CN1467289A - Method for extracting microorganism producing biological active substance from sponge cell - Google Patents

Method for extracting microorganism producing biological active substance from sponge cell Download PDF

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Publication number
CN1467289A
CN1467289A CNA021325626A CN02132562A CN1467289A CN 1467289 A CN1467289 A CN 1467289A CN A021325626 A CNA021325626 A CN A021325626A CN 02132562 A CN02132562 A CN 02132562A CN 1467289 A CN1467289 A CN 1467289A
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sponge
microorganism
active substance
produce
biologically active
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CN1252246C (en
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王书锦
胡江春
薛德林
马成新
张卫
张海涛
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Institute of Applied Ecology of CAS
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Institute of Applied Ecology of CAS
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Abstract

A process for separating the microbe able to generate bioactive substance from sponge cells includes such steps as choosing healthy living sponge from sea, washing in aseptic condition, taking the internal cell of body, cutting and grinding to obtain fresh liquid extract of sponge cells, plate diluting, smearing and culturing in thermostat for 1-15 days, slant storage of monocolony and testing the bioactive substance.

Description

A kind ofly can produce the biologically active substance method of microorganism from the sponge cellular segregation
Technical field
The present invention relates to microbe separation technology, specifically a kind of from sponge cellular segregation generation biologically active substance method of microorganism, it is to obtain new fresh and alive sponge multicellular animals from the ocean, and separation energy produces the biologically active substance method of microorganism such as anticancer, antitumor, antibiotic of high level from the inner cell of sponge.
Technical background
Sponge is the most original, minimum etc. multicellular animals, and inorganization does not have the organ differentiation, is life on earth origin and the individual branches of evolving, and is the invertebrates in the ocean.Domestic and international studies show that for many years, surpassing 10% secondary metabolite in the sponge all has cytotoxicity, be biologically active substance, and this ratio has only 2% in other marine organisms of ocean, and plant on land and microorganism are then far below 1%.Therefore, the research of active substance in the sponge is subjected to countries in the world scientist's attention.Whether but up to the present, sponge can produce the machine-processed not clear of active substance, relevant with the symbiosis microorganism of depositing in the sponge cell, does not also have conclusion so far, and this mechanism is very complicated.According to according to a preliminary estimate, the microorganism that symbiosis in the sponge cell is deposited accounts for 40% of sponge volume, Univ Maryland-Coll Park USA finds: have complicated microbe groups in the sponge, anti-cancer active matter in the sponge is that the Institute of Micro-biology that is deposited by its symbiosis produces, and from sponge, separate obtain the bright tangerine look of strain micrococci can ferment produce with sponge in the same active substance (Diketopiperagines).People such as Althoff have determined also that to the research of DNA the symbiosis of Rhodobacter and sponge Halichondria panicea deposits relation.In addition, Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences has obtained the microorganism strains that agricultural antibiotic and antitumor lead compound isoreactivity material are produced in many strains from the film sponge cell of the Bohai Sea.At present, how can separation and purification from the sponge cell obtain its symbiotic microorganism, produce the sponge microorganism of active substance, and utilize these precious resources, research marine microorganism pharmacy, agricultural antibiotic or the like has become the focus of domestic and international concern, adopt traditional conventional separation method at present, do not add Chen Haishui, sponge, can only obtain the microorganism in the small part sponge cell, can not obtain the genus kind group of microbial diversity in the sponge ecosystem.So far domestic and international none ideal still separates the report of method of microorganism in the sponge cell.
