CN115927506A - Preparation method of azotobacter polysaccharide for saline-alkali soil - Google Patents
Preparation method of azotobacter polysaccharide for saline-alkali soil Download PDFInfo
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- CN115927506A CN115927506A CN202211741420.6A CN202211741420A CN115927506A CN 115927506 A CN115927506 A CN 115927506A CN 202211741420 A CN202211741420 A CN 202211741420A CN 115927506 A CN115927506 A CN 115927506A
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Abstract
The invention relates to a preparation method of saline-alkali soil azotobacter polysaccharide, which separates and purifies soil to obtain azotobacter, wherein the strain is named azotobacter (azotobacter)Azotobacter salinestris) As101; the GenBank registration number is OK445517. By the method, the yield of extracellular polysaccharide extracted from the nitrogen-fixing bacteria in the saline-alkali soil is 6.34g/L, and the polysaccharide content is 70.45%. AzotobacteriaAzotobacter salinestris(As 101) sodium ion adsorption 21.501. + -. 0.9023mg/ml, sodium ion removal capacity 71.67. + -. 0.9555%. In order to further open the adsorption of extracellular polysaccharide to heavy metal and provide a material basis for saline-alkali soil improvement application, the obtained azotobacter As101 can be used in the fields of environmental management, medicines and the like. For the future, more microorganisms are presentStructural analysis of sugars provides technical support. The method has the advantages of reasonable design, simple process, convenient operation, high purity, low preparation cost, easy large-scale production, popularization and application and the like.
Description
Technical Field
The invention relates to a preparation method of azotobacter polysaccharide in saline-alkali soil, in particular to preparation of azotobacter for producing high-yield extracellular polysaccharide by separation, purification and fermentation.
Background
In recent years, environmental pollution, particularly heavy metal pollution of soil, has made grain safety threatened greatly. Heavy metals enter soil and are difficult to convert and decompose due to special physicochemical properties of the heavy metals, and after long-term accumulation, the heavy metals have stronger toxicity and longer harm. Heavy metals are absorbed by grain crops and then are converted and accumulated in the grain crops, and after the grain crops with the heavy metals exceeding the standard are ingested by human beings, the human health is harmed by the heavy metals; under the background, the research on the heavy metal pollution of the soil in the grain planting area is concerned by scholars. Meanwhile, the utilization of saline-alkali soil resources and the improvement of salinized cultivated land are one of important links of agricultural production development in areas with saline-alkali soil. The total area of Xinjiang saline-alkali soil is 2 181.4 multiplied by 104hm 2 Occupying saline-alkali soil of China (9 913 x 104 hm) 2 ) 22.01% of the area; at 407.84 × 104hm 2 Of the arable area, the area compromised by different degrees of salinization accounts for 30.12%.
The microbial extracellular polysaccharide is synthesized by microbes in the growth process and secreted out of cells to form an active biomembrane matrix, and has the functions of gelatinizing, flocculating, emulsifying, absorbing, forming a film, protecting and the like. Due to their diverse Biological activities, they are also called Biological Response Modifiers (BRMs). The excellent physical and chemical properties and rheological characteristics make the microbial exopolysaccharide widely used in various fields of production and life. And (3) industrial production: polysaccharides are mainly used in industry as thickeners, suspending agents, etc., and are also produced into gels as flocculants, binders, film formers, lubricants, drag reducers, etc. Pullulan can be formed into fibers similar to nylon or rayon by dry or wet spinning. Xanthan gum has a good suspending effect on insoluble solids and oil droplets. The xanthan gum sol molecules can form a super-bonding banded spiral copolymer to form a fragile gel-like net structure, so that the form of solid particles, liquid drops and bubbles can be supported, and the emulsion stabilizer has strong emulsification stability and high suspension capacity; hyaluronic Acid (HA), also called hyaluronic acid, is essentially a mucopolysaccharide with relatively low content in normal human body, but with greater action, mainly existing between human epidermis and dermis, promoting cell adhesion through protein binding. The skin moisturizing cream is also a good moisturizing product and has a good moisturizing effect on aged skin; agriculture: certain specific properties of microbial polysaccharides, including their derivatives, are useful in agriculture as an additive ingredient to pesticides