阅读说明：本技术 一种放线菌抗菌次生代谢产物的制备方法 (Preparation method of antibacterial secondary metabolite of actinomycetes ) 是由 张新军 曹舰艇 于 2019-03-05 设计创作，主要内容包括：本发明提供一种放线菌抗菌次生代谢产物的制备方法,涉及微生物技术领域,所述方法首先将产抗菌次生代谢产物的放线菌接种于液体培养基中培养,得到菌体发酵液；所述液体培养基的原料包括酵母提取物、葡萄糖和麦芽提取物和水；所得菌体发酵液接种于固体培养基发酵,得到活性发酵产物；所述固体培养基的原料包括介质支持物、酵母膏和蛋白胨和水；以乙酸乙酯浸提所述活性发酵产物,固液分离得到乙酸乙酯提取物,去除乙酸乙酯,得到放线菌抗菌次生代谢产物。本发明提供的制备方法能够明显增加放线菌抗菌次生代谢产物的积累,提高代谢产量；相对于单纯的液态发酵放线菌而言能耗低,发酵过程无需持续通入氧气。(The invention provides a preparation method of an antibacterial secondary metabolite of an actinomycete, which relates to the technical field of microorganisms, and the method comprises the steps of firstly inoculating the actinomycete producing the antibacterial secondary metabolite into a liquid culture medium for culture to obtain a thallus fermentation liquor; the raw materials of the liquid culture medium comprise yeast extract, glucose and malt extract and water; inoculating the obtained thallus fermentation liquor to a solid culture medium for fermentation to obtain an active fermentation product; the raw materials of the solid culture medium comprise a medium support, yeast extract, peptone and water; and leaching the active fermentation product by using ethyl acetate, carrying out solid-liquid separation to obtain an ethyl acetate extract, and removing the ethyl acetate to obtain the actinomycete antibacterial secondary metabolite. The preparation method provided by the invention can obviously increase the accumulation of antibacterial secondary metabolites of the actinomycetes and improve the metabolic yield; compared with the pure liquid fermentation actinomycetes, the method has low energy consumption, and oxygen does not need to be continuously introduced in the fermentation process.)

1. A preparation method of an antibacterial secondary metabolite of actinomycetes comprises the following steps:

(1) inoculating actinomycetes producing antibacterial secondary metabolites into a liquid culture medium for culturing for 65-80 h to obtain a thallus fermentation liquor;

the liquid culture medium comprises, by weight, 2-8 parts of yeast extract, 2-8 parts of glucose, 8-20 parts of malt extract and 800-1000 parts of water;

(2) inoculating the thallus fermentation liquor to a solid culture medium for fermentation to obtain an active fermentation product;

the raw materials of the solid culture medium comprise, by weight, 70-150 parts of a medium support, 1-6 parts of yeast extract, 7-20 parts of peptone and 80-120 parts of water;

the medium support is selected from one or more of rice, sorghum or wheat;

(3) and leaching the active fermentation product by using ethyl acetate, carrying out solid-liquid separation to obtain an ethyl acetate extract, and removing the ethyl acetate to obtain the actinomycete antibacterial secondary metabolite.

2. The method according to claim 1, wherein the culturing in step (1) is accompanied by stirring.

3. The method according to claim 1 or 2, wherein the pH of the liquid medium in the step (1) is 7.0 to 7.4.

4. The method according to claim 1, wherein the inoculum size of the fermentation broth of the bacterial cells in step (2) is 0.12 to 0.5 ml/kg.

5. The method for preparing a solid medium according to claim 1 or 4, wherein the method for preparing a solid medium in the step (2) comprises the steps of:

s1, mixing the yeast extract, peptone and water, and adjusting the pH value to 7.5 to obtain a premix;

s2, mixing the premix with a medium support, and standing for 6-10 hours to obtain a mixture;

s3, sterilizing the mixture, and shaking until any two particles of the medium support are not adhered to each other to obtain the solid culture medium.

6. The method according to claim 1, wherein the ethyl acetate is extracted 2 to 4 times in the step (3).

7. The method according to claim 1 or 6, wherein in the step (3), the volume ratio of the ethyl acetate to the solid fermentation product is 1:1 when the ethyl acetate is leached.

