JP2701278B2 - Lipid fermentation productivity improver - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an agent for improving the fermentative productivity of lipids from molds or algae comprising polyetherester. [Prior Art] Fermentative production of lipids by microorganisms has been conventionally carried out in a number of studies using yeasts, but their productivity is low, and their practical use has been regarded as difficult. Recently, it has been known that molds and algae, which are higher microorganisms than yeasts, have the ability to ferment and produce physiologically active lipids containing highly unsaturated fatty acids such as γ-linolenic acid, arachidonic acid, and eicosapentaenoic acid. Have been. Conventionally, natural sources of polyunsaturated fatty acids include evening primrose oil (containing γ-linolenic acid), egg yolk oil (containing arachidonic acid), sardine oil (containing eicosapentaenoic acid, etc.), and the like. It is only used as a health food because of its surface and difficulty in purifying and purifying it as a physiologically active substance. By seeking the source of these polyunsaturated fatty acids from microorganisms, expectations regarding supply stability and the easiness of purification and purification thereof have been found, and have attracted attention as physiologically active substances. However, from the viewpoint of production cost, the productivity of lipid per unit volume of fermentation liquor is low, and thus the productivity of the target polyunsaturated fatty acid is low at present. On the other hand, JP-A-62-3791 and JP-A-62-6694 propose that a surfactant such as polyoxyethylene sorbitan monooleate is used in the production of lipids. Since the activator foams during use because of its water solubility, it is necessary to use an antifoaming agent in combination, and there is a drawback that a sufficient effect is not exhibited unless a combination of the surfactant and the antifoaming agent to be used is selected. [Problems to be Solved by the Invention] The present invention, by adding to the fermentation liquor of a polyunsaturated fatty acid-containing lipid-producing microorganism, without using other additives such as an antifoaming agent, contains a highly unsaturated fatty acid-containing An object of the present invention is to provide a lipid fermentation productivity improving agent for the purpose of improving lipid productivity. [Means for Solving the Problems] The present inventors have conducted intensive studies on an agent for improving the fermentation productivity of lipids by microorganisms, and as a result, have taken measures to add an insoluble polyether ester compound to the fermentation medium of microorganisms. The inventors have found that this object can be achieved, and have completed the present invention. That is, the present invention is a lipid fermentation productivity improver comprising the compound represented by the general formula (I). (Where X is an acyl group of an unsaturated fatty acid having 18 to 20 carbon atoms, R is a hydrocarbon group having 1 to 20 carbon atoms, B is a hydroxyl group of 2 to 6
Residues of glycols or polyhydric alcohols having
AO is an oxyalkylene group having 2 to 4 carbon atoms. When it contains an oxyethylene group, the oxyethylene group content is 35% by weight or less of the total amount of the oxyalkylene group, and a, b, c, l,
m and n are a ≧ 1, b ≧ 0, c ≧ 0, 2 ≦ a + b + c ≦ 6, l ≧
It is a number that satisfies 0, m ≧ 0, n ≧ 0,1 ≦ al + bm + cn ≦ 200. In the general formula (I), X is one or a mixture of two or more acyl groups of an unsaturated fatty acid having 18 to 20 carbon atoms;
If it has less than 18 carbon atoms or is an acyl group of a saturated fatty acid, the effect of the present invention is not exhibited, and those having 21 or more carbon atoms are hardly easily available. R is one or a mixture of two or more hydrocarbon groups having 1 to 20 carbon atoms, and those having 21 or more carbon atoms do not exhibit the effects of the present invention. B is a residue of glycols or polyhydric alcohols having 2 to 6 hydroxyl groups. If the number of residues exceeds 6, the viscosity of the compound which does not dissolve in the culture solution becomes extremely high, and handling becomes difficult. In addition, 1 is included in the present invention because it has substantially the same structural formula as compound 2. AO is one or a mixture of two or more oxyalkylene groups having 2 to 4 carbon atoms. When the oxyethylene group is contained, its content is 35% by weight or less of the total amount of the oxyalkylene group. If the content of oxyethylene groups exceeds 35% by weight, it dissolves in the culture solution and foams, which is inconvenient. A, b, c, l, m, n are numbers satisfying the following conditions, a ≧ 1, b ≧ 0, c ≧ 0,2 ≦ a + b + c ≦ 6, l ≧
