JPH01174392A - Production of antibiotic substance - Google Patents

Abstract

PURPOSE:To obtain an antibiotic substance useful as an antibacterial agent, etc., against Gram-positive bacteria, etc., in high purity and yield, by fermenting and cultivating a microorganism capable of producing a macrolide based antibiotic substance using a fermentative culture medium consisting of higher fatty acids or esters thereof as a main carbon source. CONSTITUTION:A microorganism (e.g., Streptomyces narbonensis varietas josamyceticus) is cultivated using a fermentative culture medium consisting of higher fatty acids or esters thereof (e.g., soybean oil) as a main carbon source at 29 deg.C temperature for 7 days by a shaking culture method. Thereby a macrolide based antibiotic substance (e.g., josamycin) in which a main biosynthetic substrate of an aglycone is acetyl unit is obtained from the fermentation liquor.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a method for producing macrolide antibiotics.

More specifically, the present invention relates to a method for producing macrolide antibiotics, which uses a fermentation medium in which the main carbon source is a higher fatty acid or its nistyl derivatives.

(Prior Art and Problems to be Solved by the Invention) Macrolide antibiotics have antibacterial activity against a wide range of pathogenic bacteria, mainly belonging to Durham positive bacteria.

It is known to exhibit strong antibacterial activity, especially against staphylococci, other Gram-positive bacteria, mycoplasma, and the like. It is also well known that the mechanism of antibacterial action of these antibiotics is inhibition of protein synthesis on ribosomes. Furthermore, one of these antibiotics, josamycin, is known to be difficult to induce resistant bacteria, extremely effective against respiratory infections, and has low toxicity. It is.

These macrolide antibiotics are produced by actinomycetes, but these producing bacteria themselves also show positive Gram staining and are susceptible to strong growth inhibition due to small concentrations of their own produced antibiotics.

The present inventors learned in the course of basic research on josamycin fermentation that the producing bacteria are strongly inhibited by the growth of josamycin itself, and that it is extremely difficult to acquire resistance to the self-produced product. Furthermore, as shown in FIG. 1, even josamycin added to the production bacterial culture system at a concentration of only 0.5 rrIg/mt inhibited growth by 50% under neutral pH conditions.

I learned that the self-resistance value, which means the maximum product concentration at which production bacteria can grow, is approximately 1''g/ml.

Furthermore, as shown in Figure 2, in actual josamycin fermentation, the concentration of antibiotic produced and accumulated is closely linked to the amount of bacterial growth, and not only is there no antibiotic production except during times of increased growth, but there is not enough antibiotic production from the middle of the fermentation. It was also observed that bacterial cell growth and antibiotic production were interrupted despite the consumption of carbon sources.

Based on these facts, it was analyzed that it is difficult to accumulate high concentrations of antibiotics without removing the product inhibition caused by the antibiotics and increasing the amount of bacterial cell growth.

In order to avoid this growth inhibition during josamycin fermentation using a general medium system that uses carbohydrates as the main carbon source, we aim to select and use strains that are more self-resistant to the antibiotics produced, and to reduce the effects of antibiotics. Slightly acidic pH that can be moderated somewhat
We have investigated methods for fermentation in the environment, as well as methods for increasing the amount of antibiotic produced by combining growth-promoting substances, etc., and have used these methods for fermentation of josamycin.

It was not possible to obtain a sufficiently satisfactory production efficiency.

(Solution Means) In view of the above circumstances, the inventors of the present invention should increase the production and accumulation of these macrolide antibiotics. The inventors have discovered that the production and accumulation of macrolide antibiotics can be dramatically increased by using the method, and have completed the present invention.That is, the present invention is a fermentation method in which the main carbon source is higher fatty acids or their esters. This is a method for producing macrolide antibiotics characterized by using a culture medium.

