CN1252246C - Method for extracting microorganism producing biological active substance from sponge cell - Google Patents

Abstract

The invention relates to a method for isolating microorganisms capable of producing biologically active substances from sponge cells, that is, isolating from the cells of the most primitive multicellular animal sponges in the ocean that can produce relatively high levels of biologically active substances such as anticancer, antitumor, and antibacterial substances microbiological approach. Specific operations: (1) select healthy and living sponge organisms from the ocean, wash them under sterile conditions, cut the inner cell body of the sponge into small pieces, and grind to obtain fresh sponge cell extracts; (2) clean the fresh sponge Cell extract, using the plate dilution method, on the plates of isolated marine fungi, bacteria or actinomycetes culture medium added with 0.005-0.05% sponge, smear and scrape the plates, and then put them into the incubator for cultivation; (3) After culturing for 1 to 15 days, the pure microorganisms that grow single colonies on the plate are transferred to the slant for preservation, and the biologically active substances are tested and verified. The invention has simple operation, ideal result and can obtain the required bacterial strain.

Description

A method for isolating microorganisms capable of producing biologically active substances from sponge cells

technical field

The present invention relates to microorganism separation technology, specifically a kind of method for separating and producing the microorganism of biologically active substance from sponge cell, and it is to obtain fresh and living sponge multicellular animal from ocean, separates from sponge inner cell and can produce higher The content of anticancer, antitumor, antibacterial and other biologically active substances in microorganisms.

technical background

Sponge is the most primitive and lowest multicellular animal, without organization and organ differentiation, it is an independent branch of the origin and evolution of life on earth, and it is an invertebrate in the ocean. Studies at home and abroad for many years have shown that more than 10% of the secondary metabolites in sponges are cytotoxic and biologically active substances, while this ratio is only 2% in other marine organisms in the ocean, and plants and microorganisms on land is well below 1%. Therefore, the research on the active substances in the sponge has been paid attention to by scientists all over the world. But so far, the mechanism by which sponges can produce active substances is unclear. Whether it is related to the microorganisms that coexist with sponge cells has not been concluded so far. This mechanism is very complicated. According to preliminary estimates, the symbiotic microorganisms in the sponge cells account for about 40% of the sponge volume. The University of Maryland found that there are complex microbial groups in the sponge, and the anticancer active substances in the sponge are produced by the symbiotic microorganisms. , and a strain of Micrococcus bright orange can be fermented to produce the same active substance (Diketopiperagines) as in the sponge. Studies of DNA by Althoff et al. also identified a symbiotic relationship between the Rhodobacter and the sponge Halichondria panicea. In addition, Shenyang Institute of Applied Ecology, Chinese Academy of Sciences has obtained a number of microbial strains producing active substances such as agricultural antibiotics and anti-tumor lead compounds from Bohai Sea membrane sponge cells. At present, how to separate and purify the symbiotic microorganisms and sponge microorganisms that produce active substances from sponge cells, and use these precious resources to study marine microbial pharmaceuticals, agricultural antibiotics, etc., has become the focus of attention at home and abroad. Conventional separation methods, without adding aged seawater and sponges, can only obtain a small number of microorganisms in the sponge cells, and cannot obtain the genus and species communities of microbial diversity in the sponge ecosystem. So far, there is no report on an ideal method for isolating microorganisms in sponge cells at home and abroad.

Contents of the invention

The object of the present invention is to provide a method for isolating microorganisms capable of producing biologically active substances from sponge cells.

In order to achieve the above object, the technical solution adopted in the present invention is to operate as follows:

(1) Select healthy and live sponge organisms from the ocean (choose healthy, live, and free from diseases and insect pests), wash them under sterile conditions, take the inner cell body of the sponge and cut them into small pieces of 1 to 5 mm, and grind for 3 to 5 mm. 5 minutes to obtain a fresh sponge cell extract; the cleaning uses alcohol or sterile saline;

(2) For the fresh sponge cell extract, use the plate dilution method to dilute the solution at a concentration of 10 ^-1 to 10 ^-7 respectively, and add 0.005 to 0.05% of the sponge to separate marine fungi, bacteria or actinomycete culture medium. Scrape the flat plate on the plate, and then put it into an incubator at 25-30°C for cultivation;

(3) After culturing for 1 to 15 days, move the pure (uniform growth) microorganisms that grow single colonies on the plate to the inclined plane for preservation, in order to carry out the identification of the biological activity of secondary metabolites, and the identification method adopts conventional techniques;

The identification of the biological activity of the secondary metabolite is to detect and verify whether it produces anti-cancer, anti-tumor, antibacterial and other biologically active substances under laboratory conditions;

The concentration of the plate dilution solution for isolating fungi in sponge cells is 10 ^-2 to 10 ^-3 ; the concentration of the plate dilution solution for isolating bacteria in sponge cells is 10 ^-5 to 10 ^-7 ; The plate dilution concentration of the actinomycetes isolated from the sponge cells is 10 ^-1 to 10 ^-2 ; the solution used for dilution is sterile water;