and feed, such as thickening, suspended solids, film forming, and stable emulsification. Pullulan, also known as pullulan or pullulan. It is colorless, odorless, easily soluble in water, and strong in plasticity, and can be made into film. The pullulan aqueous solution has strong adhesiveness and coating property and can be used for seed coating, and when the crop seeds are mechanically sowed, the sowing amount and the sowing time can be saved, and the labor force can be saved. The pullulan also has better formability and can be used for forming granulated fertilizers and pesticides; food: the chemical composition of the hydrophilic colloids widely used in the food industry is mostly polysaccharides and derivatives, except for gelatin. They are widely distributed in nature. Polysaccharides are used in the food industry as food additives, anticoagulants, preservatives, etc. Industrial applications have been obtained for gellan gum, xanthan gum, dextran, seaweed gum, carrageenan, agar, and the like. The medicine is as follows: because polysaccharides are the most abundant polymers in nature and the special properties thereof, the polysaccharides are very long applied to the field of medicine, such as starch, dextrin and other polysaccharides, have no toxicity and side effects, can be degraded by amylase, are ideal excipients of medicines, agar is also very long widely applied as a microbial culture medium, and mucilaginous polysaccharide in Plantago ovata (Plantago ovata) seeds has very strong water absorption, and is used for treating ascites in folks. With the intensive research on the physicochemical properties, structures and pharmacological actions of polysaccharides, it is found that polysaccharides have many new uses in the medical field. And (3) environmental management: research reports that the microbial polysaccharide has the function of adsorbing heavy metals and salts.
Azotobacter is a non-symbiotic diazotrophic bacterium that is usually present in neutral or slightly alkaline soils, includes airborne dust, and is very abundant in the rhizosphere and phyllospheric regions of plants. Members of the genus azotobacter, free-living, gram-negative, aerobic and heterotrophic in nature. It is well known that azotobacteria produce large amounts of Exopolysaccharides (EPS), often appearing as large slime-like colonies when isolated from soil habitats. EPS protects cells from desiccation and predation by protozoa or from phage attack. This polysaccharide also protects the penetration of toxic metal ions inside the cell and nitrogen fixation in high oxygen concentration environments. The important group of azotobacter PGPR (plant growing rhizobacterium) plays a plurality of roles in improving the stress resistance of crops, promoting the growth potential of the crops, increasing the yield, maintaining the soil health, improving the soil ecological environment, repairing the pollution of water resources and realizing the sustainable agricultural development due to the well-known nitrogen nutrition function, and particularly shows great potential value in the aspect of improving the heavy metal contaminated soil. Azotobacteria have specific resistance to zinc and other heavy metals such as chromium and iron. The bacteria can adsorb heavy metal compounds for detoxification, making them relatively insoluble and less harmful. Therefore, the microorganism is more advantageous to repair the soil. The microbial remediation cost is low, organic matters and nutrient substances in the soil can be retained while pollutants are removed, and the function of producing agricultural products cannot be lost by the soil. Compared with a chemical soil conditioner, the microbial fertilizer has the advantages of improving soil and increasing agricultural output value.
In conclusion, the microbial exopolysaccharide has the pharmacological effects of enhancing immunity, inhibiting tumors, reducing blood sugar, reducing blood fat, resisting aging and the like. The microbial exopolysaccharide has wide application prospect and commercial value, and at present, the microbial exopolysaccharide has already obtained some favorable achievements in the aspect of being used for restoring environmental pollution, but has larger development space in the aspect of developing and utilizing the single important active ingredient polysaccharide structure of the nitrogen-fixing bacteria exopolysaccharide. In order to meet development requirements and market demands, on the basis of seeking a polysaccharide structure with better quality and higher yield, the research and development of an extraction and purification method of the extracellular polysaccharide of azotobacter and the application thereof are very necessary. The method has low cost, and is suitable for extraction method of Azotobacter salinistis with high total polysaccharide content, multiple monosaccharide species, and high polysaccharide yield.
