CN105838639B - A method of exocellular polysaccharide is produced using the red spirillum of outer sulphur - Google Patents
A method of exocellular polysaccharide is produced using the red spirillum of outer sulphur Download PDFInfo
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Abstract
The present invention relates to a kind of methods using the red spirillum production exocellular polysaccharide of outer sulphur.The different aerobe fermentation culture of illumination anaerobism is carried out for fermenting microbe for red spirillum LBD plants of sulphur other than this method, by controlling fermentation condition, biomass (the optical density OD of the fermentation culture medium obtained containing exocellular polysaccharide500nm) >=5, and in fermentation culture medium exocellular polysaccharide content up to 6mg/ml or more.Exocellular polysaccharide is produced using this method, yield of extracellular polysaccharide is high, and performance is stablized, and industrialization is easy to.
Description
Technical field
The invention belongs to the fermenting and preparing biological technical field of microbial polysaccharide, it is related to utilizing the red spirillum of outer sulphur for a kind of
The method for producing exocellular polysaccharide.
Background technique
Microbial exopolysaccharides include be adhered to cell surface capsular polysaccharide (Capsular polysaccharide,
) and the exocellular polysaccharide (Exopolysaccharides, EPS) that is secreted into medium of synthesis CPS.Extracellular polysaccharide of bacteria is in addition to it
Itself has important biological significance, such as protect bacteria from extreme environment stress, the invasion that inhibit lysozyme and bacteriophage,
It is outer to store glycogen energy and enhancing metal ion tolerance etc., also has the function of improving animal and human immunity function.
It is reported that there are 49 category, 76 kinds of microorganisms to be proved to can produce exocellular polysaccharide, and the thin of exocellular polysaccharide can be generated
Bacterium is rare, and only tens kinds.Have now found that acetobacter (Acetobacter), streptococcus (Streptococcus), vacation
Monad (Pseudomonas), bacillus (Bacillus sp.), Sphingol single-cell (Sphingomnas), galactococcus
(Lactococcus) and the strains such as lactobacillus (Lactobacillus) can produce polysaccharide, some bacterial strain exocellular polysaccharide structures are
It is identified.Although the exocellular polysaccharide structure and its function that different strains generate are different, biosynthesis is by way of almost the same, mainly
It is related to three assembly, multimerization and transport processes.It is different according to polysaccharide primary structure, homogeneity polysaccharide and heterogeneous more can be divided into
Sugar.The main chain of homogeneity polysaccharide is made of identical monosaccharide, is combined with non-saccharide substituent group by covalent bond, several frequently seen homogeneity
Polysaccharide is exactly that monosaccharide combines to be formed with non-saccharide substituent group, such as cellulose, glucan.The monosaccharide type for forming heterogeneous polysaccharide is logical
It is often 2-4 kind, monosaccharide not of the same race constitutes duplicate unit, constitutes the main chain of heterogeneous polysaccharide, can be according to different in repeating part
The composition of monosaccharide type or substituent group is to divide different heterogeneous polysaccharide.
Microbial polysaccharide can realize industrialized production by submerged fermentation, be the high-tech product of modern biotechnology.Such as
What enhances the practicability of exocellular polysaccharide, and excavating its potential value is emphasis concerned by people in recent years.Currently, micro- life in the world
The every annual of object polysaccharide yield is increased with 10% or so speed, and some strong functional polysaccharide annual growths newly developed reach
30% or more, at present the exocellular polysaccharide of large-scale industrial production mainly have xanthan gum, can obtain so, gellan gum, pyrenomycetes
Glucan and dextran etc..Because of the specific physical chemical property of microbial polysaccharide, often by as some additive applications in
The multiple fields such as food, medicine and environmental protection, such as emulsifier, film forming agent, suspending agent, thickener, gelling agent, stabilizer and lubricant
Deng.According to statistics, whole world microbial polysaccharide creates up to hundred million dollars of 50-100 of economic value every year.