In terms of percentage by weight, the medium components of the fungus in the isolated sponge cells are: 1% glucose, 0.5-2.0% soluble starch, 0.1% KH ₂ PO ₄ , 0.1% MgSO ₄ 7H ₂ O, 0.5-1.0% peptone , sponge 0.005～0.05%, agar 2%, surplus is old seawater or artificial seawater, pH6.0～6.5; In the described culture medium of every 1000ml, add 1% Bengal red (RoseBengal) aqueous solution 3.3ml before sterilization; When the melted medium is poured on the plate, add 0.1-1.0ml of 0.5-2% streptomycin solution and 100-500μl of ciprofloxacin to each 100ml of medium;

In terms of percentage by weight, the medium components for separating bacteria from sponge cells are: 0.5-2.0% soluble starch, 0.5% beef extract, 0.5% yeast extract, 0.005-0.05% sponge, 2% agar, and the balance is old sea water or artificial Seawater, pH7.0～7.2;

In terms of weight percentage, the medium components for isolating actinomycetes from sponge cells are: 0.5-2.0% soluble starch, 0.1-1.0% sucrose, 0.5% mannitol, 0.1% KNO ₃ , 0.5% K ₂ HPO ₄ , MgSO ₄ 7H ₂ O 0.5%, FeSO ₄ 7H ₂ O 0.01%, sponge 0.005-0.05%, agar 2%, the rest is old sea water or artificial sea water, pH 7.0-7.4; Add 1 ml of 3% potassium dichromate solution to the medium, then add 0.5 ml of 1% streptomycin solution, and 100-500 μl of ciprofloxacin.

The present invention has following beneficial effect:

1. apply the present invention to isolate the method for producing biologically active substance microorganisms from sponge cells, all added sponge 0.005～0.05% in the separation medium, the separation effect is very good, and add Bengal red and streptomycin when separating fungi, Potassium dichromate, streptomycin and ciprofloxacin are added to actinomycetes, so the average success rate is above 90%.

2. The operation of the present invention is simple, and the desired bacterial strain can be obtained.

Detailed ways

The present invention is described in further detail below in conjunction with embodiment.

Example 1

The method for isolating fungi producing biologically active substances from sponge cells can be operated in accordance with the following steps:

(1) Clean the healthy living sponge organisms from the ocean immediately, and use alcohol to disinfect the surface of the sponge; use aseptic technique to cut off the outer layer of the sponge, and take the inner cell body of the sponge and cut it into small pieces of 1 to 5 mm square , grind with aseptic quartz sand, add aseptic saline (NaCl 0.85%) in aseptic grinder, grind for 5 minutes, obtain fresh sponge cell extract;

(2) For the extract of fresh sponge cells, use the plate dilution method to dilute the solution at a concentration of 10 ^-2 and 10 ^-3 respectively, spread the plate on the following plate for the separation of sponge fungus culture medium, and culture it in a 25°C incubator after coating ;

(3) After cultivating for 1 week, single bacterial colonies are picked and purified, and then transplanted to the slope for preservation, and the identification of the biological activity of the secondary metabolite is carried out; the identification of the biological activity of the secondary metabolite is whether it is produced under laboratory conditions Anticancer, antitumor, antibacterial and other biologically active substances are tested and verified;

Its composition by weight percentage of isolated sponge fungus culture medium is as follows:

Glucose 1%, soluble starch 1%, KH ₂ PO ₄ 0.1%, MgSO ₄ ·7H ₂ O 0.1%, peptone 0.5%, sponge 0.05%, agar 2%, and the balance was aged sea water, pH 6.0; The seawater is seawater that has been left standing for a certain period of time;

For every 1000ml of this medium, add 3.3ml of 1% Rose Bengal aqueous solution before sterilization; when the melted medium is poured on the plate, before use: add 0.5ml of 1% streptomycin solution and cyclopropane to every 100ml of medium Floxacin 300μl.

In this embodiment, the success rate of isolating fungi in sponge cells is more than 90%.

Example 2

The difference from Example 1 is:

For the fresh sponge cell extract, use the plate dilution method to dilute the solution at a concentration of 10 ^-5 , 10 ^-6 , and 10 ^-7 respectively, and spread the plate on the following plate for separating the sponge bacteria culture medium, and put it in a 28°C incubator after coating After culturing for 3 days, single colonies were picked and purified, and then transplanted to a slant for storage to identify the biological activity of secondary metabolites.

The composition of the isolated sponge bacteria culture medium is as follows by weight percentage:

Soluble starch 1%, beef extract 0.5%, yeast extract 0.5%, sponge 0.01%, agar 2%, the balance is old sea water, pH 7.0.

In this embodiment, the success rate of isolating the bacteria in the sponge cells is over 95%.

