CN105002122B - A kind of culture medium of screening and culturing rhamnolipid producing strains - Google Patents

A kind of culture medium of screening and culturing rhamnolipid producing strains Download PDF

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CN105002122B
CN105002122B CN201510505685.XA CN201510505685A CN105002122B CN 105002122 B CN105002122 B CN 105002122B CN 201510505685 A CN201510505685 A CN 201510505685A CN 105002122 B CN105002122 B CN 105002122B
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parts
culture medium
rhamnolipid
powder
acid
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CN105002122A (en
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类延乐
张培
纪恒新
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Baizalai (qingdao) Food Co Ltd
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract

The invention discloses a kind of culture medium of screening and culturing rhamnolipid producing strains, including 40～50 parts of fishbone powder, 10～20 parts of aminoacid powder, 15～25 parts of biochemical fulvic acid potassium, 5～10 parts of alginates, 5～10 parts of vegetable polysaccharidess, 1～2 part of trace element, 1～2 part of vitamin, 200～350 parts of water.Contain marine active composition fishbone powder and alginate in the culture medium of the present invention, after effectively assembling with vegetable polysaccharidess, function bacterium, aminoacid, make three strains producing biosurfactants become the strain of advantage in enrichment process, and cause the pseudomonass for producing rhamnolipid to become dominant bacteria；And, can significantly improve the culture efficiency of pseudomonass.

Description

A kind of culture medium of screening and culturing rhamnolipid producing strains
Technical field
The invention belongs to microbiological culture media preparing technical field, and in particular to a kind of screening and culturing rhamnolipid producing strains Culture medium.
Background technology
Rhamnolipid (RL) is to study a kind of more biosurfactant at present, with emulsifying, solubilising, reduces interface The functions such as tension force, low toxicity, easily biological-degradable, in fields such as petrochemical industry, oil exploitation, biological medicine, food and environmental conservation There is very big application prospect.At present in world wide, the product rhamnolipid highest strain of most study is Pseudomonas aeruginosa (Pseudomonas aeruginosa), and various Pseudomonas aeruginosas can generate rhamnolipid using different carbon source, its system It is standby mainly by fermentable, then extract from fermentation liquid and obtain.In microbe oil production, rhamnolipid is that one kind has very much The oil displacement agent of effect, Pseudomonas aeruginosa are generally considered the effective efficiency bacterium of microbe oil production.Domestic Jing fermentables are obtained The rhamnolipid yield for arriving is relatively low, and from fermentation liquid, the complex process of separation and Extraction rhamnolipid, production cost are higher, limit Its large-scale production and application.As disclosed in CN1891831A, RL maximum productions are only 23g/L, when enter steel etc. and disclose in the patent Fermentation liquid in RL mass concentration be 30～40g/L.Therefore, the rhamnolipid yield in fermentation liquid is improved, reduces being extracted into This, to the popularization and application of rhamnolipid highly significant, key is that the strain and fermentation technology of producing rhamnolipid with high yield.
But in nature, screening is produced the Pseudomonas aeruginosa of rhamnolipid and has randomness, especially in the micro- life of oil Thing exploits field, using the method and already present screening technique that refer on current document, screens Aerugo from reservoir formation water Pseudomonass have randomness, it is impossible to pointedly filter out Pseudomonas aeruginosa, and poor selectivity, screening rate are low.
The content of the invention
The technical problem to be solved is for the deficiencies in the prior art, there is provided a kind of screening and culturing Mus The culture medium of Lee's glycolipid producing strains, effectively can filter out Fructus rhamni (Rhamnus davurica Pall.) from the bacterium source containing rhamnose producing strains using the culture medium Glycolipid producing strains, improve screening rate；Also, with the culture medium culturing
The present invention is achieved through the following technical solutions：
A kind of culture medium of screening and culturing rhamnolipid producing strains, including 40～50 parts of fishbone powder, 10～20 parts of aminoacid Powder, 15～25 parts of biochemical fulvic acid potassium, 5～10 parts of alginates, 5～10 parts of vegetable polysaccharidess, 1～2 part of trace element, 1～2 part Vitamin, 200～350 parts of water.