In the prior art, exocellular polysaccharide is prepared using bacterium, yield is lower, and so far there is not yet using the red spiral shell of outer sulphur
The report of bacterium production polysaccharide.Therefore, it needs to find a kind of microorganism fungus kind that can efficiently produce exocellular polysaccharide at present.
Summary of the invention
The technical problem to be solved by the present invention is to solve the above shortcomings of the prior art and to provide a kind of outer red spirillum bacterium of sulphur
Strain, the bacterial strain are the photosynthetic bacteria of extracellular polysaccharide, carry out the heterotrophic fermentation culture of illumination anaerobism, born of the same parents by fermenting microbe of the bacterial strain
Exo polysaccharides yield is high, and performance is stablized, and industrialization is easy to.
For this purpose, first aspect present invention provides a kind of red spirillum bacterial strain of outer sulphur, it is red spirillum LBD plants of outer sulphur
(Ectothiorhodospira strain LBD), deposit number are as follows: CGMCC NO.11520 is preserved in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center is (referred to as: CGMCC；Address: in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Institute of microbiology, the academy of sciences, state), preservation date: on October 20th, 2015.The bacterial strain is the photosynthetic bacteria of extracellular polysaccharide.
Second aspect of the present invention provides a kind of exocellular polysaccharide, by bacterial strain as described in the first aspect of the invention through illumination
Anaerobism heterotrophic fermentation culture generates.
Third aspect present invention provides a kind of exocellular polysaccharide suspension, is the fermentation culture medium containing exocellular polysaccharide.
The fermentation culture medium is to be obtained using bacterial strain as described in the first aspect of the invention through illumination anaerobism heterotrophic fermentation culture.It is described
The optical density OD of fermentation culture medium500nm≥5.Exocellular polysaccharide content >=6mg/ml in the fermentation culture medium.
The 4th aspect of the present invention provides a kind of preparation method of exocellular polysaccharide comprising fermenting microbe is seeded to hair
The step of illumination fermented and cultured is carried out in ferment culture medium, wherein the fermenting microbe is obtained by corresponding bacterial strain by seed culture
?；The corresponding bacterial strain of fermenting microbe is homologous with the 16S rRNA of the red spirillum bacterial strain of outer sulphur described in first aspect present invention
Property is at least 90% bacterial strain；It is preferred that the corresponding bacterial strain of fermenting microbe is and the red spirillum of outer sulphur described in first aspect present invention
The homology of the 16S rRNA of bacterial strain is at least 95% bacterial strain；Further preferably prepare the phase of exocellular polysaccharide photosynthetic bacteria strain
The bacterial strain answered is the outer red spirillum bacterial strain of sulphur described in first aspect present invention.
According to the method for the present invention, the fermentation medium is in terms of 1L water, including the following components in 1L water:
In some embodiments of the invention, the carbon source material includes in sodium acetate, sodium citrate, sodium lactate and glycerol
One or more.The nitrogen source includes one or more of ammonium salt, urea, nitrate and peptone.
In other embodiments of the invention, the pH value of the fermentation medium is 6.5-8.0；It is preferred that the fermentation training
The pH value for supporting base is 6.5-7.2.
According to the method for the present invention, fermenting microbe is inoculated into fermentation medium in the form of seed liquor, described with volume basis
The inoculum concentration of seed liquor is 15%-30%；The inoculum concentration of preferred seed liquid is 15%-20%.
In some embodiments of the invention, the strain Colony Forming Unit in the seed liquor be (30-50) ×
108CFU/mL。。
In some embodiments of the invention, the temperature of the fermented and cultured is 30-45 DEG C；It is preferred that the illumination fermentation training
Feeding temperature is 30-35 DEG C.
It in some embodiments of the invention, is incandescent lamp and/or sunlight for the light source of illumination；The illumination fermentation
The intensity of illumination of culture is 2000-8000Lx.
