JPS60259192A - Production of microbial lipid - Google Patents

Abstract

PURPOSE:To increase the amounts of lipid with a high unsaturated fatty acid content and microbial cells and obtain the titled lipid by cultivating a microorganism, e.g. Mortierella isabellina, etc. in a culture medium containing acetic acid (salt) added thereto and a carbohydrate as a carbon source. CONSTITUTION:A strain, e.g. Mortierella isabellina (IFO 7824), Mortierella vinacea (IFO 6738), Mortierella nana (IFO 8794), Mortierella ramanniana (IFO 8287) or Mortierella ramanniana var. anglispora (IFO 8187), is cultivated in a culture medium containing acetic acid (salt) added thereto in an amount of e.g. 0.1-20g/ ladded thereto and a carbohydrate, e.g. glucose, as a carbon source, preferably at 3.0-6.0pH, and the aimed lipid is obtained from the resultant microbial cells.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention involves culturing strains of Isabelina, Vinacea, Nana, Lamaniana, and Lamaniana anglispora belonging to the genus Morteierella in a medium containing carbohydrates as a carbon source, and growing bacterial cells with a high lipid content in the medium. By adding acetic acid or acetate to the culture medium during the process, the amount of bacterial growth, the lipid content in the bacterial cells (oil, fat, etc.), polar lipids (phospholipids, glycolipids, etc.)] 2. Highly productive lipids to be collected This relates to the production method.

The present inventors have already cultivated filamentous fungal strains of Isabelina, Vinacea, Nana, and Lamaniana angrispora belonging to the genus Morteierella in a medium containing a high concentration of Y-carbohydrate as a carbon source. It has been found that lipids can be produced at high density and with high productivity by collecting lipids from the bacterial cells. However, when a filamentous fungal strain of the same species, also belonging to the genus Morteierella, is cultured in a medium with a high concentration of carbohydrate as a carbon source, a high concentration of γ-lylunic acid-containing lipids is produced at high density, and the cultured bacteria Body! l) It has been found that r-'J phosphoric acid-containing lipids or γ-phosphoric acid can be produced with high productivity.

The above inventions each relate to the production of lipids (oils and fats, etc.) by microorganisms (filamentous fungi) in place of animals and plants.
In the latter case, it is particularly an alternative to the method for producing 0θnotb-lyrinnic acid or its containing lipids.

The present inventors further cultivated filamentous fungal strains of Isabelina, Vinacea, Nana, Lamaniana, and Lamaniana anglispora belonging to the genus Morteierella in a medium containing carbohydrates as a carbon source, and produced lipids by collecting lipids from the cells obtained. By adding acetic acid or acetate to the culture medium as an additive or auxiliary carbon source, the content of lipids in dried bacterial cells can be increased by 10 to 10% compared to when no acetic acid is added under the same culture conditions.
300% increase, and at the same time an increase in the amount of bacterial cell proliferation is observed, resulting in a markedly higher lipid productivity. The present invention has been completed based on the discovery that the That is, the present invention involves culturing strains of Isabelina, Vinacea, Nana, Lamaniana, and Lamaniana angrispora belonging to the genus Morteierella in a medium containing carbohydrates as a carbon source, and cultivating bacteria with a high content of one substance in the medium. By adding acetic acid or acetate to the medium, we aim to significantly increase the growth rate of bacterial cells and the lipid content in the bacterial cells. This is a highly productive lipid production method that collects lipids, glycolipids, etc.).

The bacteria used in the present invention are Morteiherella (Mort-i
erella ) genus Isabellina
a) (I FO7824;7884,7873,8
183.8309) Vinacea (
I P'06738), Nana (TF
O8794), Rama = 7 f (ramaniana
) (IFO8287), Ramani-ana var, anglisp
ora) (IFO8187). All of the above-mentioned bacteria are filamentous bacteria stored at the Fermentation Research Institute and listed in the ■FO Catalog (Bacterial Strain Catalog).

Examples of carbohydrates used as carbon sources for the medium for culturing the filamentous fungi include glucose, fructose, saccharose, molasses, starch, and wood saccharification liquor. The carbohydrate is preferably used in an amount of 20 to 400 g per liter of culture medium. Examples of nitrogen sources include various inorganic nitrogen sources such as ammonium nitrate, ammonium sulfate, ammonium chloride, and ammonium phosphate, or urea, peptone,
Organic nitrogen sources such as yeast extract and corn/steve liquor are used. Examples of inorganic salts include KH2PO4, 2HPO4, NaCl, FeSO4,7+ (20, M
gSO4-7H20, ZnSO4.71-120, etc. are used. Add trace elements and other nutritional sources as needed.

