JPH04179487A - Method for fermentation of simultaneous production of squalene and ethanol - Google Patents

Abstract

PURPOSE:To industrially mass-produce squalene which is a raw material for squalane useful as a base oil, etc., for cosmetics according to a fermentation method by culturing a microorganism capable of producing the squalene under anaerobic conditions. CONSTITUTION:A microorganism capable of producing squalene is cultured under anaerobic conditions such as a nitrogen gas atmosphere. Saccharomyces cerevisiae is preferably used as the microorganism capable of producing the squalene. The squalene is produced in high yield and ethanol is simultaneously produced by culturing the Saccharomyces cerevisiae under the anaerobic conditions.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing squalene and ethanol, and more particularly to a method for simultaneously producing squalene and ethanol using microorganisms.

Squalene is the raw material for squalane, which is used in cosmetic base oils. Squalene is an unsaturated hydrocarbon composed of c,D H5O, and squalane can be obtained by hydrogenating this squalene.

Squalene is also used as a health food.

(Prior art and problems to be solved) Squalene is extracted and refined from shark liver oil produced from the livers of deep-sea sharks such as merganser sharks and kingfishers, but since sharks are caught from the wild, the catch is extremely unstable. Therefore, uncertainty in the supply of squalene and large fluctuations in the price were unavoidable. Additionally, it is said that shark liver oil from sharks caught in the waters around Japan cannot be used because it contains chemicals that cannot be refined due to marine pollution.

Therefore, a method for producing squalene that does not use natural materials as a raw material is desired, and a method for producing squalene using microorganisms has been devised. One of them is a method using filamentous fungi of the genus Morteierella (Japanese Patent Publication No. 59-20356), but this method has the drawback of low productivity in terms of culturing filamentous fungi. As another method, a method using bacteria has been proposed (Japanese Patent Application Laid-Open No. 61-212290), but this method also has a low content of bacteria per cell at a maximum of L48n+g/g cells, and has not been put to practical use. In addition, both are aerobic fermentations.

On the other hand, ethanol production by fermentation is widely practiced worldwide, but the problem with this method is cost reduction, and various ideas have been developed.

For example, the use of a novel Saccharomyces cerevisiae yeast as disclosed in JP-A No. 62-65679 is said to be industrially advantageous due to its high ethanol productivity and flocculating properties. Furthermore, JP-A-62-126986 describes that efficient fermentation production of ethanol was achieved by devising an apparatus.

On the other hand, as in the present invention, producing squalene under anaerobic conditions and simultaneously producing squalene and ethanol using microorganisms are both completely unknown and new.

(Means for Solving the Problems) The present invention was made for the purpose of establishing an industrial method for producing squalene in view of the current state of technology.6 As a result of various studies to achieve the above purpose, microorganisms We obtained the knowledge that it is possible to produce squalene during the production of ethanol from the metabolic pathway of By producing squalene that does not use unstable and unstable natural products as raw materials, and by co-producing squalene, which is useful and expensive, with ethanol, the cost issue was also resolved.

With the aim of solving these problems, the present inventors conducted various studies and found that squalene exists widely in living organisms as an intermediate metabolite of eukaryotes such as molds and yeast, and that squalene exists widely in living organisms as an intermediate metabolite of eukaryotes such as molds and yeast. - It was discovered that Cereviche has a strong squalene production capacity and is suitable for squalene production.

As a result of further investigation, we focused on the metabolism of squalene within microbial cells. In other words, squalene is an intermediate product in the metabolic pathway that converts acetyl-CoA into squalene via mevalonic acid, which ultimately leads to ergosterol, in the cells of euphytoorganisms such as molds and yeast. be. The present inventors newly focused on the fact that squalene is converted to squalene epoxide by the enzyme squalene epoxidase in this metabolic pathway.

Focusing on this point, the present inventors have found that by creating an anaerobic atmosphere with little oxygen in the environment of microorganisms, production of the enzyme squalene epoxidase is suppressed, resulting in accumulation of squalene within the microbial cells. Furthermore, since ethanol is also accumulated outside the bacterial body, we considered that it might be possible to co-produce ethanol and squalene. We succeeded in producing squalene and squalene at the same time, and confirmed for the first time that the above estimation is actually possible.