Disclosure of Invention
The invention aims to provide a preparation method of saline-alkali soil Azotobacter polysaccharide, which separates and purifies soil to obtain Azotobacter, wherein the strain is named As Azotobacter salinestris (Azotobacter salinestris) As101; the GenBank registration number is OK445517. By the method, the yield of extracellular polysaccharide extracted from the nitrogen-fixing bacteria in the saline-alkali soil is 6.34g/L, and the polysaccharide content is 70.45%. Azotobacter salinistis (As 101) has sodium ion adsorption of 21.501 + -0.9023 mg/ml and sodium ion removal capacity of 71.67 + -0.9555%. In order to further open the adsorption of extracellular polysaccharide to heavy metal and provide a material basis for saline-alkali soil improvement application, the obtained azotobacter As101 can be used in the fields of environmental management, medicines and the like. Provides technical support for the structural analysis of the microbial polysaccharide in the future. The method has the advantages of reasonable design, simple process, convenient operation, high purity, low preparation cost, easy large-scale production, popularization and application and the like.
The invention relates to a preparation method of saline-alkali soil Azotobacter polysaccharide, which separates and purifies soil to obtain Azotobacter, wherein the strain is named As Azotobacter salinaris As101, and the specific operation is carried out according to the following steps:
a. separation and purification: taking 10g soil sample, adding into 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution for 10 -1 、10 -2 、10 -3 、10 -4 And 10 -5 Uniformly coating 100 mu L of diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a thermostat with the temperature of 37 ℃ and the humidity of 55% for culturing for 5-7d, selecting a single colony which grows faster and has obvious colony morphology, and continuously scribing and purifying to obtain the single colony with consistent morphology, wherein the Ashby solid culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate and 1000mL of distilled water, and sterilizing at 1 × 105Pa for 30min;
b. and (3) fermentation: b, picking the azotobacter obtained by separating in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium using carbon sources of lactose, sucrose, mannitol, mannose or glucose, culturing for 24-48 hours at a pH value of 7-12 at a rotating speed of a shaking table of 170rpm, transferring the inoculum size of 5 percent into 1L of azotobacter culture medium using carbon sources of lactose, sucrose, mannitol, mannose and glucose, culturing for 48-120 hours at a pH value of 7-12 at a culture temperature of 35 ℃ to obtain fermentation liquor, wherein the azotobacter culture medium: 20.0g of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water at 1 x 105Pa for sterilization for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernatant, and concentrating the supernatant to 1/3 by using a rotary evaporator;
d. c, adding the glacial ethanol into the concentrated solution obtained in the step c according to the volume ratio of 1;
e. and d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent according to the volume ratio of 1.
The invention relates to a preparation method of azotobacter polysaccharide in saline-alkali soil, which is used for identifying azotobacter As101 obtained by separating and purifying soil:
DNA of each strain was extracted by using a TIANGEN genomic DNA extraction kit (Lot # W9104; cat. # DP 302-02). After the DNA is detected and adjusted to a proper concentration, the DNA is used as a template, a target fragment is amplified by adopting a 16S rRNA universal primer 27F/1492R, and the sequence of the DNA is shown in the specification, wherein the sequence is shown in the specification, and is shown in the specification: AGAGTTTGATCCTGGCTCAG;1492R: GGTTACCTTGTTACGACTT. The PCR amplification system is as follows: 1.5 muL of template, 1 muL of upstream primer and downstream primer respectively, 25 muL of 2 taq PCR Green Mix, and supplementing sterile water to the total volume of 50 muL; the PCR amplification condition is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ for 7min; and (4) sending the PCR amplification product to Shanghai workers for sequencing. After the gene sequence of the 16S rRNA of the strain is sequenced, similarity comparison is carried out on an NCBI website, and the sequence is submitted to a GenBank database to obtain a strain sequence registration number.