In the present invention, macrolide antibiotics whose main biosynthetic substrate of the aglycone moiety is an acetyl unit are a group of 16-membered ring macrolides represented by josamycin. classes, deltamycins, midecamycins,
Maridomycins, platenmycins, etc. can also be exemplified.

The macrolide antibiotic-producing bacteria used in the method of the present invention may be any bacteria that can produce macrolide antibiotics whose main biosynthetic substrate for the aglycone moiety is an acetyl unit. Examples of such bacteria include Streptomyces narbonensis varietas josamiticus.
narbonensis var.

josamyceticus), Streptomyces halsdi
halsLedii), Streptomyces ambofaciens
faciens) + Streptparticillium kitasatoensis (St-reptoverticill)
Streptomyces d.ium kitasatoensis), Streptomyces d.
eltae), Streptomyces mycaro
-faciens), Streptomyces hygro
scopicus), Streptomyces platensis subspecies malvinus (Strept.
omyces platensis 5ubsp, ma
lvinus), etc.

9 Higher fatty acids or their esters that can be used as the main carbon source of the fermentation medium include fats and oils of animal and plant origin, higher fatty acids, glycerin esters of higher fatty acids, and lower alcohol varnishes. 9 For example, liver fat, Beef tallow, whale oil, chicken oil.

Animal fats and oils such as fish oil, bran oil, soybean oil, and coconut oil.

Vegetable oils and fats such as cottonseed oil, rapeseed oil, corn oil, olive oil, peanut oil, and lauric acid.

myristic acid, palmitic acid, stearic acid.

Oleic acid, linoleic acid, varunic acid, etc., and glycerin esters of these fatty acids.

Methanol esters or ethanol esters are particularly preferably used. These oils and fats may be used alone or in combination, and are not limited to these exemplified fats and oils.

The method of the present invention is carried out by aerobic fermentation using a conventional liquid medium. The medium composition uses higher fatty acids or their esters as the main carbon source, but also carbohydrates,
Lower fatty acids, organic acids, alcohols, esters, etc. may be added as supplements. In addition, the nitrogen source is selected from those of animal and plant origin, organic compounds, inorganic compounds, etc.

Used in combination. Metal salts, surfactants such as antifoaming agents, acids, alkalis, etc. may also be added to the medium as necessary. Further, in the production method of the present invention, the fermentation method such as punch or continuous fermentation method and the fermentation period are not limited. In addition, the amount of oil and fat used is greater than the amount of each of the other carbon sources and nitrogen sources, and is usually used in the range of 1% to 30% based on the weight of the medium.

Preferably it is in the range of 2 to 20%.

Fermentation is carried out at a temperature suitable for the growth of the actinomycetes used and for fermentation production.
Using a fermentation device that can provide pH, dissolved oxygen concentration, etc.
After inoculation, fermentation is carried out in a suitable fermentation environment according to conventional methods. Fermentation medium concentration 9 The fermentation period varies depending on the oxygen supply capacity of the fermenter and the fermentation method such as batch, semi-batch, semi-continuous, continuous, etc.

The fats and oils in the fermentation medium are gradually converted into fatty acids by the action of lipase produced by the actinomycetes used.

When provided to a culture medium as a higher fatty acid, it is assimilated and utilized by bacteria as is, promoting the production and accumulation of macrolide antibiotics. The fermented liquor that has accumulated the antibiotic at a high concentration is discharged from the fermentation apparatus and subjected to isolation and purification operations.

Microscopically, the target macrolide antibiotic is contained in large amounts in the oily fraction of the fermentation liquid, but by taking advantage of the lipophilic and basic properties of the macrolide antibiotic, it can be produced with good yield (and It can be isolated and collected as a highly pure substance, i.e. acidic pH.

After using heating treatment, filtration agent, dilution, etc. as necessary, the membrane,
By selectively using appropriate operations such as filtration and sedimentation, the produced bacterial cells and residual oily fraction are separated, and the clear aqueous phase containing the antibiotic is collected. From this point on, the desired antibiotic can be obtained through membrane methods, resin operations, extraction operations, concentration, precipitation or crystallization, drying, etc.