The present invention was made based on this new and useful knowledge, and its basic technical idea is to produce squalene by maintaining microorganisms under anaerobic conditions. According to microorganisms that also have the ability to produce ethanol, it is possible to simultaneously produce ethanol as well as squalene.

The present invention will be explained in detail below.

The present invention utilizes the squalene metabolic system described above, and therefore any microorganism that has such a metabolic pathway can be used. And at that time,
If the microorganism has the ability to produce ethanol, it can produce squalene and ethanol at the same time as described above by maintaining it under anaerobic conditions.

Examples of such microorganisms include Saccharomyces cerevisiae.
iae), and by culturing it anaerobically, squalene is accumulated within the bacterial cells and ethanol is secreted and accumulated outside the bacterial cells. For industrial implementation, it is preferable to select and use a strain with higher productivity.

To carry out the present invention, such microorganisms are inoculated into a nutrient medium containing assimilated carbon and nitrogen sources and cultured under anaerobic conditions.

Carbon sources include glucose, sucrose, starch,
Preference is given to using blackstrap molasses, fructose, glycerin and other carbohydrates.

Nitrogen sources include oatmeal, yeast extract, peptone, gluten meal, cottonseed flour, soybean meal,
In addition to cornstarch liquor, dried yeast, wheat germ, peanut flour, chicken bone meal, etc., ammonium salts (
Inorganic and organic nitrogen compounds such as ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acids, etc. can also be used advantageously.

Although it is advantageous to use these carbon sources and nitrogen sources in combination, it is not necessary to use pure sources. This is because impure products contain growth factors and trace elements.

If necessary, in addition to vitamins, inorganic salts such as the following may be added to the medium: sodium carbonate,
Potassium carbonate, sodium phosphate, potassium phosphate, sodium chloride, potassium chloride, calcium chloride, sodium iodide, potassium iodide, magnesium salt, copper salt,
Cobalt salt etc.

A culture medium is prepared using the necessary amounts of each of these necessary components, and at that time, the composition of the medium for ethanol fermentation can also be fully utilized. After putting this into a fermenter and inoculating it with microorganisms, nitrogen gas and other Under anaerobic conditions such as an inert gas atmosphere or air-blocking conditions, with or without stirring at about 10 to 1100 rpm, at the growth temperature of microorganisms (about 20 to 45 °C, although it varies depending on the microorganism), as necessary. Cultivate for a period of time (for example, 10 to 200 hours in the case of yeast).

Although the desired purpose can be achieved even if the culture is carried out as described above, if the culture is carried out while adding vitamins, especially thiamine and/or maintaining the sugar concentration, the yield of ethanol and squalene will be reduced. In particular, the yield of squalene increases. The sugar concentration differs depending on the bacteria used, but in the case of S. cerevisiae, it is preferably maintained at 0.3 g/l or more, preferably 0.5 g/Ω or more, and more preferably 1 g712 or more.

The sugar concentration may be maintained as appropriate according to conventional methods; for example, a fed-batch culture method in which a nitrogen source and a carbon source are simultaneously supplied based on a pH-stat method is one of the preferred methods. Specifically, ammonium hydroxide, ammonium water, etc. are added to adjust the pH and serve as a nitrogen source, and at the same time, a carbon source is also added during cultivation. As a result, the sugar concentration in the medium is maintained constant. This makes it possible to efficiently produce ethanol and squalene, especially squalene, which has been difficult to produce in the past.

After completion of the culture, the target substance is separated and recovered from the culture (microbial cells, culture solution containing the microorganisms, culture solution from which the microorganisms have been removed, culture filtrate from which the culture medium of the microorganisms has been removed, etc.). . Separation and recovery of the target substance may be performed according to conventional methods.

Although it differs depending on the microorganism used, when S. cerevisiae is used, ethanol is usually secreted and produced outside the microorganism, and squalene is accumulated inside the microorganism. therefore,
After the cultivation is completed, the bacterial cells are separated from the culture solution, ethanol is recovered from the culture solution by distillation, and squalene is extracted from the bacterial cells using a solvent. As the extraction solvent, use a low polar solvent such as N-hexane or petroleum ether if the bacterial cells are dried, or methylene chloride, methanol, or It is efficient to use a mixed solvent such as ethanol/N-hexane. In order to take out the squalene dissolved in the solvent alone, the solvent is removed by distillation, and the resulting crude squalene is distilled under reduced pressure to obtain highly pure squalene. If necessary, the bacterial cells may be destroyed using enzymes, ultrasound, etc. prior to extraction.