Detailed description of the invention
Fig. 1 shows the red spirillum LBD plants of molecular evolutionary trees based on 16S rDNA of outer sulphur of the invention.
Fig. 2 shows the effect of the red spirillum LBD plants of growths of the external sulphur of different carbon source.
Fig. 3 shows the effect of the red spirillum LBD plants of extracellular polysaccharide of the external sulphur of different carbon source.
Culture presevation
Red spirillum LBD plants of outer sulphur (Ectothiorhodospira strain LBD) is had by more than green hundred biology exploitation of Guangdong
Limit responsible company separation, identification, China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation (referred to as:
CGMCC；Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica) carry out preservation, preservation day
Phase: on October 20th, 2015, deposit number: CGMCC NO.11520.
Specific embodiment
To keep the present invention easier to understand, below in conjunction with embodiment, the present invention will be described in detail, these embodiments are only
Serve illustrative, it is not limited to application range of the invention, unmentioned specific experiment method in the following example, usually
It is carried out according to routine experiment method.
As previously mentioned, in the prior art, it is lower to prepare exocellular polysaccharide normal yield using bacterium.The present inventor's warp
Numerous studies are crossed, screen and find a kind of red spirillum bacterial strain of outer sulphur, which is the photosynthetic bacteria of extracellular polysaccharide, extracellular more
Candy output is high, and performance is stablized.The present invention is based on what above-mentioned discovery was made.
Therefore, the outer red spirillum bacterial strain of sulphur involved in first aspect present invention is the photosynthetic bacteria of extracellular polysaccharide, is adopted
It is obtained with following Screening of Media and culture, the composition of the culture medium is following (in 1000L deionized water):
The present invention screening for produce the red spirillum bacterial strain of outer sulphur of exocellular polysaccharide method the following steps are included:
(1) it is formed according to above-mentioned culture medium and prepares culture medium, and use sodium hydroxide and hydrochloric acid solution by the first of culture medium
Beginning pH value is adjusted to 7.0.Be packed into prepared fluid nutrient medium 100ml with 250ml triangular flask, after the closed sealing of sealed membrane
Sterilize 20min under high temperature (121 DEG C) high pressure (0.15MPa), then makes after ultraviolet radiation sterilization 20min in clean bench
With.The agar of 1.8% (w/v) is added in liquid medium, is poured into after autoclave sterilization dissolves cold in culture dish
But, corresponding solid medium tablets are made.
(2) normal saline solution that 1.0g shrimp culture pond bed mud is put into after 10mL sterilizing is weighed, is fullyd shake after ten minutes
Stand 2 hours, take 5 milliliters of supernatant to be inoculated in 10 milliliters of liquid culture mediums, at 35 degree of temperature, under 40W incandescent light photograph into
Row illumination Anaerobic culturel.Brownish red is presented in culture after 5 days, and 5 milliliters of sampling inoculates cultivates in 10 milliliters of culture mediums, repeats 3
After secondary, culture is diluted to 10 respectively using normal saline solution after sterilizing-3、10-4With 10-5After gradient, respectively take 100 μ l right
Uniform coated plate on the solid medium answered, sealed with wax it is closed be placed on 35 DEG C of incubators, 40W incandescent light photograph under carry out illumination
Anaerobic culturel.The aubergine photosynthetic bacteria monoclonal colonies grown can be observed in solid culture primary surface through culture in 5 days, use
Transfer needle picking monoclonal colonies are reinoculated in liquid screening medium and cultivate 5 days, then carry out applying plate screening photosynthetic thin
Bacterium pure culture, is so repeated 5 times, and has filtered out the photosynthetic bacteria of one plant of extracellular polysaccharide.
