JPS5953033B2 - Microbial culture method - Google Patents

Description

Detailed Description of the Invention The present invention involves culturing microorganisms using hydrocarbons or hydrocarbon derivatives as the main carbon source, by adding glycolipids produced by yeast of the genus Torulopsis to the culture medium. Regarding the method.

Fermentation production research using hydrocarbons or their derivatives as the main carbon source has made remarkable progress in recent years, with the production of microbial proteins reaching a stage of practical use, as well as various amino acids, nucleic acid-related substances, vitamins, and organic It is widely used in the production of physiologically active substances such as acids, carbohydrates, and antibiotics.
It has become possible to replace most of the carbohydrates produced by fermentation using carbohydrates as the main carbon source with hydrocarbons.

On the other hand, hydrocarbons or their derivatives either have carbon and hydrogen as their only constituent elements, or contain these as their main constituent elements and some other elements, such as oxygen and halogens. The amount of effective carbon per unit weight is about twice that of the quality of carbon, and as a fermentation carbon source,
It has clear advantages over carbohydrates.

However, when fermentation is carried out using hydrocarbons or derivatives thereof as carbon sources, the following problems arise due to their physical and chemical properties.

One of these is that hydrocarbons or their derivatives do not contain oxygen in their molecules, or even if they do contain a small amount, they require a very large amount of oxygen to be supplied in order to grow microorganisms. Another reason is that hydrocarbons and their derivatives are insoluble in water, so the uptake mode by microorganisms is different from that of water-soluble carbon sources such as carbohydrates, and the uptake rate is low, and the growth rate is generally low. It is said that this is about half of that from carbohydrate carbon sources.

The first problem of oxygen supply is being solved in recent years, due to remarkable advances in culture engineering, by devising and improving fermenters that can supply oxygen efficiently.

However, the second problem, the efficient uptake of hydrocarbons or their derivatives by microorganisms, is thought to depend on the frequency of simple mechanical contact between the water-insoluble substrate and the microorganisms. Efforts have been concentrated on devising mechanical and physical emulsification and dispersion methods, and (ii) physicochemical emulsification and dispersion methods using surfactants, but both can only be applied to limited microorganisms.

These artificial methods of forcing microorganisms to incorporate water-insoluble substrates such as hydrocarbons or their derivatives ignore the physiological properties of the microorganisms themselves, and are not very successful in practice. Met.

These results suggest that the uptake mechanism of water-insoluble substrates by microorganisms is mainly based on the physiological properties of the microorganisms themselves.As a result of our studies from this perspective, the present inventors found that: It has been found that microorganisms that utilize water-insoluble substrates have the following unique mechanisms for taking in water-insoluble substrates.

In other words, i. the microorganism itself has an organization on its cell surface that has a high affinity for water-insoluble substrates, and ii.
), and actively incorporates substances that have the function of

Mechanism i is considered to be an original function of the microorganism and cannot be adjusted artificially, but mechanism ii can be adjusted by understanding and controlling the substance.

As a result of further study based on the above considerations, the present inventors found that
When culturing microorganisms using hydrocarbons or their derivatives as the main carbon source, if a glycolipid represented by the following general formula (I) is added to the medium, these carbon sources will be taken up quickly and the growth of the microorganisms will be inhibited. The present invention was completed based on the discovery that the present invention can be promoted.

(In the formula, R1 and R2 are H or -COCH3, R3 is H or -CH3, and R4 has 12 to 16 carbon atoms when R3 is H.
When R3 is -CH3, it represents a saturated or unsaturated hydrocarbon group having 11 to 15 carbon atoms.

) The glycolipid represented by the general formula (I) is derived from Torulopsis (
Certain yeasts of the genus Torulopsis, such as Torulopsis magnoliae (T, magnoliae)
, Torulopsis apicola (T. apicola)
, Torulopsis grotspengiersseri , Torulopsis X-bonbicola (T, bombicola), etc. (J, F, T, 5
pencer et al.

Canadian Journal of Ch.
emistry 39, 846 (1961)).