Preferably, the preparation method of described fishbone powder is：Fishbone is crushed after drying 2～3h at 50～60 DEG C, then is ground 200～300nm is milled to, fishbone powder after sterilizing, is obtained.
Preferably, described aminoacid powder includes aspartic acid, poly-aspartate, glutamic acid and glycine, its weight ratio For 1:1:1:1.
Preferably, described alginate is sodium alginate, potassium alginate, calcium alginate with weight ratio as 2:1:1 mixing Mixture.
Preferably, described vegetable polysaccharidess be shitosan, dextrin, carrageenan, in pectin any one or it is two or more The mixture mixed with arbitrary proportion.
Preferably, described trace element be nicotinic acid, Folic Acid, pantothenic acid, in Fe, Mn, Cu, Zn, Se any one or two The mixture mixed with arbitrary proportion more than kind.
Preferably, described vitamin is VA, VD3、VE、VB2、VB12In any one or it is two or more arbitrarily comparing The mixture that example is mixed.
Contain marine active composition fishbone powder and alginate in the culture medium of the present invention, with vegetable polysaccharidess, function bacterium, ammonia After base acid is effectively assembled, make three strains producing biosurfactants become the strain of advantage in enrichment process, and cause to produce Mus The pseudomonass of Lee's glycolipid become dominant bacteria；And, can significantly improve the culture efficiency of pseudomonass.
Specific embodiment
The culture medium of the present invention is specifically described with reference to embodiment：
Embodiment 1：
A kind of culture medium of screening rhamnolipid producing strains, including 420g fishbone powder, 160g aminoacid powder (aspartic acids 40g, poly-aspartate 40g, glutamic acid 40g and glycine 40g), 160g biochemical fulvic acid potassium, 80g alginate (sodium alginates 40g, potassium alginate 20g, calcium alginate 20g), 60g vegetable polysaccharidess (shitosan 10g, oligochitosan 10g, starch 10g, dextrin 10g, Carrageenan 10g, pectin 10g), 15g trace element (nicotinic acid 2g, Folic Acid 1g, pantothenic acid 0.5g, Fe 3g, Mn 2g, Cu 2g, Zn 4g, Se 0.5g), 15g vitamin (VA 3g, VD3 4g、VE 3g、VB2 1g、VB124g)；Above-mentioned raw materials add 2L after mixing Water, stirs and obtains final product I culture medium.
The preparation method of described fishbone powder is：Fishbone washing is dried after removing surface mud and attachment at 50～60 DEG C Dregs are ground into after dry 2～3h, then dregs are delivered to be ground in grinder 200～300nm, after sterilizing fishbone powder.
The I culture medium of preparation is removed into oil removal by the beautiful coarse filtration that carries out of sieve of 100 mesh, 300 mesh, 500 mesh step by step；So Mixed cellulose ester membrane again through 5 microns carries out fine straining afterwards, and fine straining liquid is used directly to inoculation；Mixed cellulose ester membrane fine straining liquid With preferable sterilization functions, it is to avoid autoclave sterilization culture medium and destroy the nutrition of composition in culture medium.
Embodiment 2：
Prepare II culture medium；Formula, preparation method, filter method are same as Example 1, different addition 480g Fishbone powder and 160g aminoacid powder (aspartic acid 30g, poly-aspartate 30g, glutamic acid 30g and glycine 30g).
Comparative example 1：
Prepare A culture medium；Formula, preparation method, filter method are same as Example 1, except that not containing 420g fishbone powder described in embodiment 1.
Comparative example 2：
Prepare B culture medium；Formula, preparation method, filter method are same as Example 1, except that not containing 80g alginates described in embodiment 1.
Confirmatory experiment one：
The Screening of Media rhamnolipid producing strains prepared using embodiment 1：
Comprise the following steps：
(1) strain source：With oil field reservoir formation water as strain source, North China Mongolia was collected on 03 02nd, 2015 The Tenggeer Formation of woods rises the oil field reservoir formation water of 2,3 sub-layers of the 3rd substratum of a pars infrasegmentalis, removes the visible mechanical admixture of naked eyes.