Inventor has been observed that the monoclonal colonies feature of the bacterial strain for red, mellow and full, neat in edge；Pass through microscope
Microscopy (1000 times of amplification) discovery, the cell individual form of the bacterial strain are rod-shaped, long 4 microns of cell, wide 0.9 micron of left side
It is right；Extracted genomic DNA is divided by PCR amplification with the Molecular Identification based on 16s rDNA sequencing, the Photosynthetic bacterium strain
Class status is bacterium circle, Proteobacteria, deformation Gammaproteobacteria, coloring Zoopagales, outer sulphur Rhodospirillaceae, Ectothiorhodospira
(Ectothiorhodospira sp.), the red spirillum of outer sulphur, based on above-mentioned, the bacterial strain is identified to be named as the red spirillum LBD of outer sulphur
Strain (Ectothiorhodospira strain LBD).The bacterial strain is common in China Committee for Culture Collection of Microorganisms
Microorganism center preservation (referred to as: CGMCC) carries out preservation, deposit number: CGMCC NO.11520.
The preparation method of exocellular polysaccharide according to the present invention can be understood as a kind of extracellular using the red spirillum production of outer sulphur
The method of polysaccharide comprising fermenting microbe is seeded in fermentation medium the step of carrying out illumination fermented and cultured, wherein described
Fermenting microbe is obtained by corresponding bacterial strain by seed culture.
As it is known by one skilled in the art, currently, being often used 16S Ribosomal RNA (16S rRNA) in the world
The Molecular Identification of bacterium is carried out, therefore can be compared in the comparison of similitude that obtain its homologous with 16S rRNA
Property.So fermentation strain used in the present invention is not limited to field separation strains used in the present invention, 16s rRNA gene
It is to encode the corresponding DNA sequence dna of rRNA on bacterial chromosome, is present in the germy DNA sequence of institute.Fig. 1 is shown
Red spirillum LBD plants of molecular evolutionary trees based on 16S rDNA of outer sulphur of the invention.
Therefore, in the present invention, the corresponding bacterial strain of fermenting microbe is and the red spirillum of outer sulphur described in first aspect present invention
The homology of the 16S rRNA of bacterial strain (red spirillum LBD plants of i.e. outer sulphur) is at least 90% bacterial strain；It is preferred that fermenting microbe is corresponding
Bacterial strain be it is same with the 16S rRNA of the red spirillum bacterial strain of outer sulphur described in first aspect present invention (red spirillum LBD plants of i.e. outer sulphur)
Source property is at least 95% bacterial strain；The corresponding bacterial strain of further preferred fermenting microbe be with it is outer described in first aspect present invention
The red spirillum bacterial strain of sulphur (red spirillum LBD plants of i.e. outer sulphur).I.e. under the premise of not changing red spirillum LBD plants of outer sulphur of 16S rRNA,
Those skilled in the art can red spirillum LBD plants by the outer sulphur simply screened or mutagenesis is of the invention, obtain with the present invention outside
The bacterial strain of the red spirillum LBD plants of 16S rRNA very high homology of sulphur, and obtain correspondingly with the same or similar extracellular polysaccharide function
The mixture of the bacterial strain seed liquor of energy.
According to the present invention, the fermented and cultured is the heterotrophic fermentation culture of illumination anaerobism, it means that if using aerobic hair
Ferment can not just realize extracellular polysaccharide well.
According to the method for the present invention, the fermented and cultured is the standing for fermentation culture that strain dissociates, and fermenting microbe is with seed liquor
Form be inoculated into fermentation medium.With volume basis, the inoculum concentration of the seed liquor is 15%-30%.Preferred seed liquid
Inoculum concentration is 15%-20%.The inoculum concentration of further preferred seed liquor is 20%.
In some embodiments of the invention, in the seed liquor, strain Colony Forming Unit be (30-50) ×
108CFU/mL。
The present inventor also investigates intensity of illumination and fermentation temperature, it is found that the temperature of the illumination fermented and cultured is
30-45℃.It is preferred that the temperature of the illumination fermented and cultured is 30-35 DEG C.Light source for illumination is incandescent lamp and/or the sun
Light, the intensity of illumination of the illumination fermented and cultured are 2000-8000Lx；Fermented and cultured yield of extracellular polysaccharide obtained all compared with
It is high.