8. The method according to claim 1 or 6, wherein in the step (3), the leaching time is preferably 30-60 min.

Technical Field

The invention relates to the technical field of microorganisms, in particular to a preparation method of an antibacterial secondary metabolite of actinomycetes.

Background

The fermentation of secondary metabolites with antibacterial activity of actinomycetes is usually carried out by liquid fermentation with a certain liquid culture medium, and the fermentation is carried out in a shaking table by a triangular flask in a laboratory, so that the antibacterial active products are accumulated in fermentation liquor, and the active products are extracted by a certain purification means. Generally, the amount of active product accumulated in the fermentation broth is very small, and because the concentration of active ingredients in the fermentation broth is very low, the loss of active ingredients in the subsequent purification process is large, and some active ingredients with low content cannot be separated. Therefore, a method capable of rapidly extracting a large amount of antibacterial active secondary metabolites produced by actinomycetes is lacked at present.

Disclosure of Invention

The invention provides a preparation method of an antibacterial secondary metabolite of an actinomycete, aiming at overcoming the defects of low yield, high separation difficulty and high energy consumption of the existing method for preparing the antibacterial secondary metabolite of the actinomycete, wherein the obtained antibacterial secondary metabolite can be increased by more than 10 times, and the energy consumption is low and the yield is high.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of an antibacterial secondary metabolite of an actinomycete, which comprises the following steps:

(1) inoculating actinomycetes producing antibacterial secondary metabolites into a liquid culture medium for culturing for 65-80 h to obtain a thallus fermentation liquor;

the liquid culture medium comprises, by weight, 2-8 parts of yeast extract, 2-8 parts of glucose, 8-20 parts of malt extract and 800-1000 parts of water;

(2) inoculating the thallus fermentation liquor to a solid culture medium for fermentation to obtain an active fermentation product;

the raw materials of the solid culture medium comprise, by weight, 70-150 parts of a medium support, 1-6 parts of yeast extract, 7-20 parts of peptone and 80-120 parts of water;

the medium support is selected from one or more of rice, sorghum, wheat, rice bran or rice husk;

(3) and leaching the active fermentation product by using ethyl acetate, carrying out solid-liquid separation to obtain an ethyl acetate extract, and removing the ethyl acetate to obtain the actinomycete antibacterial secondary metabolite.

Preferably, the culturing in step (1) is accompanied by stirring.

Preferably, the pH of the liquid culture medium in the step (1) is 7.0-7.4.

Preferably, the inoculation amount of the bacterial fermentation liquid in the step (2) is 0.12-0.5 ml/kg.

Preferably, the preparation method of the solid medium in the step (2) comprises the following steps:

s1, mixing the yeast extract, peptone and water, and adjusting the pH value to 7.5 to obtain a premix;

s2, mixing the premix with a medium support, and standing for 6-10 hours to obtain a mixture;

s3, sterilizing the mixture, and shaking until any two particles of the medium support are not adhered to each other to obtain the solid culture medium.

Preferably, in the step (3), the leaching times of the ethyl acetate are 2-4 times.

Preferably, in the step (3), the volume ratio of the ethyl acetate to the solid fermentation product in the ethyl acetate leaching is 1: 1.

Preferably, in the step (3), the leaching time is preferably 30-60 min.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a preparation method of an antibacterial secondary metabolite of an actinomycete, which comprises the steps of inoculating the actinomycete producing the antibacterial secondary metabolite into a liquid culture medium to culture for 65-80 hours to obtain a thallus fermentation liquor; the raw materials of the liquid culture medium comprise yeast extract, glucose and malt extract and water; inoculating the thallus fermentation liquor to a solid culture medium for fermentation to obtain an active fermentation product; the raw materials of the solid culture medium comprise a medium support, yeast extract, peptone and water; the medium support is selected from one or more of rice, sorghum, wheat, rice bran or rice husk; and leaching the active fermentation product by using ethyl acetate, carrying out solid-liquid separation to obtain an ethyl acetate extract, and removing the ethyl acetate to obtain the actinomycete antibacterial secondary metabolite. The preparation method provided by the invention can obviously increase the accumulation of antibacterial secondary metabolites of the actinomycetes and improve the metabolic yield; meanwhile, the solid culture medium in the method provided by the invention adopts rice, sorghum and the like as medium supports, not only plays a role of supporting media, but also can provide carbon sources and nutrient substances for the actinomycetes, provides a large amount of growth space for the actinomycetes, and is beneficial to the growth of the actinomycetes hyphae and the accumulation of secondary metabolites. Compared with the pure liquid fermentation actinomycetes, the preparation method has low energy consumption, and oxygen does not need to be continuously introduced in the fermentation process.