Summary of the invention
The object of the present invention is to provide a kind of from the sponge cell separation energy produce the biologically active substance method of microorganism.
To achieve these goals, the technical solution used in the present invention is for operating as follows:
(1) from the ocean, chooses sponge organism healthy, that live (choosing healthy, that live, no disease and pest), under aseptic condition, clean, get sponge interior detail cell space and be cut into 1～5mm fritter, ground 3～5 minutes, obtain fresh sponge cell extract; Alcohol or stroke-physiological saline solution are adopted in described cleaning;
(2), adopt plate dilution method, respectively with 10 to fresh sponge cell extract -1～10 -7Concentration dilution liquid on the plate that all is added with the separating marine fungi of 0.005～0.05% sponge, bacterium or actinomycetes substratum, is coated with strike-off stick, puts into then in 25～30 ℃ of insulation cans and cultivates;
(3) cultivate after 1～15 day, plate is grown pure (the growth unanimity) microorganism of single bacterium colony move to slant preservation, in order to carrying out the bioactive evaluation of secondary metabolite, authentication method adopts routine techniques;
The bioactive evaluation of described secondary metabolite is to being that it does not produce biologically active substance such as anticancer, antitumor, antibiotic and carries out detection validation under laboratory condition;
The described dull and stereotyped diluent concentration that is used for separating sponge cell fungi is 10 -2～10 -3The described dull and stereotyped diluent concentration that is used for separating sponge cell bacterium is 10 -5～10 -7It is described that to be used for separating the actinomycetic dull and stereotyped diluent concentration of sponge cell be 10 -1～10 -2Wherein diluting used solution is sterilized water;
By weight percentage, the medium component of fungi is in the described separation sponge cell: glucose 1%, Zulkovsky starch 0.5～2.0%, KH 2PO 40.1%, MgSO 47H 2O 0.1%, peptone 0.5～1.0%, sponge 0.005～0.05%, agar 2%, surplus are Chen Haishui or artificial seawater, pH6.0～6.5; In the described substratum of every 1000ml, add 1% rose-bengal (RoseBengal) aqueous solution 3.3ml before the sterilization; When being poured on to face on the flat board, the thawing substratum adds 0.5～2% Streptomycin sulphate solution, 0.1～1.0ml in every 100ml substratum of time spent, and Ciprofloxacin 100～500 μ l;
By weight percentage, the medium component that separates bacterium in the sponge cell is: Zulkovsky starch 0.5～2.0%, extractum carnis 0.5%, and yeast extract paste 0.5%, sponge 0.005～0.05%, agar 2%, surplus are Chen Haishui or artificial seawater, pH7.0～7.2;
By weight percentage, actinomycetic medium component is in the separation sponge cell: Zulkovsky starch 0.5～2.0%, sucrose 0.1～1.0%, N.F,USP MANNITOL 0.5%, KNO 30.1%, K 2HPO 40.5%, MgSO 47H 2O 0.5%, FeSO 47H 2O 0.01%, sponge 0.005～0.05%, agar 2%, surplus are Chen Haishui or artificial seawater, pH7.0～7.4; Face the time spent, in the described substratum of 300ml of heating and melting, add 3% potassium bichromate solution 1ml, add 1% Streptomycin sulphate solution 0.5ml again, and Ciprofloxacin 100～500 μ l.
The present invention has following beneficial effect:
1. use the present invention and from the sponge cell, separate generation active substances method of microorganism, in isolation medium, all added sponge 0.005～0.05%, separating effect is fine, and when separating fungi, add rose-bengal and Streptomycin sulphate, add potassium bichromate, Streptomycin sulphate and Ciprofloxacin during actinomycetes, so average success ratio is more than 90%.