To obtain higher exocellular polysaccharide yield, research is optimized to fermentation medium in the present inventor, the results showed that
Fermentation medium is formulated with according to consisting of conducive to fermented and cultured and extracellular polysaccharide, and the fermentation medium is with 1L water
Meter, including the following components in 1L water:
In above-mentioned culture medium, EDTA-2Na can be understood as being optionally added ingredient, be added the ingredient be conducive to improve it is extracellular
The yield of polysaccharide.
Likewise, in above-mentioned culture medium, compound VB(vitamin B compound) can be understood as being optionally added ingredient, and being added should
Ingredient is conducive to improve the yield of exocellular polysaccharide.
The vitamin B compound is not particularly limited in the present invention, such as it can be commercially available vitamin B compound
Compound preparation, main ingredient contained by every tablet of vitamin B compound are as follows: vitamin B13 milligrams, vitamin B21.5 milligrams, vitamin
B60.2 milligram, 10 milligrams of niacinamide, 1 milligram of calcium pantothenate；Auxiliary material are as follows: starch, calcium sulfate and magnesium stearate.
For the yield for further increasing exocellular polysaccharide, further research is optimized to culture medium in inventor, using not
Same carbon source, such as sodium acetate, sodium citrate, sodium lactate and glycerol carry out the optimization of carbon source (with the sodium lactate of 2.5g/L containing organic
Subject to carbon).The result shows that above-mentioned carbon source material be conducive to produce exocellular polysaccharide, wherein it is preferable to sodium acetate and/or
Sodium citrate.More preferably sodium acetate.
Using different nitrogen sources, the optimization of nitrogen source is carried out (with 1.2g/ including ammonium salt, urea, nitrate and peptone
Subject to the ammonium chloride nitrogen content of L).The result shows that above-mentioned nitrogen source is conducive to produce exocellular polysaccharide, wherein more preferred
It is ammonium salt and/or peptone.More preferably ammonium salt.
In some embodiments of the invention, the ammonium salt includes ammonium chloride, ammonium sulfate, ammonium hydrogen sulfate, ammonium nitrate, carbonic acid
At least one of ammonium and ammonium hydrogen carbonate；It is preferred that the ammonium salt is ammonium chloride and/or ammonium nitrate.
In some embodiments of the invention, the nitrate includes in sodium nitrate, potassium nitrate, ammonium nitrate and calcium nitrate
It is one or more of；It is preferred that the nitrate is ammonium nitrate.
In some embodiments of the invention, the initial pH value of fermentation medium is adjusted using sodium carbonate and hydrochloric acid solution,
The initial pH value of the fermentation medium is 6.5-8.0；It is preferred that the initial pH value of the fermentation medium is 6.8-7.2.
According to certain embodiments of the present invention, the preparation method of exocellular polysaccharide according to the present invention further includes seed training
Feeding step: the picking outer red spirillum LBD plants of monoclonal colonies of sulphur provided by the present invention are inoculated into 10ml fluid nutrient medium,
After cultivating 5d under the conditions of 30-35 DEG C of temperature, illumination 2000-8000Lx, first generation fermenting microbe is obtained.
The step of above-mentioned seed culture of the invention, can also be according to the demand of fermentation inoculum concentration, and in first time, culture terminate
Afterwards, fermenting microbe obtained is transferred to 50ml fluid nutrient medium again, cultivates 5d under the same terms, obtains a greater amount of
Two generation fermenting microbes, can be further improved the yield of exocellular polysaccharide in this way.
Term of the present invention " optional " is meant optionally, is referred to addition or is added without, also refer to include or not
Include.
Invention is for " water " during culture medium or fermented and cultured, in the case where not specifying, refer to through
The water that 0.45 μ membrane filtration obtains.