Squalene can be identified by mass spectrometry and nuclear magnetic resonance spectroscopy, and squalene can be quantified by gas chromatography.

(Examples) The present invention will be described below with reference to Examples, but the present invention is not limited thereto.

Example 1 Two ethanol-producing yeast strains were cultured to produce squalene and ethanol using a small fermenter with a 5Q capacity as a culture vessel and a medium having the composition shown below.

Dust Storm Soglucose 100g #1 (NH4)
2So45g/12 to 82P041g/l MgSO4・7H201g#I CaCL・2820 0, 17
g/Q Yeast Extract 0.5g/l Inositol 0.1g#! Vitamin mixture (3 LmQ/l ``j''''
(B/100m12, biotin
0.35 Calcium pantothenate 5
0 Pyridoxal hydrochloric acid 25 Thiamine hydrochloric acid 100 Nicotinic acid amide 100
Add 5ac, which is an ethanol-producing yeast, to this.
charo+ayces ceravisiae AT
CC26603 and SaCcharomyces ce
revisiae IFO216, and stirred at 30°C, no ventilation, and 84 rpm at 1100 rpm.
After culturing for a period of time, the results shown in Table 1 were obtained.

Table 1 Squalene production amount C+++g/g-cell)
0.35 8.81 Dry bacterial cell concentration (g/1
2) 4.135 4.25
As is clear from the above results, it was confirmed that there are differences in squalene production ability even among the same ethanol-producing yeasts, and Satucharomyces cerevisiae strain IF0216 was selected as a particularly excellent squalene-producing bacterium.

Example 2 (1) Ethanol-producing yeast was cultured under anaerobic conditions to produce squalene and ethanol using a small fermenter with a capacity of 2.3Q and a medium having the composition shown below as a culture vessel.

1. Glucose 30g/l (N) 14)
2S04 5g/lKH2P047
g/l MgSO, 77H2O1/l CaCL 2H200, 17g/Ill Yeast Extract 0.5g #1 Inositol
O, 1g/ρ vitamin mixture (Li 1mQ/
l3°) 4sunoq (mg/l.., 9) Biotin 0.35 Calcium pantothenate 50 Pyridoxal hydrochloric acid 25 Thiamine hydrochloric acid 100 Nicotinic acid amide 100 That is, a medium with the above composition was placed in a 2.3Q jar fermenter. 2Ω, and the ethanol-producing yeast Saccharomyces cerevisiae was added to this.
IFO216 was inoculated and cultured for 78 hours at pH 5.0, temperature 30°C, no ventilation, and stirring at 1100 rp, and the temporal changes in bacterial cell concentration, sugar concentration, squalene content, and alcohol concentration were measured. The results shown in Figure 1 and Table 2 were obtained.

Table II As shown, 48 hours after the start of culture, the ethanol concentration and squalene concentration (maximum value 12 mg/g-cell) increased, and the sugar concentration showed Ig/l. Further, after 48 hours, there is almost no change in the ethanol concentration, but when the sugar concentration drops below 1 g/l, the squalene concentration tends to decrease, indicating that squalene production performance will decline if the sugar concentration is not maintained. is clear.

From these results, from the standpoint of industrial production of squalene, it is recommended that the sugar concentration be maintained at 0.3 g/12 or higher, preferably 0.5 g/l or higher, particularly preferably Ig/II or higher. I understand that.

(2) Under the conditions described in the previous section, except that the cultivation was initially carried out under aerobic conditions rather than anaerobic conditions.
FO0216 was cultured in a semi-batch mode. Then, after 36 hours, the conditions were changed to anaerobic conditions and anaerobic culture was carried out.
Culture was continued for 84 hours. and the bacterial cell concentration during this period.

The temporal changes in sugar concentration and squalene content were measured, and the results shown in FIG. 2 were obtained.

As is clear from the results in FIG. 2, the growth rate under aerobic conditions was μ, x=o, is, which was the same as the value under anaerobic conditions. These results revealed that the activity of the electron transport system of this bacterium is weak, making it difficult to improve the bacterial growth rate under aerobic conditions.