2. the present invention is simple to operate, can obtain required bacterial strain.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment 1
Separating the method that produces the biologically active substance fungi from the sponge cell can operate according to following steps:
(1) cleans immediately after from the ocean, choosing the sponge organism of healthy work, adopt alcohol that the sponge appearance is carried out disinfection; Use Aseptic technique, cut the cavernous body skin, get sponge interior detail cell space and be cut into the square fritter of 1～5mm, grind, add stroke-physiological saline solution (NaCl 0.85%), ground 5 minutes, obtain fresh sponge cell extract in aseptic grinding with aseptic quartz sand;
(2), adopt plate dilution method, respectively with 10 to fresh sponge cell extract -2, 10 -3Concentration dilution liquid is coated with flat board on the plate of following separation sponge fungi culture medium, be coated with to put in 25 ℃ of insulation cans and cultivated;
(3) cultivate and to choose single bacterium colony after 1 week and divide purely, then move to grow to the inclined-plane and preserve, carry out the bioactive evaluation of secondary metabolite; The bioactive evaluation of described secondary metabolite is that whether it produces biologically active substance such as anticancer, antitumor, antibiotic and carry out detection validation under laboratory condition;
Its composition is as follows by weight percentage to separate the sponge fungi culture medium:
Glucose 1%, Zulkovsky starch 1%, KH 2PO 40.1%, MgSO 47H 2O 0.1%, peptone 0.5%, sponge 0.05%, agar 2%, surplus are Chen Haishui, pH6.0; Described old seawater is the seawater that leaves standstill for some time;
In this substratum of every 1000ml, add 1%Rose Bengal aqueous solution 3.3ml before the sterilization; Be poured on the flat board when melting substratum, face the time spent: add 1% Streptomycin sulphate solution 0.5ml and Ciprofloxacin 300 μ l in every 100ml substratum.
The success ratio that present embodiment separates fungi in the sponge cell is more than 90%.
Embodiment 2
Difference from Example 1 is:
To fresh sponge cell extract, use plate dilution method, respectively with 10 -5, 10 -6, 10 -7Concentration dilution liquid is coated with flat board on the plate of following separation sponge bacteria culture medium, be coated with to put in 28 ℃ of insulation cans cultivation 3 days, chooses single bacterium colony and divides purely, then moves to grow to the inclined-plane and preserves, and carries out the bioactive evaluation of secondary metabolite.
It is composed as follows by weight percentage to separate the sponge bacteria culture medium:
Zulkovsky starch 1%, extractum carnis 0.5%, yeast extract paste 0.5%, sponge 0.01%, agar 2%, surplus are Chen Haishui, pH7.0.
The success ratio that present embodiment separates bacterium in the sponge cell is more than 95%.
Embodiment 3
Difference from Example 1 is:
After from the ocean, choosing the sponge organism of healthy work, adopt physiological saline that the sponge appearance is carried out disinfection;
To fresh sponge cell extract, use plate dilution method, respectively with 10 -1, 10 -2Concentration dilution liquid is coated with flat board on the plate of following separation sponge actinomycetes substratum, be coated with to put in 30 ℃ of insulation cans after 2 weeks of cultivation, chooses single bacterium colony and divides purely, then moves to grow to the inclined-plane and preserves, and carries out the bioactive evaluation of secondary metabolite.
It is composed as follows by weight percentage to separate sponge actinomycetes substratum:
Zulkovsky starch 1%, sucrose 0.5%, N.F,USP MANNITOL 0.5%, KNO 30.1%, K 2HPO 40.5%, MgSO 47H 2O 0.01%, FeSO 47H 2O 0.01%, sponge 0.01%, agar 2%, surplus are Chen Haishui, pH7.0～7.2;
Face the time spent, in the 300ml substratum of heating and melting, add 3% potassium bichromate solution 1ml, add 1% Streptomycin sulphate solution 0.5ml again, and Ciprofloxacin 500 μ l.
Actinomycetic success ratio is more than 70% in the present embodiment separation sponge cell.
The also available artificial seawater of old seawater of the present invention substitutes.