Other than fermentation culture medium of the present invention containing exocellular polysaccharide is illumination is carried out for fermenting microbe for red spirillum LBD plants of sulphur
Anaerobism heterotrophic fermentation culture obtains, by controlling fermentation condition, the biomass of the fermentation culture medium obtained containing exocellular polysaccharide
(optical density OD500nm>=5) the exocellular polysaccharide content in fermentation culture medium after, and by 0.45 μ membrane filtration removing thallus can
Up to 6mg/ml or more.The fermentation culture medium containing exocellular polysaccharide is rich in protein, vitamin, somatomedin and immune factor
Etc. nutritional ingredients, fishes and shrimps can be added to, in animal and fowl fodder, improve cultivated animals immunity, promote growth, the function such as enhancing body colour
Energy；In addition the fermentation culture medium for containing exocellular polysaccharide can be also used for purifying aquaculture environment, for example, can be used for flocculating setting water
Particulate matter in body reduces the content of hydrogen sulfide, ammonia nitrogen, nitrite nitrogen, nitric nitrogen in water etc..
Term " exocellular polysaccharide " of the present invention can be understood as carrying out illumination anaerobism heterotrophic fermentation using the red spirillum of outer sulphur
A kind of microbial polysaccharide caused by cultivating, rich in the nutrition such as protein, vitamin, somatomedin and immune factor at
Point, it is outer in addition to glycogen energy and enhancing metal ion tolerance etc. can be stored, also there is raising animal and human immunity function
Effect.Therefore, purification microbial polysaccharide can further be extracted for human health care's product or bio-pharmaceuticals.
Exocellular polysaccharide is measured using following methods in the present invention:
3. the measurement of optical density is existed using Gold S54 ultraviolet specrophotometer (Shanghai Ling Guang Technology Co., Ltd.)
The optical density of fermentation culture medium is measured under 500nm wavelength, which can reflect out bacterium cell concentration；Heretofore described fermentation training
Support the optical density OD of object500nmIt is able to reflect red spirillum LBD plants of the growing state of outer sulphur.
4. the measurement of total reducing sugar measures total sugar content using dinitrosalicylic acid (DNS) colorimetric method, the specific steps are as follows:
(1) glucose Standard for Sugars solution (0.05mg/mL) is prepared:
1. weighing in the glucose sugar 50mg of 80 DEG C of drying in oven；
2. 50mL deionized water dissolving glucose sugar is added in beaker, it is transferred in 100mL volumetric flask, moisturizing constant volume, mixes
It is even stand-by.
(2) color developing agent 3 is prepared, 5- dinitrosalicylic acid (DNS):
1,6g flaky sodium hydrate solid is weighed, is dissolved in 20mL distilled water and is made into 2M NaOH solution；Into above-mentioned solution
3, the 5- dinitrosalicylic acid of 0.5g is added；It weighs 13g sodium potassium tartrate tetrahydrate solid to be dissolved in 50mL deionized water, with second step
Solution mixes together；Weighing 0.5g crystalline phenol and 0.5gNa2SO3Above-mentioned solution is added；Above-mentioned solution is settled to 100mL, in
Dark place saves backup.
(3) standard curve is made
1. taking 8 clean tubes and numbering；
2. the glucose Standard for Sugars for sequentially adding different content into 8 test tubes according to total sugar content standard curve sample-adding table is molten
Liquid, deionized water and DNS reagent；
3. excellent test tube will be added to be put into water-bath, boiling water bath 3 minutes, reacts mixed liquor sufficiently, use flowing water later
It is cooling；
4. adding deionized water 17mL into each test tube；
5. reading each concentration standard liquid at 540nm on microplate reader (FC type matches Mo Feishier Instrument Ltd.)
Absorbance value simultaneously records, and first reads and record every time the absorbance value of blank plate before reading；
6. being ordinate by the difference of abscissa, sample and blank plate absorbance value of concentration of glucose, standard curve is drawn；
(4) total sugar content in sample is calculated
It brings the absorbance value of analyte sample fluid into calibration curve formula, calculates its concentration, contain to calculate total reducing sugar
Amount.