In this case, the glycolipids produced are of the following formulas (I) and (I
It is a mixture consisting of a compound represented by I) and an isomer (III) of formula (II) with different sugar lactone bonding positions.

Hereinafter, these will be collectively referred to as sophorolipids (
(sometimes abbreviated as SL).

Formula (I): ■-a; R1=R2=COCH3 ■-b; R
1-COCH3, R2=H I-C; R□=H, R2-C0CH3 I-d; R1=R2=H (in the formula, R3 and R4 are the same as above) II-a; R1-R2=C0CH3 II- b: R1=COCH3, R2=HII-
C; R1=H, R2-C0CH5 II d ; R1= R2= H Sophorolipids can be extracted, for example, from the culture solution of Torulopsis bombicola with an organic solvent that dissolves glycolipids, such as ethyl acetate, chloroform, or ethyl ether. , crude sophorolipids are obtained by separating sophorolipids by taking advantage of their tendency to precipitate in a culture solution, and if the culture solution is contaminated with components such as fat-soluble substances, the sophorolipids are not dissolved and lipids are removed. It is desirable to purify by washing several times with an organic solvent that dissolves only soluble substances, such as n-hexane, petroleum ether, etc.

In the present invention, the sophorolipid mixture containing the glycolipid represented by formula (I) may be added as is, or the glycolipid represented by formula (I) may be separated from the sophorolipid mixture and used.

The glycolipid represented by formula (I) is, for example, a component (mainly composed of formulas (II) and (III)) that precipitates by dispersing and dissolving the above sophorolipid mixture in water and leaving it to stand. (sometimes referred to as water-insoluble sophorolipids) and components dissolved in water (mainly consisting of glycolipids represented by formula (I), hereinafter sometimes referred to as water-soluble sophorolipids). It can be separated from the mixture by fractionation.

Fractionation can generally be carried out by dispersing and dissolving the sophorolipid mixture in water, but in order to shorten the treatment time, the sophorolipid mixture can be forcibly placed in warm water by ultrasonication etc. Then, the water-insoluble sophorolipits are precipitated by standing at a low temperature, separated into filtrate and precipitate by filtration, etc., the filtrate is distilled under reduced pressure to remove most of the water, and dried under vacuum. It is appropriate to obtain a solid form of sophorolipid.

Alternatively, the sophorolipid mixture is dissolved in an aqueous solution of a lower alcohol such as methanol or ethanol, and heated with an alkaline reagent such as sodium hydroxide or potassium hydroxide to remove the acetyl ester bond on the machine without decomposing the glycosidic bond of the sophorolipid. It is also possible to cleave (hydrolyze) only the lactone bond and convert almost the entire amount into a glycolipid represented by formula CI-d (hereinafter sometimes referred to as acid sophorolipid) for use.

The mechanism of promoting the uptake of hydrocarbons or derivatives thereof by microorganisms by the glycolipid represented by formula (I) of the present invention is the effect of emulsifying and dispersing hydrocarbons or derivatives thereof, which are water-insoluble substrates, which ordinary synthetic surfactants have. It turns out that the functionality is completely different.

That is, polyoxyethylene alkyl (C2-02°) ether, polyoxyethylene alkyl (C8C9) phenyl ether, polyoxyethylene, which are nonionic synthetic surfactants conventionally used in n-paraffin media. Higher fatty acid (0□2 C1) ester, sorbitan higher fatty acid (C12-C18) ester, polyoxyethylene sorbitan higher fatty acid (C1□-018) ester, polyoxyethylene sorbitol higher fatty acid (
C1□-C□8) ester, higher fatty acid (C12-01
8) Monoglycerides and higher fatty acids (C1°-C18
) Even if sucrose ester or the like is added under the same conditions, the microorganism-promoting effect like the glycolipids of the present invention cannot be obtained.

Hydrocarbons or derivatives thereof used as the main carbon source include any that are generally used as water-insoluble carbon sources, but hydrocarbons having 8 to 20 carbon atoms or derivatives thereof are suitable; In particular, n-alkanes having 10 to 18 carbon atoms are suitable, and have a remarkable promoting effect.

Hydrocarbons may be used singly or as mixtures containing them, such as petroleum fractions (e.g. kerosene, kerosene fraction, etc.)
But of course it's a good thing.