0, m ≧ 0, n ≧ 0,1 ≦ al + bm + cn ≦ 200, and when the total amount of the oxyalkylene group exceeds 200, the viscosity becomes extremely high and handling becomes difficult. In the general formula (I), if the acyl group of the unsaturated fatty acid having 18 to 20 carbon atoms represented by X has at least one double bond or triple bond, the effect of the present invention can be obtained. The oxyalkylene group represented by AO in the general formula (I) includes an oxyethylene group, an oxypropylene group,
An oxybutylene group and an oxytetramethylene group;
As the hydrocarbon group contained in, methyl group, ethyl group,
Allyl group, propyl group, isopropyl group, butyl group, tert-butyl group, amyl group, isoamyl group, hexyl group, heptyl group, 2ethylhexyl group, octyl group, nonyl group, decyl group, undecyl group, lauryl group, tridecyl group , Myristyl group, isocetyl group, stearyl group,
Isostearyl group, oleyl group and acryldodecyl group; glycols or polyhydric alcohols having 2 to 6 hydroxyl groups having B as a residue include ethylene glycol, propylene glycol, butylene glycol and hexylene; Glycols, alkylene glycols having 8 to 18 carbon atoms, glycols such as neopentyl glycol, glycerin, diglycerin, trimethylolethane, trimethylolpropane, 1,3,5 pentanetriol, erythritol, pentaerythritol, dipentaerythritol, etc. And polyhydric alcohols. The rate of addition of these productivity enhancers to the medium is 0.01 to 10
%, Preferably 0.05 to 5%. The effect is small when the addition is less than 0.01%, and the effect hardly increases when the addition exceeds 10%. The microorganisms having lipid fermentation productivity used in the present invention are molds or algae other than Mortierella spp., And specifically include Penicillium spp., Aspergillus spp. Genus (Rhizopus), Cunninghamell
a), genus Entomophrora, genus Circinella, genus Rhizomucor, genus phycomyces, genus Gongrone
lla), Cnidiobolus, Lipomyces, Lipomyces, Backsella, Spirulina, Pythium, Pheneromyces, and the like. The culture conditions for lipid fermentation production used in the present invention are not particularly limited, and ordinary media and conditions under which various microorganisms can grow are used. For example, hydrocarbons such as glucose, molasses, saccharified starch, and alkanes can be used as the carbon source. As other additives, generally known ones are used as necessary, and the temperature, pH, aeration and reaction time may be in accordance with the basic conditions. [Effects of the Invention] By using the present invention, it is possible to improve the productivity of fermentation of lipids by microorganisms. [Production Examples] The lipid fermentation productivity improver A used in the examples was produced as follows. Take 62g of ethylene glycol and 5g of potassium hydroxide in a pressurized reactor, under nitrogen gas atmosphere, 130-140 ° C, 0.5-3kg
The addition reaction was carried out while gradually introducing 225 g of ethylene oxide at / cm ² (gauge pressure). After injecting the whole amount, stirring was continued at 110 to 120 ° C. for 1 hour, and 1600 g of propylene oxide was added.
The addition reaction was carried out by gradually pressurizing the mixture at 10 to 120 ° C and 0.5 to 3 kg / cm ² (gauge pressure). After injecting the whole amount, stirring was continued at 110 to 120 ° C. for 2 hours. Thereafter, potassium hydroxide was neutralized with hydrochloric acid, and salts produced as by-products were removed by filtration. The hydroxyl value of the obtained polyalkylene glycol was 63.3. Next, 886 g of the obtained polyalkylene glycol, 282 g of oleic acid, 5.8 g of paratoluenesulfonic acid and 600 g of toluene were placed in a four-necked flask equipped with a nitrogen gas blowing tube, a thermometer, a stirrer, a cooling tube, and a reflux device. Then, the esterification reaction was carried out at 85 to 95 ° C. while refluxing the solvent while blowing nitrogen gas. Generated water is calculated value (18
The reaction was stopped when g) was reached, and the mixture was washed three times with 500 g of saturated saline. Thereafter, dehydration was performed at 110 to 120 ° C. under a reduced pressure of 20 to 50 mmHg for 2 hours. Aside from the by-produced salt, a lipid fermentation productivity improver A (hydroxyl value 0.9, saponification value 62.1), which is a polyalkylene glycoldiolate, was obtained. By the same method, productivity improvers BF used in the examples were produced. [Examples] The present invention will be described with reference to examples. The examples shown here are for explaining the effect of the lipid fermentation productivity improver and do not limit the genus of the microorganism and the culture conditions thereof. Example 1. Entomophthora exteiaris (ATCC32890) was cultured using the following peptone-glucose medium as a basal medium. Glucose 20g Peptone 5g Yeast extract 2g KH ₂ PO ₄ 1g MgSO ₄・ H ₂ O 0.5g Productivity enhancer A Predetermined amount of medium total 1L Productivity enhancer A is C ₁₇ H ₃₃ COO (PO) ₁₃ (EO) ₆ ( in PO) ₁₃ COC ₁₇ H _33, wherein the _{PO: C 3 H 6 O,} EO: C 2 H 4 O, C 17 H 33 CO: an acyl group of oleic acid. One platinum loop of Entomophthora exitearis stored on peptone-glucose-agar slope was inoculated into 6 ml of the medium in a test tube, and cultured at 28 ° C. for 3 days under shaking conditions.