The heterotrophic fermentation culture of illumination anaerobism is carried out for fermenting microbe for red spirillum LBD plants of sulphur other than according to the method for the present invention, lead to
Cross control fermentation condition, biomass (the optical density OD of the fermentation culture medium obtained containing exocellular polysaccharide500nm>=5), and pass through
The exocellular polysaccharide content in fermentation culture medium after 0.45 μ membrane filtration removal thallus is up to 6mg/ml or more.Utilize this method
Exocellular polysaccharide is produced, yield of extracellular polysaccharide is high, and performance is stablized, easy to industrialized production.
Embodiment
Embodiment 1: influence of the different carbon source to the optical density and exocellular polysaccharide content of fermentation culture medium is investigated.
1. the preparation of seed liquor
(1) fluid nutrient medium is prepared: by the fluid nutrient medium of seed culture in terms of 1L water, including following in 1L water
Component:
(2) inoculation and seed culture: the red spirillum LBD plants of monoclonal colonies of outer sulphur of picking screening are inoculated into the above-mentioned liquid of 10ml
In body culture medium, after cultivating 5d under the conditions of 30-35 DEG C of temperature, illumination 2000-8000Lx, by 1st generation zymophyte obtained
Kind is transferred to 50ml fluid nutrient medium again, and 5d is cultivated under the same terms, and it is spare to obtain second generation fermenting microbe (seed liquor).
2. preparing fermentation medium
(1) fermentation medium is prepared, fermentation medium is in terms of 1L water, including the following components in 1L water:
3. inoculation and fermented and cultured
Sodium carbonate and hydrochloric acid solution is used to adjust initial pH value as 6.8.Prepared liquid training is packed into 15ml centrifuge tube
Support base 10ml.Sterilize under high temperature (121 DEG C) high pressure (0.15MPa) 20min after sealing, then ultraviolet in clean bench
After line radiation sterilization 20min, fermenting microbe (seed liquor) is accessed thereto.
Under the conditions of 30-35 DEG C of temperature, illumination 2000-8000Lx, it is respectively adopted different carbon source substance, including sodium acetate,
Sodium lactate or glycerol carry out fermented and cultured.
It measures the optical density of the fermentation culture medium of different time and removes the fermentation after thallus by 0.45 μ membrane filtration
Total sugar content in culture investigates influence of the different carbon source to the optical density and exocellular polysaccharide content of fermentation culture medium, knot
Fruit sees Fig. 2 and Fig. 3.
Fig. 2 shows the effect of the red spirillum LBD plants of growths of the external sulphur of different carbon source；Fig. 3 shows the external sulphur of different carbon source
The effect of red spirillum LBD plants of extracellular polysaccharide.From figures 2 and 3, it will be seen that being used for using sodium acetate or sodium lactate as carbon source
The illumination anaerobism heterotrophic fermentation culture of red spirillum LBD plants of outer sulphur, the optical density of fermentation culture medium obtained is higher, exocellular polysaccharide
Content is also higher, wherein using sodium acetate as the optical density of the carry out fermented and cultured culture obtained containing exocellular polysaccharide of carbon source and
Exocellular polysaccharide content highest, optical density OD500nm>=5, exocellular polysaccharide content >=6mg/ml.
Embodiment 2: outer red spirillum LBD plants of the sulphur of industrialization culture.
In embodiment 2 preparation of seed liquor and exocellular polysaccharide to isolate and purify process same as Example 1.
Fermentation medium is in terms of 1L water in embodiment 2, including the following components in 1L water:
Initial pH value is adjusted to 7.2 with sodium carbonate and hydrochloric acid solution, and the liquid prepared and sterilized is packed into 2.5L culture bottle
Culture medium 2L, and access fermenting microbe (seed liquor) thereto.