In addition, hydrocarbons with unsaturated bonds (alkenes, α-
The microorganism-promoting effect of the present invention can also be seen when using as the main carbon source a derivative having oxygen (alcohol, fatty acid, fatty acid ester, etc.), or a derivative having a halogen atom (alkyl halide, etc.).

It is appropriate that these carbon sources be present in the medium at 0.2 to 10% wt/v.

The glycolipid represented by formula (I) is present in the medium at 0.0027
It was found that adding %wt/v or more is effective.

Therefore, when adding water-soluble sophorolipids, it is effective at this amount or more, and when adding a mixed vIJ (sophorolipid mixture) containing water-soluble sophorolipids, the content is 7.
Since the amount may vary, it is effective to use an amount greater than the above amount in terms of water-soluble sophorolipid.

The growth promoting effect will improve if the amount added is increased, but in practice it is 0.0027-0.2%w as a water-soluble sophorolipid.
It is better to add t/v.

In culturing the microorganism according to the present invention, the other culture conditions can be the usual conditions employed in microbial culture using hydrocarbons or derivatives thereof as the main carbon source.

For example, peptone, yeast extract, meat extract, corn steep liquor, amino acids, casamino acids, ammonium nitrate, etc. can be used as nitrogen sources, and potassium,
Commonly used inorganic ions such as sodium, magnesium, and phosphorus can also be used.

The culture temperature may be a normal temperature, but a temperature of about 18 to 37°C is suitable.

The effect of promoting the growth of microorganisms using hydrocarbons or derivatives thereof as the main carbon source by adding the glycolipid represented by formula (I) is widely seen in microorganisms that assimilate hydrocarbons or derivatives thereof. Among yeasts, an example of a hydrocarbon-assimilating yeast belonging to Ascomycete yeast is Metschnikowia pulhierima belonging to the genus Metshnikowia.
cherrima), Pichia farino which belongs to the genus Pichia
sa), etc., and there is also a Ustei Rajina L/su (U
An example of a hydrocarbon-assimilating yeast belonging to the yeast family S. stilaginales is Rhodosporidium (Rhodosporidium).
There are yeasts of the genus Sporidium, and examples of hydrocarbon-assimilating yeasts that belong to the non-sporulating yeasts include Candida mogi (that belong to the genus Candida).
Candida mogii), Candida lipolytica (Candida 1ipolytica)
)/1,1nia7° Torulopsis bombicola (Torulopsis) belonging to the genus Torulopsis
sis bombicola), Torulopsis
iesseri), Torulopsis ahicola (To
rulopsis apicola), Torulopsis
Torulopsis magnoli
ae) etc.

According to the present invention, the uptake of hydrocarbons or derivatives thereof, which are conventional carbon sources, is slow, and therefore the growth rate of microorganisms is slow.
As a result, the time required for culturing was greatly shortened.

In order to better understand the present invention, reference examples,
Although the present invention will be explained using test examples and examples, the present invention is not limited to these scopes.

Reference Example 1 Preparation of Sophorolipid Add 500 g of glucose and 25 g of yeast extract to 51 deionized water, dissolve uniformly, and add 500 g of safflower oil.
The medium to which g was added was placed in a 101 volume jar fermenter and sterilized in the jar fermenter to prepare a medium solution.

Torulopsis bombicola AT was then added to a shake flask containing 50 ml of a culture solution prepared with 2.5 g of previously sterilized glucose and 0.25 g of yeast extract.
CC22214 was inoculated and cultured with shaking at a temperature of 25°C for 2 days, and the culture solution in which sufficient bacteria had grown was used as the total inoculated bacterial solution.
The jar fermenters described above were inoculated aseptically.

After inoculation, temperature: 30°C, stirring number: 300 rpm, aeration volume: 1
7 under culture conditions of vvm (air volume equal to medium per minute).
Fermentation was carried out for one day.

After fermentation is complete, take out the fermented liquid and make a 101 volume inner diameter 15
The sophorolipid fraction in the fermentation broth was transferred to a cylindrical phase separation tube with a diameter of 50 cm and subjected to sedimentation fractionation at 50°C.