The above-mentioned filamentous fungi are usually cultured in a liquid medium by shaking culture, aerated agitation culture, or the like. The pH of the medium is 30-60
The culture is usually carried out for about 2 to 10 days. In history,
Add acetic acid or acetate (alkali metal salts such as sodium salts and potassium salts) to the culture conditions such as carbon source concentration in the initial stage of culture, in the preculture medium in the case of aerated agitation culture, or in the intermediate stage of culture. 0.1~ depending
The bacterial cells are cultured by adding 20 ji/l of the medium. In this way, bacterial cells with a high content of lipids with a high content of unsaturated fatty acids such as γ-lylunic acid are produced by themselves in the culture. Therefore, it is preferable to collect lipids with a high content of unsaturated fatty acids such as γ-lylunic acid from the bacterial cells. When separating bacterial cells from a culture, the hyphae do not spread very much and are cultured in extremely small units (1 to 10 cells). It has become clear that this product has the advantage of reducing the water content (water content of about 60%). Lipids with a high content of unsaturated fatty acids such as γ-lylunic acid are collected by conventional methods such as solvent extraction.

Thus, according to the present invention, lipids with a high content of unsaturated fatty acids such as r-lylunic acid can be produced with high productivity,
The present invention is excellent as a method for producing r-') phosphoric acid-containing lipids and a method for modifying fish lipids. The present invention is particularly excellent as a method for producing r-+) phosphoric acid-containing lipids, which are currently collected from plant seeds. ! Incidentally, γ-lylinic acid (18:3 (6,9,12)) is a fatty acid (essential fatty acid) that cannot be synthesized in the body of mammals and is required as a feed, along with linoleic acid. This is because γ-lylphinic acid is a precursor that becomes bishomo-r-lyllunic acid in the body and then arachidonic acid, and bishomo-γ-lylinic acid and arachidonic acid are respectively prostaglandins, El + Fl cl, and F2.
+F2. This is because it plays an extremely important physiological role in living organisms. Therefore, it is clear that γ-lylunic acid-containing lipids can be used as pharmaceuticals.

Next, examples of the present invention will be shown, but the present invention is not limited thereto.

Example 1゜Glucose 309, KH2PO42,! i',
MgSO3・7Hρ0.3g, NaC10.1g, Fe
SO4,7H7H2O10, CaCl22H20LoI
niiiI, Cu5O,-5H200,2Tn9,Z
nSO4・7H201, Orng, M n C12・4
H201, Om? ,'I”hiamine-HCIJ2
rng, D-Biotin O,o 2 mg as a nitrogen source (NH4) 28043 g, (C/N ratio (carbon atom weight in glucose/nitrogen atom weight in nitrogen source) is 2
．． 2.2) was mixed with 10100O of deionized water, p)(
A medium was prepared by adding a predetermined amount of acetic acid or acetate (sodium salt, potassium salt) based on a synthetic medium adjusted to 4.6. Pour 400ml of this synthetic medium into 11 Erlenmeyer flasks, inoculate each with the bacterial strain, and culture at 30°C.
Shaking culture was performed at 150 rl; 1 m for 7 days at C.

After culturing, the bacterial cells were collected by the mouth filtration method or centrifugation method. A part of it was accurately weighed to determine the moisture content and placed in a constant temperature bath at 120°.
The glue solution was dried at C, the water content was determined, and the remaining bacterial cells were extracted for lipids. Extraction of lipids from bacterial cells was performed by adding chloroform-methanol (2:IV/V) to the remaining wet bacterial cells.
) was added and homogenized in the presence of glass peas to simultaneously crush the bacterial cells and extract the lipids.

In order to perform the extraction completely, this was repeated five times and all the extracts were collected. After the above extract was purified by the partition washing method of II'olch, the solvent was distilled off under reduced pressure and the total lipids were measured by gravimetric method. Using this method, when acetic acid or acetate salt (f, thorium Hanawa potassium salt) was added to various types of Morteierella filamentous fungi, the amount of bacterial cell growth and fatty acid composition Table 1 shows the results regarding changes in deafness.