This was transferred to a 200 ml medium in a 500 ml flask, and cultured for 3 days under a rotating stirring condition. 2 ml of this culture was inoculated into 200 ml of a medium in a 500 ml flask, and cultured at 28 ° C. for 5 days with rotary shaking. After the culture, the medium was suction-filtered to obtain wet cells. After adding 100 ml of methanol to the wet cells and dispersing them well, suction filtration was carried out to obtain methanol-washed cells, which were further mixed with 70 ml of a chloroform / methanol mixture (2: 1 v / v) and glass beads.
100 ml was added and homogenized for 10 minutes. The filtrate was desolvated to determine the amount of lipid. Table 1 shows the relationship between the amount of the productivity improver A obtained here and the lipid yield. Example 2 In Example 1, the productivity improver B having the following structure was used in place of the productivity improver A. C ₁₈ H ₃₅ O (PO) ₂₄ (EO) ₃ COC ₁₇ H ₃₃ where C ₁₈ H ₃₅ O: oleyl alcohol residue PO: C ₃ H ₆ O, EO: C ₂ H ₄ O C ₁₇ H ₃₃ CO: olein It is an acyl group of an acid. Other conditions were the same as in Example 1. Table 2 shows the relationship between the amount of the productivity improver A obtained here and the lipid yield. Example 3 A productivity improver C having the following structure was used in place of the productivity improver A in Example 1. Here, PO: C ₃ H ₆ O, EO: C ₂ H ₄ O, C ₁₇ H ₃₃ CO: an acyl group of oleic acid R ₃ CO: an acyl group of a linole group. Other conditions were the same as in Example 1. Table 3 shows the relationship between the amount of the productivity improver C obtained and the lipid yield. Example 4 A productivity improver D having the following structure was used in place of the productivity improver A in Example 1. Here _{PO: C 3 H 6 O,} EO: C 2 H 4 O, C 17 H 33 CO: an acyl group of oleic acid. Other conditions were the same as in Example 1. Table 4 shows the relationship between the amount of the productivity improver D obtained and the lipid yield. Example 5. Lipomyces lipofa (ATCC 10742) was cultured using the following peptone-glucose medium as a basal medium. Glucose 20 g Peptone 5 g Yeast extract 2 g KH ₂ PO ₄ 1 g MgSO ₄・ H ₂ O 0.5 g Productivity enhancer E Predetermined medium total 1 L Productivity enhancer E is C ₃ H ₆ O (PO) ₁₈ (BO) ₁₃ COC in ₁₉ H _31, wherein the _{PO: C 3 H 6 O,} BO: C 4 H 8 O, C 19 H 31 CO: an acyl group of arachidonic acid. One platinum loop of Lipomyces lipofa, stored on a slope of peptone-glucose-agar, was inoculated into 6 ml of the medium in a test tube, and cultured at 28 ° C. for 3 days under shaking conditions. This is 50
The medium was transferred to a 200 ml medium in a 0 ml flask, and cultured for 3 days under a rotating stirring condition. Add 2 ml of this culture to a 500 ml flask
0 ml of the medium was inoculated and cultivated at 28 ° C. for 5 days with rotation and shaking. After the culture, the medium was suction-filtered to obtain wet cells. After 100 ml of methanol was added to the wet cells and dispersed well, suction filtration was carried out to obtain methanol-washed cells, which were further mixed with 70 ml of a chloroform / methanol mixture (2: 1 v / v) and glass beads.
100 ml was added and homogenized for 10 minutes. The filtrate was desolvated to determine the amount of lipid. Table 5 shows the relationship between the amount of the productivity improver E obtained and the lipid yield. Example 6 In Example 6, the productivity improver F having the following structure was used in place of the productivity improver E. R ₂ COO (PO) ₃₄ COR ₂ where R ₂ CO is an acyl group PO: C ₃ H ₆ O of soybean fatty acid. Other conditions were the same as in Example 5. Table 6 shows the relationship between the amount of the productivity improver F obtained and the lipid yield.

Claims (1)

(57) [Claims] An agent for improving fermentation productivity of lipid comprising the compound represented by the general formula (I). (Where X is an acyl group of an unsaturated fatty acid having 18 to 20 carbon atoms, R is a hydrocarbon group having 1 to 20 carbon atoms, B is a hydroxyl group of 2 to 6
Residues of glycols or polyhydric alcohols having
AO is an oxyalkylene group having 2 to 4 carbon atoms. When it contains an oxyethylene group, the oxyethylene group content is 35% by weight or less of the total amount of the oxyalkylene group, and a, b,
c, l, m and n are a ≧ 1, b ≧ 0, c ≧ 0, 2 ≦
a + b + c ≦ 6, l ≧ 0, m ≧ 0, n ≧ 0, 1 ≦ al + bm
It is a number that satisfies + cn ≦ 200. )