If necessary, several hundred ml of ethyl acetate was added to speed up the sedimentation fractionation of sophorolipids.

Next, the sophorolipid fraction that has settled in the lower layer is extracted,
Most of the water contained was distilled off using a rotary evaporator, the sophorolipid concentrate was extracted with 750ml of ethyl acetate, the ethyl acetate extract was dehydrated with anhydrous sodium sulfate, and most of the ethyl acetate was distilled off using a rotary evaporator. After washing and extracting twice with 200 ml of n-hexane, the sophorolipids insoluble in n-hexane were spread into a film in a petri dish and dried under reduced pressure in a vacuum dryer until a constant weight was reached.

This operation yields about 380 g of sophorolipid (formula (I)
A mixture of the products prepared by , , Ⅲ) and (III), whose fatty acid moiety mainly has 16 and 18 carbon atoms, was obtained.

In addition, if the amount of fermentation liquid is 11 or less, the sedimentation fractionation operation using a phase separation tube among the above operations is omitted, and the ethyl acetate layer obtained is directly extracted with an equivalent amount of ethyl acetate, and the resulting ethyl acetate layer is treated with the above method. Similar sophorolipids can also be obtained.

Reference Example 2 Preparation of Water-Soluble Sophorolipid 3 g of the sophorolipid obtained in Reference Example 1 was placed in a 500 ml flask, 300 ml of deionized water was added, and the mixture was ultrasonically dispersed using an ultrasonic transmitter.

2. Leave to stand at 4°C for 5 days, divide into a solution to remove the precipitate, and dry the precipitate under vacuum to form an amorphous white solid.
8g was obtained.

This was designated as water-soluble sophorolipid.

After distilling off most of the water from the filtrate under reduced pressure, it was vacuum dried.
0.8 g of an amorphous yellow solid was obtained.

This was designated as water-soluble sophorolipid.

Reference Example 3 Preparation of Acid Sophorolipid 5 g of the sophorolipid obtained in Reference Example 1 was placed in a 100 m round bottom flask, and then 10 ml of water and 25 ml of ethanol were added.
After adding 1 ml of the solution and uniformly dissolving it, 0.5 g of potassium hydroxide was added and the mixture was allowed to reflux for 2 hours.

After the reaction was completed, the mixture was washed with a dilute aqueous sulfuric acid solution, the precipitated potassium sulfate was separated, and the P solution was distilled off under reduced pressure using a rotary evaporator to obtain 4.3 g as a residue.

This reaction material was analyzed by silica gel thin layer chromatography [developing agent, chloroform-methanol-acetic acid (70:25:5), l
When this is carried out, a single spot with an Rf value of 0.33 is produced which develops a bluish-green color using the Anthrone coloring reagent.

Next, dissolve the entire amount of this substance in 5 ml of chloroform,
Filled with 50g of silica gel, inner diameter 1cm, length 50cm
Chloroform-ethanol (chloroform 100 parts to 0 parts, ethanol 0 to 100 parts
Chromatographic separation was performed using the elution solvent of part), and the elution fraction corresponding to an Rf value of 0.33 was collected in the above thin layer chromatography analysis, and the solvent was distilled off to obtain 4.1 g of purified product.

This purified product is a white solid substance, which is acidosophorolipid.

When this acido-sophorolipid was subjected to infrared spectrum analysis using the KBr tablet method, a wide absorption band originating from the hydroxyl group of sugar from 3380 to 3200 cm' was observed, and a wide absorption band at 2800 cm' was observed.
It gives a strong absorption band originating from aliphatic methylene and methyl groups around -1, an absorption band originating from free carboxyl groups at 1700 cm-1, and an absorption band characteristic of glucobylanose rings from 900 to 750 cm''.

In nuclear magnetic resonance spectroscopy analysis in pyridine solvent, an absorption spectrum of a double bond characteristic of unsaturated fatty acids around 65.5, a wide absorption spectrum originating from the sugar structure over δ3.5 to 5.0, and a wide absorption spectrum originating from the sugar structure around δ1 .1 to 1.6, a strong absorption spectrum derived from methylene groups was given.