Using red spirillum LBD plants of sulphur outside this culture medium industrialization culture after outdoor solar irradiation culture 6d, object light is cultivated
Density OD500nmIt can reach 5 or more.Red spirillum LBD strain 120 hours using sulphur outside this culture medium culture, fermented and cultured optical density
OD500nmIt can reach 5 or more, and removing by 0.45 μ membrane filtration in the fermentation culture medium after thallus exocellular polysaccharide concentration can be with
Reach 6mg/ml or more.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, made any modification, equivalent replacement or improvement etc. be should all be included in the protection scope of the present invention.
Claims (17)
1. a kind of red spirillum bacterial strain of outer sulphur is red spirillum LBD plants of outer sulphur (Ecothiorhodospira strain LBD), protect
Hiding number are as follows: CGMCC NO.11520.
2. bacterial strain according to claim 1, which is characterized in that the bacterial strain is the photosynthetic bacteria of extracellular polysaccharide.
3. application of the bacterial strain according to claim 1 or 2 in the preparation of exocellular polysaccharide, the exocellular polysaccharide are passed through by bacterial strain
Illumination anaerobism heterotrophic fermentation culture generates, exocellular polysaccharide content >=6mg/ in fermentation culture medium that the fermented and cultured obtains
ml。
4. a kind of exocellular polysaccharide suspension is the fermentation culture medium containing exocellular polysaccharide；The fermentation culture medium is using such as
Bacterial strain of any of claims 1 or 2 is obtained through illumination anaerobism heterotrophic fermentation culture；The optical density OD of the fermentation culture medium500nm
≥5；Exocellular polysaccharide content >=6mg/ml in the fermentation culture medium.
5. a kind of preparation method of exocellular polysaccharide comprising fermenting microbe is seeded in fermentation medium and carries out illumination fermentation training
Feeding step, wherein the fermenting microbe is obtained by corresponding bacterial strain by seed culture；The corresponding bacterial strain of fermenting microbe is
The outer red spirillum bacterial strain of sulphur as claimed in claim 1 or 2.
6. according to the method described in claim 5, it is characterized in that, the fermentation medium in terms of 1L water, is included in 1L water
Following components:
Wherein, every compound VBContained composition is as follows: vitamin B13 milligrams, vitamin B21.5 milligrams, vitamin B60.2 milligram,
10 milligrams of niacinamide, 1 milligram of calcium pantothenate；Auxiliary material are as follows: starch, calcium sulfate and magnesium stearate.
7. according to the method described in claim 6, it is characterized in that, the carbon source material includes sodium acetate, sodium citrate, lactic acid
One or more of sodium and glycerol.
8. the method according to the description of claim 7 is characterized in that the nitrogen source includes ammonium salt, urea, nitrate and egg
One or more of white peptone.
9. the method according to any one of claim 5-8, which is characterized in that the pH value of the fermentation medium is
6.5-8.0。
10. according to the method described in claim 9, it is characterized in that, the pH value of the fermentation medium is 6.5-7.2.
11. the method according to any one of claim 5-8, which is characterized in that fermenting microbe is in the form of seed liquor
It is inoculated into fermentation medium, with volume basis, the inoculum concentration of the seed liquor is 15%-30%.
12. according to the method for claim 11, which is characterized in that with volume basis, the inoculum concentration of the seed liquor is
15%-20%.
13. according to the method for claim 11, which is characterized in that the strain Colony Forming Unit in the seed liquor is
(30-50)×108CFU/mL。
14. the method according to any one of claim 5-8, which is characterized in that the temperature of the illumination fermented and cultured
It is 30-45 DEG C.
15. according to the method for claim 14, which is characterized in that the temperature of the illumination fermented and cultured is 30-35 DEG C.
16. according to the method for claim 14, which is characterized in that the light source for illumination is incandescent lamp and/or sunlight.
17. according to the method for claim 14, which is characterized in that the intensity of illumination of the illumination fermented and cultured is 2000-
8000Lx。
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