured for 28 hours while aeration was performed at a culture temperature of 30 ° C., a stirring speed of 400 rpm, and 0.5 vvm in the same manner as in Example 1, but the mycelium grew in the form of pulp.
20 kg of dissolved palm oil (once heated at 60 ° C. and then cooled to 35 ° C.) and 15 g of lipase A (manufactured by Amano Enzyme Co., Ltd.) are added, and the culture temperature is 30 ° C., the stirring speed is 400 rpm, and 0.5 vvm. The culture was carried out with aeration. The relationship between the reaction time and the amount of methyl ketones produced is shown in FIG. In addition, the production | generation amount (%) of the methyl ketone in FIG. 3 represents the weight (%) with respect to the weight of the added coconut oil.
As shown in FIG. 3, in Comparative Example 3 in which the mycelium grew in a pulp shape, the conversion efficiency of fats and oils to methyl ketone was 1/2 or less compared to Example 1 in which the mycelium grew in a pellet shape, Conversion efficiency was poor.

Example 3 (using penicillium as a microorganism producing methyl ketones)
10 g of water was added to 50 g of bread crumbs, sterilized at 120 ° C. for 15 minutes, 40 ml of sterilized water was added, 1 g of Penicillium roqueforti IFO 4622 culture cultured in the same medium was inoculated, and cultured at 30 ° C. for 4 days. A culture was obtained.
Separately, 129 g of potassium monophosphate, 12 g of sodium hydrogen phosphate · 12H ₂₀ 18 g, 166 g of crystalline glucose, 112 g of yeast extract, 500 g of coconut oil, and 9075 g of water are put into a 50 L jar fermenter, and sterilized by heating at 121 ° C. for 20 minutes. After performing, it cooled to 30 degreeC and was set as the liquid culture medium. To this liquid medium, 50 g of the previously cultured solid culture suspended in 250 g of sterile water (2.4 × 10 ⁶ conidia / ml) was added. At this time, the number of conidia was 7.0 × 10 ⁴ / ml. When the cells were cultured for 26 hours while aeration was performed at a culture temperature of 30 ° C., a stirring speed of 400 rpm, and 0.5 vvm, the mycelium grew in a pellet form, and the average particle size was about 0.7 mm.
20 kg of dissolved palm oil (once heated at 60 ° C. and then cooled to 35 ° C.) and 15 g of lipase A (manufactured by Amano Enzyme Co., Ltd.) are added, and the culture temperature is 30 ° C., the stirring speed is 400 rpm, and 0.5 vvm. The culture was carried out with aeration. The relationship between the reaction time and the amount of methyl ketones produced is shown in FIG. In addition, the production | generation amount (%) of the methyl ketone in FIG. 4 represents the weight (%) with respect to the weight of the added coconut oil.
As shown in FIG. 4, when Penicillium roqueforti IFO 4622 was used as the microorganism, the same results as in Example 1 were obtained.

It is the figure which showed the relationship between the culture | cultivation time at the time of adding mycelia after a mycelium forms pellets, and the production amount of methyl ketones. Example 1 It is the figure which showed the relationship between the culture | cultivation time at the time of extending reaction time further in FIG. 1, and the production amount of methyl ketones. (Example 2) It is the figure which showed the relationship between the culture | cultivation time at the time of adding coconut oil to the place where a mycelium grew like a pulp, and the production amount of methyl ketones. (Comparative Example 1) Pen. It is the figure which showed the relationship between the culture | cultivation time and the production amount of methyl ketones when a coconut oil is added after a mycelium forms a pellet using roqueforti. (Example 3)

Claims (4)

A liquid culture medium is inoculated with a solid culture of a fungus belonging to the genus Aspergillus or Penicillium that can produce methyl ketone, at a concentration of 10 ³ to 10 ⁵ / ml in the liquid medium. And after forming the pellet-like mycelium so that the average particle size is 0.5 to 3 mm, adding 2 to 4 times the amount of fats and oils by weight to the liquid medium, and further culturing A method for producing methyl ketone. The microorganisms belonging to the genus Aspergillus are Aspergillus oryzae, Aspergillus tamari, Aspergillus parasiticus, Aspergillus awamoris (Asp. Kawachii), Asp. Niger, or Aspergillus pheonicis, and microorganisms belonging to the genus Penicillium are Penicillium P. (Pen. Roqueforti), Penicillium p. .Cammberti), Penicillium casei (Pen.casei) or Methyl ketone The method of claim 1 which is Nishiriumu-Janshineramu (Pen.janthinellum). The method for producing methyl ketone according to claim 1 or 2 , wherein lipase is further added at the time of adding fats and oils to the liquid medium. The method for producing methyl ketone according to any one of claims 1 to 3 , wherein the fat is triglyceride containing lauric acid, capric acid, and caprylic acid as main constituent fatty acids.

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