From the results in Table 1, for the same strain, when acetic acid or acetate salts (sodium salt, potassium salt) are added, the bacterial cell growth rate is always higher than when it is not added, and in many cases more than twice the value is obtained. I can see that it is being done. This was even more evident with regard to the amount of lipid production, which increased 15 to 6 times. Although in some cases the lipid content did not change much, in many cases it was observed that the 10-20 content increased. These facts indicate that bacterial cells formed in response to the consumption of glucose 100.9
The bacterial cell coefficient and lipid coefficient expressed as f1g) and lipid content (I) were significantly increased compared to the case where acetic acid or acetate was not added, respectively, and the productivity of lipids and bacterial cells was significantly increased in this system. was found to be significantly higher.

In addition, the fatty acid composition increases by adding vinegar or acetate (sodium salt, potassium salt), and the content of γ-lylunic acid increases from 1.7 to 1.7 in total unsaturated fatty acids.
An increase of 201% was observed.

The unsaturated number, which indicates the number of unsaturated bonds in one fatty acid, also increased considerably, so it is clear that the increasing unsaturation of unsaturated fatty acids is occurring at the same time as the increase in unsaturated fatty acids. be. Koq has an effect on lipid modification in the sense that the addition of acetic acid or acetate increases the amount of unsaturated fatty acids containing γ-lylunic acid, a highly unsaturated acid, in its production direction. That is confirmed.

A7824 --4.35 2.01 HM sodium sodium 5 8,83 4.38#
7873 - - 5.54 2.19 Sodium acetate 5 9,95 5.72 7884 - - 5
．． 55 2.2]〃 Sourium acetate 5 '7,0
8 4.04〃 Potassium acetate 5 6,30 3.8
4〃 Acetic acid 1 7,30 3.15 #8183 --- 9.08 3.37〃 Sodium acetate 5 9.98 4.65 #8309 ---
5.25 1.30 Sodium acetate 5 10,9
0 6.038 6738, - -- 5.03 1
．． 85 Sodium acetate 5 10,10 5.19
C8794--4,522,33 Sodium acetate 5 9,15 4.421) 8
287 --3.65 1.49 Sodium acetate
5 5,20 2.75r> 8187 - - -
3.80 0.71 Sodium acetate 5 8,33
4.31 Table -1 45,86,714,68,071!1 1.0349
．． 6 14,6 29,5 10,7 75.1 1.
0740.2 7.3 ] 8.2 4,7 63. '
,) (1,8457,5]9,1 33.2 6,7
65.0 +, 1 (139,97,418,5305
9,0(1,7556,913,523,64,267
41 (1,886+, 3 12,8 21.0 4.2
()9.8 0.8942.8 10.5 21.9
4,0 64. :(08537,211,230,3
4,2646 (18246,615533,27973
710024,7' 4,3 17.5 5,6 68
．． 4 041:(55,320,136,310,(1
88,51,: (i) 36.8 6.2 ] 6.8
3.2 5 G, 3 0.6751.4 17,3 3
3.7 3,6 64.5 0.8: (51,37,8
15,15358,+0.7848.3 14,8
30.5 6.1 7+, 9(]i) 540.9 5,
0 12.2 5,8 64.6 0.8+52.9
9,2 17.3 7,4 68.0 0.9618.
8 2,4 12.7 9.1 6], 7 f), 82
51.8 14.4 27.7 9.2 77.8 1
．． 00 In addition, in Table 1, Hake, Morteierreisaberina (11ortiere11a 1sabe),
], ina ), 13 is Morteierella lavinacea (
Mortierella vinacea), C is
Mortierellanana
), D is 'Mort
J, erellaramaniana) and E indicate molten glucose, and the unsaturation coefficient indicates the average number of unsaturated bonds in one fatty acid.

Example 2 Glucose 60g, KH2PO42g, M! 9S04
・7I] 200.3g, NaC10,1,g, -i'
Fivto extract 0.2g, yeast extract 0.
2g, peptone 0.1g, FeSO4.7 H2O10
mg, Caclz H2I42010”f/, cus
o4・5H2゜0.2m9, Mn504-41-1□0
1.0 9.9.
2) 2CO) and ammonium sulfate ((NH4)zSO
4) was added so that the C/N ratio (weight of carbon atoms in the carbon source/weight of nitrogen atoms in the nitrogen source) was approximately 60, and 10,100 O of deionized water [mixed mfr happy extract was used for prohibited fishing at a shore inspection source]. When the concentration of a certain carbohydrate (glucose) was increased, the medium components were increased accordingly, and when the nitrogen source was changed to urea, etc., the medium was adjusted to maintain the same C/N ratio.