Next, 0.1 g of this purified product was added to 10 ml of 5N HCl.
- A reflux reaction was carried out in a methanol solution for 1 hour to form methyl glycoxide and hydroxymethyl ester, and each of them was quantitatively analyzed by graphic analysis on Gas Chroma 1. As a result, the ratio of methyl glycoxide and hydroxy fatty acid methyl ester was 2 moles to 1. The molar ratio was confirmed.

From the above results, it was supported that this purified product was an acid sophorolipid.

Test Example 1 The sophorolipid obtained in Reference Example 1 was used at 0.75 wt/v% in a dry medium for carbon compound assimilation test for yeast (Bact.

Nitrogen base: Difco Lab
, Inc. (Michigan, USA)) and 5 v/v% n
-0.064wt in a sterile medium containing hexadecane
/v% concentration using aseptic technique, and then torulopsis bombicola (Toru
lopsis bombicola) KSM-36 NokoTnia knee was taken with a platinum loop, and adjusted in physiological saline so that the absorbance was 0.5 to 0.6 when measured with an absorbance needle with a bacterial cell concentration of 650 parts m. The bacterial suspension was inoculated into the above medium at an amount of 2%, or in some cases up to 10%, and cultured at 30°C, and the growth of the bacterial cells was observed.

To observe the growth rate, collect some or all of the culture solution, adjust the pH of the collected culture solution to 2 to 3 with mineral acid, such as dilute hydrochloric acid or dilute sulfuric acid, as necessary, and then add twice the amount of acetic acid. The culture solution was washed twice with ethyl, and the aqueous phase containing the resulting bacterial cells was diluted with deionized water to a concentration that could be measured with a 55 Qnm absorbance needle, and the absorbance of the aqueous phase was measured.

Table 1 shows a comparison with the same conditions without the addition of sophorolipid.

From the results in Table 1, it can be seen that the addition of the sophorolipid mixture clearly promotes the growth of bacteria.

Test Example 2 Medium: n-hexatemin (5%) - Dry medium for carbon compound assimilation test (0.75%) - Yeast extract (0.2%)
) culture medium (hereinafter abbreviated as DNB-YE medium), other conditions were the same as in Test Example 1, and the oral changes in growth due to the addition of sophorolipid were investigated.

The results are shown in Table 2. Test Example 3 Various n-alkanes (5%) as a medium - Dry medium for carbon compound assimilation test (0.75%) - Yeast extract (0.2%)
) Culture medium was used and other conditions were the same as in Test Example 1, and the growth promoting effect of the addition of sophorolipid was investigated.

The results are shown in Table 3. Test Example 4 Using the water-soluble sophorolipid and water-insoluble sophorolipid obtained in Reference Example 2 instead of sophorolipid, culturing was carried out under the same conditions as Test Example 2 except that the number of culture days was 4 days, and the growth rate was measured. , the results shown in Table 4 were obtained.

These results show that the microbial growth promoting effect of sophorolipids is due to water-soluble sophorolipids [glycolipids represented by formula (I)].

Test Example 5 The amount of sophorolipid containing about 27% water-soluble sophorolipid (prepared in Reference Example 1) was varied,
Microorganisms were cultured under the same conditions as in Test Example 2, except that they were cultured for one day, and the growth rate was determined.

The results are shown in Table 5. This result shows that it is effective when the amount of sophorolipid added to the medium is 0.01%wt/v or more, and the effective amount of water-soluble sophorolipid added is 0.0027%wt/v based on the content in the sophorolipid.
It can be seen that the value is greater than or equal to v.

Test Example 6 n-hexadecane (2%) as medium = DNB-YE
Culture was carried out under the same conditions as in Test Example 1, except that a medium was used, and the results shown in Table 6 were obtained.

Example 1 In advance, a dry flame medium for yeast carbon compound assimilation test (B
acto-Nitrogen-base, hereinafter DN
B groans.

0.75% yeast extract (manufactured by Difco, USA), yeast extract (Ye
ast extract, hereinafter abbreviated as YE.