Claims (9)

1. one kind can produce the biologically active substance method of microorganism from the sponge cellular segregation, it is characterized in that and can operate as follows:
(1) from the ocean, chooses sponge organism healthy, that live, under aseptic condition, clean, get sponge interior detail cell space and be cut into 1～5mm fritter, ground 3～5 minutes, obtain fresh sponge cell extract;
(2), adopt plate dilution method, with 10 to fresh sponge cell extract -1～10 -7Concentration dilution liquid on the plate that all is added with the separating marine fungi of 0.005～0.05% sponge, bacterium or actinomycetes substratum, is coated with strike-off stick, puts into then in 25～30 ℃ of insulation cans and cultivates;
(3) cultivate after 1～15 day, plate is grown the pure microorganism of single bacterium colony move to slant preservation, in order to carrying out the bioactive evaluation of secondary metabolite.
2. can produce the biologically active substance method of microorganism from the sponge cellular segregation according to claim 1, it is characterized in that: alcohol or stroke-physiological saline solution are adopted in described cleaning.
3. can produce the biologically active substance method of microorganism from the sponge cellular segregation according to claim 1, it is characterized in that: the bioactive evaluation of described secondary metabolite is to being that it does not produce anticancer, antitumor, antibiotic bioactive material and carries out detection validation under laboratory condition.
4. can produce the biologically active substance method of microorganism from the sponge cellular segregation according to claim 1, it is characterized in that: the described dull and stereotyped diluent concentration that is used for separating sponge cell fungi is 10 -2～10 -3
5. can produce the biologically active substance method of microorganism from the sponge cellular segregation according to claim 1, it is characterized in that: the described dull and stereotyped diluent concentration that is used for separating sponge cell bacterium is 10 -5～10 -7
6. can produce the biologically active substance method of microorganism from the sponge cellular segregation according to claim 1, it is characterized in that: described to be used for separating the actinomycetic dull and stereotyped diluent concentration of sponge cell be 10 -1～10 -2
7. can produce the biologically active substance method of microorganism from the sponge cellular segregation according to claim 1, it is characterized in that:
By weight percentage, the medium component of fungi is in the described separation sponge cell: glucose 1%, Zulkovsky starch 0.5～2.0%, KH 2PO 40.1%, MgSO 47H 2O 0.1%, peptone 0.5～1.0%, sponge 0.005～0.05%, agar 2%, surplus are Chen Haishui or artificial seawater, pH6.0～6.5;
In the described substratum of every 1000ml, add 1% rose-bengal aqueous solution 3.3ml before the sterilization; When being poured on to face on the flat board, the thawing substratum adds 0.5～2% Streptomycin sulphate solution, 0.1～1.0ml in every 100ml substratum of time spent, and Ciprofloxacin 100～500 μ l.
8. can produce the biologically active substance method of microorganism from the sponge cellular segregation according to claim 1, it is characterized in that: by weight percentage, the medium component that separates bacterium in the sponge cell is: Zulkovsky starch 0.5～2.0%, extractum carnis 0.5%, yeast extract paste 0.5%, sponge 0.005～0.05%, agar 2%, surplus are Chen Haishui or artificial seawater, pH7.0～7.2.
9. can produce the biologically active substance method of microorganism from the sponge cellular segregation according to claim 1, it is characterized in that: by weight percentage, actinomycetic medium component is in the separation sponge cell: Zulkovsky starch 0.5～2.0%, sucrose 0.1～1.0%, N.F,USP MANNITOL 0.5%, KNO 30.1%, K 2HPO 40.5%, MgSO 47H 2O 0.5%, FeSO 47H 2O 0.01%, sponge 0.005～0.05%, agar 2%, surplus are Chen Haishui or artificial seawater, and pH 7.0～7.4;
Face the time spent, in the described substratum of 300ml of heating and melting, add 3% potassium bichromate solution 1ml, add 1% Streptomycin sulphate solution 0.5ml again, and Ciprofloxacin 100～500 μ l.
CNB021325626A 2002-07-09 2002-07-09 Method for extracting microorganism producing biological active substance from sponge cell Expired - Fee Related CN1252246C (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006081722A1 (en) 2005-02-05 2006-08-10 Dilian Institute Of Chemical Physics, Chinese Academy Of Sciences A purification and cultivation method of ocean sponge pluripotent stem cell
CN1309840C (en) * 2005-09-01 2007-04-11 上海交通大学 Combined bacterial screening method with antibiotic function synergistic enhancing effect in sponge
CN1320098C (en) * 2005-09-01 2007-06-06 上海交通大学 Blending culture for sponge and intergrowth microbe, culturing colony monitoring method
CN100358516C (en) * 2004-12-07 2008-01-02 中国科学院大连化学物理研究所 Application of rugose soft sulfo-acid in anti HIV-1 virus
CN106701579A (en) * 2016-12-27 2017-05-24 广东环凯生物科技有限公司 Rose bengal sodium agar culture medium flat plate capable of irradiation sterilization

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100358516C (en) * 2004-12-07 2008-01-02 中国科学院大连化学物理研究所 Application of rugose soft sulfo-acid in anti HIV-1 virus
WO2006081722A1 (en) 2005-02-05 2006-08-10 Dilian Institute Of Chemical Physics, Chinese Academy Of Sciences A purification and cultivation method of ocean sponge pluripotent stem cell
CN100562568C (en) * 2005-02-05 2009-11-25 中国科学院大连化学物理研究所 A kind of separation purification method of spongy archaecyte
CN1309840C (en) * 2005-09-01 2007-04-11 上海交通大学 Combined bacterial screening method with antibiotic function synergistic enhancing effect in sponge
CN1320098C (en) * 2005-09-01 2007-06-06 上海交通大学 Blending culture for sponge and intergrowth microbe, culturing colony monitoring method
CN106701579A (en) * 2016-12-27 2017-05-24 广东环凯生物科技有限公司 Rose bengal sodium agar culture medium flat plate capable of irradiation sterilization
CN106701579B (en) * 2016-12-27 2019-07-23 广东环凯生物科技有限公司 It is a kind of can irradiation sterilization Rose Bengal Sodium agar medium plate

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