After 36 hours from the start of culture, a new medium was added and aeration was stopped, and the dissolved oxygen concentration reached almost zero in about 20 minutes. Bacterial growth started again and all added sugars were consumed in 12 hours, resulting in a final bacterial cell concentration of 2.7 g/l. After shifting to anaerobic conditions, squalene was actively accumulated, reaching 17 mg/g-cell at the beginning of the stationary phase, but then rapidly decreasing.

From the above results, it was found that squalene accumulates actively in the bacterial cells during the late logarithmic growth period and the early stationary phase, and rapidly decreases when sugar in the culture solution is depleted. In other words,
For efficient production of squalene.

The necessity of anaerobic conditions and sugar was confirmed once again.

Example 3 Using a small fermenter with a capacity of 2.0Q as a culture vessel, ethanol-producing yeast S, cerevisiae IFO
216 was subjected to fed-batch culture under anaerobic conditions while controlling the sugar concentration not to fall below Ig#1.

A culture medium with the same composition as that used in Example 2 was used, but the following composition and aqueous ammonia (25.6g #l) were added as a supplementary medium to maintain the sugar concentration. A mixed medium was used in which each medium was mixed separately and/or simultaneously so that the pH of the medium was 5.0.

Replenishment medium composition Glucose 210g/ρKH, PO
47g/l MgSO4・7Hz01g/l CaCI22-28,0 0.17
g#1 yeast extract 0.5g/l inositol 0.1g/l vitamin mixture (
J 1aQ/l biotin
0.35 Calcium pantothenate 5° Pyridoxal hydrochloride 25 Thiamine hydrochloric acid 100 Nicotinic acid amide 100 That is, 1.012 of the medium used in Example 2 was put into a small fermenter with a capacity of 2.0 Q, and ethanol-producing yeast S,
cerevisiae IFO216 was inoculated into a fermenter, temperature was 25°C, nitrogen gas was aerated at 0.5ff/win, 3
The culture was carried out by stirring at 00 rpm, monitoring changes in sugar concentration by monitoring changes in pH of the medium, and appropriately introducing ammonia mixed medium to maintain pH 4.8.

Since the medium amount reached 1.4Q after 54 hours, 0.7Q
After 99 hours, the culture was terminated.
．． The amount of ethanol was 89 g, and the amount of squalene was 115+ng. The progress of the culture was as shown in Table 3.

0 0.18 5.1 0.2 10
23 2.1 6.5 9.0 3.
038 3.4 5.6 23.0
8.154 3.4 4.3 33.8
16.174 4.0 5.6 38.1
14.0 (Effects of the Invention) According to the present invention, squalene can be industrially produced in large quantities by a fermentation method using microorganisms by adopting a new system for controlling culture conditions, without relying on extraction from natural products such as deep-sea sharks. can do.

Further, according to the present invention, by using ethanol-fermenting microorganisms, it is also possible to industrially produce ethanol in addition to squalene simultaneously and in large quantities.

[Brief explanation of drawings]

Figure 1 shows S. cerevisiae IFO216.
FIG. 2 is a drawing showing the progress of culturing the same bacterium under anaerobic conditions, and FIG. 2 is a drawing showing the progress of culturing the same bacterium under aerobic conditions and then under anaerobic conditions. Agent: Patent attorney 1) Parent: Male dry bacterial cell conductivity ig/17・su') 7len k1 weight [mg/g-cell l sI
A-yari concentration (q/ll. OLn' Alcohol concentration [g/13Δ Dry cell conductivity 1 g/II/Squalene production [mg/g-cel 1st residual glucose J degree [[]/ll.

Claims (6)

[Claims] (1) A method for producing squalene, which comprises culturing squalene-producing bacteria under anaerobic conditions. (2) A method for producing squalene, which comprises culturing squalene-producing bacteria belonging to the genus Saccharomyces under anaerobic conditions and collecting squalene from the culture. (3) A method for producing squalene and/or ethanol, which comprises culturing squalene and ethanol-producing bacteria under anaerobic conditions. (4) A method for producing squalene and/or ethanol, which comprises culturing squalene and ethanol producing bacteria belonging to the genus Saccharomyces under anaerobic conditions and collecting squalene and/or ethanol from the culture. (5) Sugar concentration of the medium is 0.5 g/l or more, preferably 1 g
5. The method according to any one of claims 1 to 4, characterized in that it is maintained at or above /l. (6) The method according to any one of claims 1 to 5, characterized in that thiamine is added to the medium.