Liquid medium (hereinafter referred to as DN) containing 0.2%
It is abbreviated as B-YE medium.

) in a test tube with an inner diameter of 2 cm and a length of 20 cm.
I put in 1 each.

Next, after adding 0.5 ml of n-hexadecane, 11
Steam sterilization was performed at 5°C for 30 minutes.

Next, add sophorolipid aseptically to 10 m of previously sterilized deionized water, warm it to disperse it uniformly, and add 0.5 ml of it to the above n-hexadecane-DNB-YE area.
was added aseptically.

Torulopsis bombicola was then grown on a glucose-yeast extract-malt extract (YM medium, manufactured by Difco, USA) agar medium at 30°C for 2 days.
isbombicola) with a platinum loop and aseptically added to 10 ml of sterilized physiological saline, and the absorbance of the bacterial suspension at a wavelength of 650 nm was measured with a variable needle of 0.5 to 0. A bacterial suspension with a concentration of .6 was prepared, and 0.5 ml of the suspension was collected and added aseptically to the above-mentioned n-hexadecane-DNB-YE-ethylsophorolipid medium.

5 in a shaking culture machine with 120 reciprocating shakes per minute at a temperature of 30°C.
The bacterial cells were grown for several days.

After the culture was completed, the liquid properties of the culture solution were measured, and if necessary, the pH was adjusted to 2-3 with dilute hydrochloric acid, and ethyl acetate in an amount twice the amount of the culture solution was placed in a shaking test tube for extraction.

After performing the extraction operation twice, shake the aqueous phase of the culture solution well to make a homogeneous bacterial suspension, collect 0.5 ml of it, and add 15 ml of it.
Transfer to a volume test tube, dilute 10 times with deionized water,
The absorbance at a wavelength of 5Qnm was measured to determine the degree of bacterial growth.

The results were as shown in Table 7.

Example 2 Growth promoting effect of sophorolipid on growth of Metschnikowia yeast on non-hexadecane carbon source medium The method was carried out in the same manner as in Example 1, and the results are shown in Table 8.

Example 3 Growth promoting effect of sophorolipid on growth of Pichia yeast on n-hexadecane carbon source medium The method was carried out in the same manner as in Example 1, and the results are shown in Table 9.

Example 4 1'1 of yeast of the genus Torulopsis
- Growth promoting effect of sophorolipid on growth in hexadecane carbon source medium The method was carried out in the same manner as in Example 1, and the results are shown in Table 10.

Example 5 Growth-promoting effect of sophorolipid on growth of Candida yeast on n-hexadecane carbon source medium The method up to crab cultivation was carried out in the same manner as in Example 1, and the subsequent growth was measured from the culture solution. A certain amount of the culture solution was taken, diluted appropriately, and then cultured on YM agar medium without pour culture at 30° C. for 7 days. The number of growing colonies was calculated and determined as the degree of growth.

The results are shown in Table 11. Example 6 Growth-promoting effect of water-soluble sophorolipid on the growth of petroleum-utilizing yeast in n-hexadecane carbon source medium The water-soluble sophorolipid prepared from Reference Example 2 and the water-insoluble sophorolipid were treated according to the method of Example 1. A test was conducted to examine the growth-promoting efficacy of water-soluble sophorolipids on the growth of petroleum-utilizing yeast in an n-hexadecane carbon source medium.

The results are shown in Table 12.

Claims (1)

[Claims] 1. When culturing microorganisms using hydrocarbons or hydrocarbon derivatives as the main carbon source, a culture medium containing the following general formula (I), (wherein R1 and R2 are H or -COCH3, and R3 is H or -CH3 and R4 have 12 to 16 carbon atoms when R3 is H
represents a saturated or unsaturated hydrocarbon group, and R3 is -CH3
A method for culturing microorganisms, which comprises adding a glycolipid represented by the following formula (indicates a saturated or unsaturated hydrocarbon group having 11 to 15 carbon atoms). 2. The microorganism culturing method according to claim 1, wherein the main carbon source is an n-alkane having 10 to 18 carbon atoms. 3. The microorganism culturing method according to claim 1, wherein the microorganism is yeast.