(2) stratum water 10ml is taken respectively, is accessed the fine straining liquid of culture medium prepared by 90ml embodiments 1, is loaded 250m triangles Bottle, is placed in 37 DEG C of shaking table cultures, rotating speed 200r/min；Taking-up enrichment culture liquid, gradient dilution after culture 7d, taking extension rate is 104th, 105 bacterium solution is spread evenly across LB Agar Platings and haemolysis Agar Plating, is placed in 35 DEG C of incubator trainings Foster 36h, observes colonial morphology, selects the same strain that form clump count is in the majority or haemolysis circle is big, especially selects with following spy The bacterium colony levied：Taupe, is put down partially, and edge is irregular, and often in mutual Fusion Strain, periphery of bacterial colonies has water colo(u)r, makes training Foster base is dyed to aeruginouss or yellow green, and bacterium colony has metallic luster or has Rhizoma Zingiberis Recenss abnormal smells from the patient.
The haemolysis plating medium (g/L)：Sucrose 10, beef extract-peptone 1, yeast extract 2, agar 20, Sanguis caprae seu ovis 0.3pH 7.0～7.5.
The LB culture medium (g/L)：Sucrose 5, beef extract-peptone 0.5, yeast extract 0.2, NaCl0.2, pH7.0～7.5.
(3) inoculating loop picking target bacterium colony is inoculated in fermentation medium, 250ml triangular flasks, liquid amount 100ml, 34 DEG C 200r/min shaking table cultures.Fermentation liquid is taken out after 70h, 60 DEG C of heating in water bath 10min, 10000*g centrifugation 20min takes supernatant Respectively with method of discharge, circle surface tension SF and emulsifying coefficient EI24 test surfaces activity, select oil extraction loop diameter more than 10cm, Surface tension be less than 30mN/m, and EI24 be more than 90% strain as Producing Strain preserve.Cell fermentation liquid supernatant will be removed Liquid capillary tube point sample in thin layer chromatography TLC analysis silica gel plate on, developing solvent is chloroform: methane: acetic acid (V/VV)=65: 15: 2 in In chromatography cylinder, the silica gel plate of point sample is put into into chromatography cylinder, to be deployed dose is taken out during 1cm apart from the top of silica gel plate, and to be deployed dose is waved Developer (developer is uniformly sprayed to silica gel plate with glass aerosol apparatus after sending out：2g anthrones are dissolved in 80%H2SO4, with 80% H2SO4 constant volumes are to 1000ml) 5min colour developings are heated at 110 DEG C.Rhamnolipid produces fermented liquid and occurs Jing after launching colour developing Brown color speckle, Rf value are 0.62.Therefore, select the fungi preservation that spot and R f value is 0.62 and the shade of colour is too deep on silica gel plate.
The fermentation medium (g/L)：Oleum Glycines 60, NaNO36, yeast extract 1.5；K2HPO40.3%, CaCl20.010%, MgSO40.024%, FeSO40.01%, Na2MO40.01%, pH7.0～7.5.
Conditions of flask fermentation：The bottled 100mL seed culture mediums of 250mL triangles (LB culture medium), are placed in after inoculation rotary 36h (200r/min, 37 DEG C) is cultivated on shaking table and obtains seed liquor.The bottled 90mL fermentation medium of 250mL triangles, accesses after sterilizing 10mL seed liquor, is placed in 200r/min on rotary shaker, 37 DEG C of culture 100h.
The oil extraction circle method is as follows：Take scrubbed dose of cleaning of culture dish of a diameter of 15cm, deionized water rinsing 3 times Afterwards, 50mL deionized waters are added, 0.5mL hexadecanes are slowly added dropwise in water surface, form the oil film of thin layer, in oil film The heart is slowly added into go the cell fermentation supernatant, center oil film of 30 μ L and is pressed against surrounding and forms circle, and circle diameter is lived with surface Property agent content is directly proportional.Oil extraction loop diameter is bigger, illustrates that surfactant yield is higher.