(Effects of the Invention) According to the method of the present invention, in the fermentation of macrolide antibiotics using a medium containing higher fatty acids or their esters as the main carbon source, the target antibiotic is extremely concentrated in the fermentation liquid. It can be produced and accumulated at high concentrations. This effect is
This was particularly noticeable when higher fatty acid esters, especially vegetable oils, were used as the main carbon source of the culture medium.

The effects of the present invention when using higher fatty acids or their esters are obtained when the esters given to the medium are gradually hydrolyzed by lipase secreted by bacterial cells, or when they are given directly to the medium as higher fatty acids. In some cases, it is dissolved in the aqueous environment at a very small concentration that is in equilibrium, and is ingested by bacteria, where it is converted to acetyl-CoA via the beta-oxidation pathway within the bacteria.

This is thought to be due to the fact that it not only supplies abundant energy for biosynthesis, but also serves as a rich source of acetyl units, which are essential for the biosynthesis of the aglycone moiety of macrolide antibiotics.

Furthermore, in the production method of the present invention, the fatty acids remaining outside the bacterial cells in the culture medium have a mutual relationship with the lipid-soluble basic properties of the antibiotic produced and secreted by the producing bacteria. can be extracted and stored.

Therefore, the concentration of the product in the aqueous phase, which is the microscopic growth environment for the producing bacteria, can be lowered below the self-resistance value, and the inhibition of the produced antibiotics can be removed, resulting in even higher productivity. Able to demonstrate The above-mentioned supply of abundant biosynthetic energy and abundant essential biosynthetic substrates, as well as the release of product inhibition through extraction and dissolution and retention of the product using fatty acids, all at once, result in extremely efficient fermentation of macrolide antibiotics. It can have a remarkable effect on achieving production.

Next, the present invention will be explained in more detail by presenting two examples. Note that the present invention is not limited in any way by this example.

Examples 1 to 8 Streptomyces narbonensis vulgaraetus josamyceticus (Streptomyces narbonensis) stored frozen at -30°C in a small test tube
nsis var.

josamyceticus) with improved josamycin productivity, 1% starch, 1% glucose, 15% soybean flour,
Transferred to a 500 ml Sakaro flask containing 100 ml of seed medium consisting of 0.05% magnesium sulfate heptahydrate, 01% dipotassium hydrogen phosphate, and 05% yeast extract, and cultured at 29°G for 2 days with shaking at 140 rpm. I did it. Separately, 7% of any of the various carbon sources shown in Table 2, 2% of gluten meal, 2% of Pharmamedia, and 0.05% of magnesium sulfate heptahydrate.

50 ml of fermentation medium consisting of 01% sodium hydroxide was prepared in a 500 ml Erlenmeyer flask.

Add 1 m of the seed culture solution obtained above to these flasks.
1 was transplanted and placed on a rotary shaker at 240'rpm for 7 days at 29°C. The fermentation liquid was collected on the 5th and 7th day, and the accumulated concentration of josamycin (JM), pH, and bacterial cell amount (
pcv), apparent viscosity, etc. were measured. The results are shown in Table IK.

Table 1 Here, the josamycin concentration is determined by weighing out a predetermined amount of fermentation liquid and toluene, mixing and extracting under alkaline pH, and then centrifuging the josamycin extract obtained by silica gel TL.
It was measured by TLC-densitometry using a C plate and a developing agent (benzene/butyl acetate/methanol/water-65/19/2]/1).

Since the amount of bacterial cells cannot be accurately measured because the medium contains solid matter, put the two fermentation liquids into a small test tube.

Sediment volume ratio after centrifugation at 3000 rpm for 10 minutes (@
was expressed as a relative value of bacterial cell amount. As another indicator showing the amount of bacterial cells, the falling time was measured using a 1 mt volume (20 cm length) pipette, and the appearance was expressed as viscosity.