Detailed Description

The invention provides a preparation method of an antibacterial secondary metabolite of an actinomycete, which comprises the following steps:

(1) inoculating actinomycetes producing antibacterial secondary metabolites into a liquid culture medium for culturing for 65-80 h to obtain a thallus fermentation liquor;

the liquid culture medium comprises, by weight, 2-8 parts of yeast extract, 2-8 parts of glucose, 8-20 parts of malt extract and 800-1000 parts of water;

(2) inoculating the thallus fermentation liquor to a solid culture medium for fermentation to obtain an active fermentation product;

the raw materials of the solid culture medium comprise, by weight, 70-150 parts of a medium support, 1-6 parts of yeast extract, 7-20 parts of peptone and 80-120 parts of water;

the medium support is selected from one or more of rice, sorghum, wheat, rice bran or rice husk;

(3) and leaching the active fermentation product by using ethyl acetate, carrying out solid-liquid separation to obtain an ethyl acetate extract, and removing the ethyl acetate to obtain the actinomycete antibacterial secondary metabolite.

In order to obtain the antibacterial secondary metabolite of the actinomycetes, firstly, the actinomycetes for producing the antibacterial secondary metabolite is cultured by using a liquid culture medium, so that the quantity of the actinomycetes can be enlarged, and on the other hand, the generation of the antibacterial secondary metabolite can be stimulated more favorably by firstly carrying out liquid culture and then inoculating a solid culture medium. The solid culture medium adopted by the invention utilizes natural substances such as rice, sorghum, wheat bran and the like as medium supports, on one hand, the natural substances are used as carbon sources to provide nutrient substances generated by antibacterial secondary metabolites of actinomycetes, on the other hand, the medium supports can also play a role of supporting media, so that larger living and extending space is provided for the mycelia of the actinomycetes, but the problem that mycelia are easy to appear during liquid culture is avoided, and therefore, the solid culture medium provided by the invention is adopted for solid fermentation to obtain active fermentation products accumulated with a large amount of antibacterial secondary metabolites. The invention leaches the obtained active fermentation product by ethyl acetate and separates to obtain a mixture containing the secondary actinomycete antibacterial metabolite. As shown in the specific embodiment of the invention, the yield of the antibacterial secondary metabolite of the actinomycetes prepared by the method is 11.3 times that of the antibacterial secondary metabolite of the actinomycetes prepared by the traditional liquid fermentation method, the yield is obviously improved, and the energy consumption is lower.

Specifically, firstly, inoculating actinomycetes producing an antibacterial secondary metabolite into a liquid culture medium for culturing for 65-80 hours to obtain a thallus fermentation liquor; the liquid culture medium comprises, by weight, 2-8 parts of yeast extract, 2-8 parts of glucose, 8-20 parts of malt extract and 800-1000 parts of water. The obtained thallus fermentation liquid comprises actinomycete thallus, partial secondary metabolite and liquid culture medium thereof.

In the present invention, the antibacterial secondary metabolite-producing actinomycetes include, but are not limited to, staphylococcus aureus-resistant actinomycetes and bacillus subtilis-resistant actinomycetes.

In the invention, the liquid culture medium preferably comprises 3-5 parts by weight of yeast extract, 3-6 parts by weight of glucose, 9-12 parts by weight of malt extract and 950-980 parts by weight of water. In the invention, the pH value of the liquid culture medium is preferably 7.0-7.4, and more preferably 7.2. In the present invention, the yeast extract and the malt extract are commercially available, but the present invention is not limited thereto.