The surface tension SF measurement：Using surface tension instrument, the less explanation surface activity of surface tension is better.
The emulsifying coefficient EI24 measurements：In test tube with a scale, 7mL tests hydrocarbon (0# diesel oil) and 3mL fermentations are added Liquid supernatant, turbula shaker fully vibrate 2min, stand 24h, with emulsifying index
(Emusification index, EI24) represents the emulsifying activity of emulsifying agent.EI24 is oil-water interfaces upper level Account for the percent of total height.EI24 is bigger, and explanation surface activity is better, and stability is higher.
In the pseudomonass fermented liquid supernatant liquid for being screened, rhamnolipid content adopts colorimetric determination：
Developer：2g anthrones are dissolved in 80%H2SO4, with 80%H2SO4 constant volumes to 1000ml.
A. prepare standard curve：
B. rhamnolipid content detection in fermentation liquid：
(1) fermentation liquid is heated to 60 DEG C, is centrifuged off thalline；
(2) HCl is added to be hydrolyzed.When being hydrolyzed, temperature should be between 100～120 DEG C, and pH value should 1.5 or so, the time is 3.5 hours；
(3) supernatant being filtered to take, beta-hydroxy capric acid being removed with ethyl acetate extracting, layering takes clear liquid；
(4) in Boiling tube, (16～20mm of test tube internal diameter can make quick heat radiating after sulphuric acid addition, so as to reach good mixing Closing) anthrone-concentrated sulphuric acid for Jia 0.2% is developer 4.0mL, along test tube wall Deca sample 1.0mL, after cooling, boiling water bath is heated 20min, cools down the OD values for determining solution in 30min at 553nm afterwards；
(5) rhamnolipid content (corresponding content * of standard curve in fermentation liquid is calculated according to standard curve and extension rate 300/1000=final content g/L).
10 target bacterium colonies are selected altogether according to the colony characteristicses, and fermentation culture is respectively provided with higher surface activity, TLC During 10 plants of fermentation liquids that method analysis is obtained have the strain of surface activity, rhamnolipid producing strains are 14 plants, expand 14 plants of Mus Lee glycolipid producing strains 16S rDNA, carry out sequencing and phylogenetic analysis, show that 14 plants of bacterium belong to Rhodopseudomonass, Wherein 13 plants is Pseudomonas aeruginosa, and the bacterial strain can be in -80 DEG C of glycerol tubes preservations.
Jing rhamnolipid assays, in the bacterium of 14 plants of product rhamnolipids, one plant of yield highest is Pseudomonas aeruginosa Pseudomonas aeruginosa。
Confirmatory experiment two：
The II Screening of Media rhamnolipid producing strains prepared using embodiment 2：
The method and step of strain screening is identical with checking enforcement one, as a result as follows：
In step (3), inoculating loop picking target bacterium colony is inoculated in shaking table culture after fermentation medium, and culture 71.5h can be with Obtain oil extraction loop diameter and 30mN/m is less than more than 10cm, surface tension, and EI24 is that more than 90% strain is protected as Producing Strain Deposit.
In 10 target bacterium colonies that II culture medium is selected, 10 plants of fermentation liquids that the analysis of TCL methods is obtained have surface activity Strain in rhamnolipid producing strains be 14 plants, expand 14 plants of rhamnolipids producing strains 16S rDNA, carry out sequencing And phylogenetic analysis, show that 14 plants of bacterium belong to Rhodopseudomonass, wherein 10 plants is Pseudomonas aeruginosa；Jing rhamnolipids Assay, one Pseudomonas aeruginosa strain Pseudomonas of yield highest in the bacterium of 14 plants of product rhamnolipids aeruginosa。
Confirmatory experiment three：
The A Screening of Media rhamnolipid producing strains prepared using comparative example 1：
The method and step of strain screening is identical with checking enforcement one, as a result as follows：
In step (3), inoculating loop picking target bacterium colony is inoculated in shaking table culture after fermentation medium, and culture 120h just can be with Obtain oil extraction loop diameter and be less than 30mN/m more than 10cm, surface tension, and EI24 be more than 90% strain as Producing Strain Preserve.Compared with I culture medium (incubation time is 70h), the time required for strain of cultivating significantly extends.