Example 3

The difference from Example 1 is:

After selecting healthy living sponge organisms from the ocean, use normal saline to disinfect the appearance of the sponge;

For the extract of fresh sponge cells, use the plate dilution method to dilute the solution at a concentration of 10 ^-1 and 10 ^-2 respectively, spread the plate on the following plate for the isolation of Actinomyces spongiosa culture medium, and culture it in a 30°C incubator for 2 A week later, single colonies were picked and purified, and then transplanted to the slant for storage to identify the biological activity of secondary metabolites.

The composition of the isolated spongy actinomycetes culture medium is as follows by weight percentage:

Soluble starch 1%, sucrose 0.5%, mannitol 0.5%, KNO ₃ 0.1%, K ₂ HPO ₄ 0.5%, MgSO ₄ 7H ₂ O 0.01%, FeSO ₄ 7H ₂ O 0.01%, sponge 0.01%, agar 2 %, the balance is old sea water, pH7.0～7.2;

Just before use, add 1 ml of 3% potassium dichromate solution to 300 ml of medium that has been heated and melted, then add 0.5 ml of 1% streptomycin solution, and 500 μl of ciprofloxacin.

In this embodiment, the success rate of isolating actinomycetes in sponge cells is over 70%.

Said seawater of the present invention can also be replaced by artificial seawater.

Claims (9)

1. A method that can produce biologically active substance microorganisms from sponge cell separation, is characterized in that operating as follows: (1) Select healthy and living sponge organisms from the ocean, wash them under aseptic conditions, take the inner cell body of the sponge and cut them into small pieces of 1 to 5 mm, and grind for 3 to 5 minutes to obtain a fresh sponge cell extract; (2) For the fresh sponge cell extract, use the plate dilution method to dilute the solution at a concentration of ^10-1 to ^10-7 , and place it on a plate of a culture medium for separating marine fungi, bacteria or actinomycetes added with 0.005-0.05% sponge Scrape the flat plate, and then put it into a 25-30°C incubator for cultivation; (3) After culturing for 1 to 15 days, move the pure microorganisms that grow single colonies on the plate to the slant and store them for the identification of the biological activity of the secondary metabolites. 2. The method for isolating microorganisms capable of producing bioactive substances from sponge cells as claimed in claim 1, characterized in that: alcohol or sterile saline is used for cleaning. 3. as claimed in claim 1, can produce the method for biologically active substance microorganism from spongy cell separation, it is characterized in that: the identification of described secondary metabolite bioactivity is whether to produce anticancer, antitumor to it under laboratory condition , Antibacterial bioactive substances were tested and verified. 4 . The method for isolating microorganisms capable of producing biologically active substances from sponge cells according to claim 1 , wherein the concentration of the plate dilution solution used for isolating fungi in sponge cells is 10 ⁻² to 10 ⁻³ . 5 . The method for isolating microorganisms capable of producing biologically active substances from sponge cells according to claim 1 , wherein the concentration of the plate dilution solution used for isolating bacteria in sponge cells is 10 ⁻⁵ to 10 ⁻⁷ . 6 . The method for isolating microorganisms capable of producing biologically active substances from sponge cells according to claim 1 , wherein the concentration of the plate diluent used for isolating actinomycetes in sponge cells is 10 ⁻¹ to 10 ⁻² . 7. as claimed in claim 1, can produce the method for bioactive substance microorganism from spongy cell isolation, it is characterized in that: In terms of percentage by weight, the medium components of the fungus in the isolated sponge cells are: 1% glucose, 0.5-2.0% soluble starch, 0.1% KH ₂ PO ₄ , 0.1% MgSO ₄ 7H ₂ O, 0.5-1.0% peptone , sponge 0.005-0.05%, agar 2%, the balance is old sea water or artificial sea water, pH 6.0-6.5; In every 1000ml of the medium, add 3.3ml of 1% Bengal red aqueous solution before sterilization; when the melted medium is poured on the plate, add 0.1-1.0ml of 0.5-2% streptomycin solution in every 100ml of medium, And ciprofloxacin 100 ~ 500μl. 8. as claimed in claim 1, can produce the method for bioactive substance microorganism from spongy cell separation, it is characterized in that: by weight percentage, the substratum composition of separating bacteria in spongy cell is: soluble starch 0.5～2.0%, beef extract 0.5%, yeast extract 0.5%, sponge 0.005-0.05%, agar 2%, the balance is old sea water or artificial sea water, pH7.0-7.2. 9. as claimed in claim 1, can produce the method for bioactive substance microorganism from spongy cell isolation, it is characterized in that: by weight percentage, the substratum composition of separating actinomycetes in spongy cell is: soluble starch 0.5～2.0%, Sucrose 0.1～1.0%, Mannitol 0.5%, KNO ₃ 0.1%, K ₂ HPO ₄ 0.5%, MgSO ₄ 7H ₂ O 0.5%, FeSO ₄ 7H ₂ O 0.01%, Sponge 0.005～0.05%, Agar 2% , The balance is old sea water or artificial sea water, pH 7.0-7.4; Just before use, add 1 ml of 3% potassium dichromate solution to 300 ml of the medium that has been heated and melted, then add 0.5 ml of 1% streptomycin solution, and 100-500 μl of ciprofloxacin.