In the invention, the time for culturing in the liquid culture medium is preferably 70-75 h, more preferably 72 h; the temperature of the culture is preferably 25-32 ℃, and more preferably 28 ℃. In the present invention, the culture in the liquid medium is accompanied by stirring; more preferably, the stirring rate is preferably

After obtaining a thallus fermentation liquid, inoculating the thallus fermentation liquid to a solid culture medium for fermentation to obtain an active fermentation product; the raw materials of the solid culture medium comprise, by weight, 70-150 parts of a medium support, 1-6 parts of yeast extract, 7-20 parts of peptone and 80-120 parts of water;

in the present invention, the inoculation amount of the bacterial fermentation broth in the solid medium is preferably 0.12 to 0.5ml/kg, and more preferably 1.0 ml/L. In the invention, the fermentation temperature is preferably 25-32 ℃, and more preferably 28 ℃. In the invention, the fermentation time is preferably 10-15 d, and more preferably 12-14 d.

In the invention, the raw materials of the solid culture medium comprise 80-120 parts by weight of medium support, 2-5 parts by weight of yeast extract, 8-12 parts by weight of peptone and 90-110 parts by weight of water. In the present invention, the medium support in the solid medium is selected from one or more of rice, sorghum or wheat; the medium support selected by the invention is a natural substance, has complex nutrient components but takes starch (polysaccharide) as the main material, has the functions of providing nutrition and supporting medium, and provides a larger space for the growth of the hypha of the actinomycetes. In the present invention, yeast extract and peptone provide other nutrients such as organic nitrogen, carbon source, vitamins, etc. to actinomycetes.

In the present invention, the preparation method of the solid medium preferably comprises the steps of:

s1, mixing the yeast extract, peptone and water, and adjusting the pH value to 7.5 to obtain a premix;

s2, mixing the premix with a medium support, and standing for 6-10 hours to obtain a mixture;

s3, sterilizing the mixture, and shaking until any two particles of the medium support are not adhered to each other to obtain the solid culture medium.

The purpose of mixing yeast extract, peptone and water and adjusting the pH value is to premix for subsequent adsorption on a media support.

In the invention, in the standing process after the premix and the medium support are mixed, the medium support absorbs the premix containing the yeast extract and the peptone, the volume of the medium support expands along with the absorption of the premix, and the yeast extract and the peptone enter a nitrogen source to provide sufficient nutrition for the growth of actinomycetes under the condition of subsequent dispersion of the medium support. In the present invention, the standing time of the premix after mixing with the medium support is preferably 8 hours

The mixture is sterilized to obtain the solid culture medium, any two particles of the medium support are oscillated until the two particles are not adhered to each other, so that the smooth air among the particles of the medium support can be ensured, the growth of the mycelia of the actinomycetes is facilitated, the ventilation and the extension of the mycelia are facilitated, the step of continuously introducing oxygen into the fermentation culture medium during the conventional fermentation culture is omitted, and the energy consumption is effectively saved. The present invention is not particularly limited with respect to the sterilization method, and a sterilization method known in the art, such as high-temperature steam sterilization, may be employed.

After obtaining the active fermentation product, the invention uses ethyl acetate to leach the active fermentation product, and then the ethyl acetate extract is obtained by solid-liquid separation, and the ethyl acetate is removed, thus obtaining the actinomycete antibacterial secondary metabolite. According to the invention, the ethyl acetate is used as the leaching solvent, so that the antibacterial secondary metabolite of the actinomycetes can be improved, the antibacterial secondary metabolite in the active fermentation product is leached into the ethyl acetate through leaching, and the extraction rate is high.

In order to further improve the yield of the antibacterial secondary metabolites of the actinomycetes, ethyl acetate is preferably used for leaching for 2-4 times; the active fermentation product is mixed with ethyl acetate, preferably in equal volume ratios, each time with ethyl acetate. In the invention, the leaching time of each ethyl acetate leaching is preferably 30-60 min independently, and more preferably 40-50 min independently.

The fermentation of secondary metabolites with antibacterial activity of actinomycetes is usually carried out by liquid fermentation with a certain liquid culture medium, and the fermentation is carried out in a shaking table by a triangular flask in a laboratory, so that the antibacterial active products are accumulated in fermentation liquor, and the active products are extracted by a certain purification means. Generally, the amount of active product accumulated in the fermentation broth is very small, and because the concentration of the active ingredient in the fermentation broth is very low, the loss of the active ingredient in the subsequent purification process is large, and some active ingredients with low content cannot be separated.

The invention aims at improving the yield of the antibacterial secondary metabolite of the actinomycetes, and obtains the preparation method which can obviously improve the yield of the antibacterial secondary metabolite and reduce the energy consumption compared with the conventional liquid fermentation mode.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.