As shown in Table 1, the accumulated concentration of josamycin in the glucose control area was 0, similar to the results shown in Figure 2.
．． 5ml 1l! /kg or less. However, it was found that when higher fatty acid esters were added as the main carbon source, the accumulated concentration of josamycin increased dramatically. In particular, it was found that all the media using soybean oil, peanut oil, olive oil, rapeseed oil, corn oil, etc. gave an accumulated josamycin concentration of 4 to 5 mg/ml on the 7th day of fermentation.

In these cases, it was also confirmed that the amount of bacterial growth increased dramatically. The fermentation liquid consisting of an oil-based medium with a josamycin concentration of 4 to 5 mg/ml was several meters in diameter.
It contained oily aggregates and oily cream of m. These oily aggregates contain not only a large amount of fatty acids but also more than 6 omg/g of josamycin.

It was confirmed that the total amount of josamicone-related substances, including other 3-0-deacetyljosamycin, 4-perbutyryljosamycin, etc., was over 100 mg/g. The josamycin content of oily cream is approximately 40rr.
It was about 1 g/g.

(Reference Experimental Example) Josamycin fermentation was carried out in the same manner as in the example in a medium using various animal and vegetable oils and fats and higher fatty acids as the main carbon sources. 40,000 x g of the fermented liquid.

After centrifugation for 20 minutes, an aqueous supernatant was obtained by separating it into an oil phase and a precipitate. As shown in FIG. 3, the concentration of josamycin in the aqueous supernatant obtained here was about a fraction of the concentration of josamycin in the fermentation liquid itself. This fact indicates that in fermentation liquids that use fats and oils as the main carbon source, in addition to their utilization, josamycin from the aqueous phase, which is the microscopic environment of the producing bacteria, can be selectively extracted and retained in the oil phase. During one fermentation period, the aqueous phase product concentration is suppressed below the self-tolerance value, and through this, product inhibition in a growth-linked production system is released.

It was inferred that it further promoted antibiotic production.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the influence of josamycin itself on the growth of josamycin-producing bacteria. In the figure, the horizontal axis represents the concentration of josamycin added to the medium, and the vertical axis represents the relative value of the growth amount with the josamycin-free system as a control (100%). Medium: 2% glycerol, 0.75% peptone,
025% trimagnesium phosphate. and 0.25% yeast extract, and the pH was adjusted to 8.0 before sterilization. 50 mL of this 7.5 ml
The cells were cultured in a ml test tube for 64 hours. FIG. 2 is a diagram showing an example of josamycin fermentation in a medium system using glucose as the main carbon source. The culture medium consisted of 9% glucose, 5% soybean flour, 0.25% trimagnesium phosphate, and 0.125% yeast extract and was cultured in 50 ml in one conical flask. FIG. 3 is a diagram showing the respective josamycin concentrations of the fermentation broth obtained using an oil-based medium (horizontal axis) and its centrifuged supernatant (vertical axis). Patent applicant Yamanouchi Pharmaceutical Co., Ltd.

Claims (5)

[Claims] (1) A method for producing a macrolide antibiotic, which comprises using a fermentation medium in which the main carbon source is a higher fatty acid or its ester. (2) The production method according to claim (1), wherein the main biosynthetic substrate of the aglycone moiety is a macrolide antibiotic. (3) The production method according to claim (1), wherein the higher fatty acid or its ester is a glycerin ester or lower alcohol ester of a higher fatty acid, or an animal or vegetable oil or fat. (4) Claim No. 3 in which the animal and vegetable oils and fats are soybean oil, peanut oil, olive oil, rapeseed oil, or corn oil.
Manufacturing method described in section. (5) The production method according to claim (1), wherein the higher fatty acid or its ester is soybean oil, peanut oil, olive oil, rapeseed oil, or corn oil, and the macrolide antibiotic is josamycin.