In 10 target bacterium colonies that A culture medium is selected, 10 plants of fermentation liquids that the analysis of TCL methods is obtained have surface activity In strain, rhamnolipid producing strains are 9 plants, expand 9 plants of rhamnolipids producing strains 16S rDNA, carry out sequencing and be System evolutionary analysis, show that 9 plants of bacterium belong to Rhodopseudomonass, wherein 6 plants is Pseudomonas aeruginosa；Jing rhamnolipids are containing measurement Fixed, in the bacterium of 6 plants of product rhamnolipids, one plant of yield highest is Pseudomonas aeruginosa Pseudomonas aeruginosa.
Confirmatory experiment four：
The B Screening of Media rhamnolipid producing strains prepared using comparative example 2：
The method and step of strain screening is identical with checking enforcement one, as a result as follows：
In step (3), inoculating loop picking target bacterium colony is inoculated in shaking table culture after fermentation medium, and culture 128h just can be with Obtain oil extraction loop diameter and be less than 30mN/m more than 10cm, surface tension, and EI24 be more than 90% strain as Producing Strain Preserve.Compared with I culture medium (incubation time is 70h), the time required for strain of cultivating significantly extends.
In 10 target bacterium colonies that B culture medium is selected, 10 plants of fermentation liquids that the analysis of TCL methods is obtained have surface activity In strain, rhamnolipid producing strains are 6 plants, expand 6 plants of rhamnolipids producing strains 16S rDNA, carry out sequencing and be System evolutionary analysis, show that 6 plants of bacterium belong to Rhodopseudomonass, wherein 5 plants is Pseudomonas aeruginosa；Jing rhamnolipids are containing measurement Fixed, in the bacterium of 6 plants of product rhamnolipids, one plant of yield highest is Pseudomonas aeruginosa Pseudomonas aeruginosa.
Examples detailed above is technology design to illustrate the invention and technical characterstic, can not limit the present invention's with this Protection domain.Equivalent transformation or modification that all essence of the invention is done, all should cover in protection scope of the present invention Within.

Claims (3)

1. a kind of culture medium of screening and culturing rhamnolipid producing strains, it is characterised in that including 40～50 parts of fishbone powder, 10～20 Part aminoacid powder, 15～25 parts of biochemical fulvic acid potassium, 5～10 parts of alginates, 5～10 parts of vegetable polysaccharidess, 1～2 part of micro unit Element, 1～2 part of vitamin, 200～350 parts of water；
Described aminoacid powder is made up of aspartic acid, poly-aspartate, glutamic acid and glycine, and its weight ratio is 1:1:1:1；
Described vegetable polysaccharidess be shitosan, oligochitosan, starch, dextrin, carrageenan, in pectin any one or it is two or more The mixture mixed with arbitrary proportion；
Described trace element is nicotinic acid, Folic Acid, pantothenic acid, any one in Fe, Mn, Cu, Zn, Se, or two or more appointing The mixture that meaning ratio is mixed；
Described vitamin is any one in VA, VD3, VE, VB2, VB12, or two or more is mixed with arbitrary proportion Mixture.
2. culture medium according to claim 1, it is characterised in that the preparation method of described fishbone powder is：Fishbone is 50 Crush after 2～3h is dried at～60 DEG C, then be ground to 200～300nm, after sterilizing, obtain fishbone powder.
3. culture medium according to claim 1, it is characterised in that described alginate be sodium alginate, potassium alginate, Calcium alginate is with weight ratio as 2:1:1 mixture for mixing.
CN201510505685.XA 2015-08-17 2015-08-17 A kind of culture medium of screening and culturing rhamnolipid producing strains Expired - Fee Related CN105002122B (en)

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