When culturing this medium in 101 culture tanks, 61.3
Prepare 201 in a culture tank of 01, inoculate each strain, and incubate at a culture temperature of 30°C for a predetermined time with an aeration rate of 0.5 to 2.
．． Culture was performed using OVVm with stirring at 300 to 700 rpm. After culturing, the bacterial cells were collected by centrifugation. In addition, in order to measure the growth amount of bacterial cells, the amount of lipid production, and the carbohydrate concentration in the medium, 10
A sample of 0 mt was collected, and the bacterial cells and the medium were separated by the mouth filtration method.

Extraction and quantification of lipids from isolated bacterial cells were performed according to Example 1. The concentration of carbohydrate (glucose) in the medium from which the bacterial cells were removed was measured by high performance liquid chromatography, and the culture was terminated when the concentration reached 0.

Strain Morteierella lamaniana anglispora IF
O8187 was cultured in 101 and 301 culture vessels using glucose as a carbon source and acetic acid as an additive under various conditions. , lipid production (g/
l), lipid content (%), etc.

The results are shown in Table 2, including the addition conditions.

In Table 2, when 101 culture tank is used, the amount of sodium acetate added is 0.3~
The results of culturing with ICJjj/l and at the same time changing the addition conditions are shown in comparison with the case without addition.

When sodium acetate is added, a significant increase in lipid production and lipid content is observed regardless of the addition conditions. This means that the carbon source glucose 100,! This can also be seen from the fact that the lipid system number, which is the amount of lipid produced relative to 9, is significantly increased compared to the case without additives. Although the amount of bacterial cell growth was generally larger than in the case without additives, some variation was observed. Even with the culture results using a 3M culture tank with a glucose concentration of approximately 2o1/13 and a high concentration carbon source, bacterial cell growth was somewhat lower in both systems with 0.3 and 51/11SA added than in the case without additives. , the lipid content has increased by more than 10%, and therefore the lipid production has increased by 20%.
It was observed that the number of cases increased. This was manifested as a large increase in the number of lipid systems, indicating that the effect of SA addition was observed on lipid production by high-density culture of bacterial cells over a considerable range from 03 to 5j9/1. There is.

2 # G90 SA5 51 3 1 G90 5AIO69 4Iy G90 SA5 63 5 # G90 5AIO69,5 7〃 G85 5AIO51 8# G85 SA2.3 46 9 +' G85 SAo, 3 50 11 y G185 SA5 98 2 31.9 11.9 37.2 13.2 35.43
4.0 17.2 5Q, 7 19.1 37.836
．． 1 18.4 51.0 2Q, 5 40.140.
5 17.0 41.9 18.9 45.036.8
17.9 48.5 19.8 40.931.3
145 46.4 171 36.829.3 15.
7 53.4 18.4 34532.7 14.7
45.0 17,3 38,434.4 15,2 4
4,2 17,9 40,777.6 28,8 37
,1 15,2 40,966.8 33,8 50,
6 18,2 36.1'745 35.9 48,2
18,0 37.3 In the above coating-2, q is glucose, (J is urea, As is ammonium sulfate, S
A represents sodium acetate.

In addition, in experiment numbers 1 to 12 shown in Table 2, experiment numbers 1 and 10 are control tests, and in experiment numbers 2 to 5, no SA was added to the preculture medium. In experiment numbers 4 to 5, SA was added at 30 o'clock after the start of culture, and in experiment numbers 6 to 9 and experiment number 11.
In No. 12, S A 5 ji/11 was added to the preculture medium.

Claims (1)

[Claims] (1) Strains of Isabelina, Vinacea, Nana, Lamaniana, and Lamaniana angrispora belonging to the genus Morteierella are cultured in a medium using carbohydrates as a carbon source, and when lipids are collected from the culture, the strains are used as a medium for culturing the bacterial cells. A method for producing microbial lipids, characterized by adding acetic acid or acetate.