The invention relates to a preparation method of azotobacter polysaccharide in saline-alkali soil, which separates and purifies soil to obtain a DNA sequence of azotobacter As 101:
1gggcaagtgc ggcagctaca catgcagtcg agcggcagcg ggaccttcgg gttgccggcg
61agcggcggac gggtgagtaa tgcctaggaa tctgcctgtt agtgggggat aacgcgggga
121aactcgcgct aataccgcat acgtcctacg ggagaaagtg ggggaccttc gggcctcacg
181ctaacagatg agcctaggtc ggattagctg gttggtgggg taacggccca ccaaggcgac
241gatccgtaac tggtctgaga ggatgatcag tcacactgga actgagacac ggtccagact
301cctacgggag gcagcagtgg ggaatattgg acaatgggcg aaagcctgat ccagccatgc
361cgcgtgtgtg aagaaggtct tcggattgta aagcacttta agtcgggagg aagggctgta
421ggcgaatacc ctgcagtctt gacgttaccg acagaataag caccggctaa cttcgtgcca
481gcagccgcgg taatacgaag ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcgc
541gtaggtggtt tggtaagttg gatgtgaaag ccccgggctc aacctgggaa ctgcatccaa
601aactgcctgg ctagagtacg gtagagggtg gtggaatttc ctgtgtagcg gtgaaatgcg
661tagatatagg aaggaacacc agtggcgaag gcgaccacct ggactgatac tgacactgag
721gtgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat
781gtcgactagc cgttgggctc cttgagagct tagtggcgca gctaacgcat taagtcgacc
841gcctggggag tacggccgca aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc
901ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttacctggcc ttgacatcct
961gcgaactggg tagagatacc cgggtgcctt cgggagcgca gagacaggtg ctgcatggct
1021gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgt aacgagcgca acccttgtcc
1081ttagttacca gcgattcggt cgggcactct aaggagactg ccggtgacaa accggaggaa
1141ggtggggatg acgtcaagtc atcatggccc ttacggccag ggctacacac gtgctacaat
1201ggtcggtaca gagggttgcc aagccgcgag gcggagctaa tcccagaaaa ccgatcgtag
1261tccggatcgc agtctgcaac tcgactgcgt gaagtcggaa tcgctagtaa tcgcgaatca
1321gaatgtcgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt
1381gggttgctcc agaagtagct agtctaaccc tcgggaggac ggtaccacga gtataggg
the invention relates to a preparation method of saline-alkali soil Azotobacter polysaccharide, and application of Azotobacter salinestris As101 in adsorption of heavy metals on saline-alkali soil.
The invention finally obtains the best yield of azotobacter As101 and the fermentation conditions of polysaccharide content through a single-factor experiment: the fermentation temperature is 35 ℃, the fermentation time is 120h, the pH value is 7, the inoculation amount is 5%, the carbon source of the nitrogen-fixing culture medium is mannitol, and the test method comprises the following steps:
fermentation broth polysaccharides obtained at five different fermentation times:
under the conditions that the fermentation temperature is 35 ℃, the pH value is 7, mannitol is used as a carbon source, and the inoculation amount is 5%, the influence of extraction time factors on the sugar content and the yield is carried out for 24 hours, 48 hours, 72 hours, 96 hours or 120 hours respectively:
the extraction steps are as follows:
pretreatment: centrifuging the fermentation liquor which is fermented for 24h, 48h, 72h, 96h or 120h at 7000rpm for 10min, taking the supernatant, and concentrating to 1/3 by using a rotary evaporator;
alcohol precipitation: concentrating the obtained fermentation liquor for 24h, 48h, 72h, 96h or 120h in different time periods, adding glacial ethanol for alcohol precipitation according to the volume ratio of 1;
and (3) purification: dissolving the obtained precipitate for 24h, 48h, 72h, 96h or 120h in different time periods with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1;
and (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution by gradient to 0, 30, 60, 90, 120, 150 and 180 mu g/ml, preparing the obtained azotobacter As101 extracellular polysaccharide into 200 mu g/ml concentration, adding 50 mu L of the obtained azotobacter As101 extracellular polysaccharide into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, standing for 10min at 90 ℃, taking out enzyme labeling solution with 632nm to measure the absorption wavelength, drawing a standard curve, and calculating the sugar content of the crude polysaccharide.
Fermentation broth polysaccharides obtained under six different pH conditions:
under the conditions that the fermentation temperature is 35 ℃, the fermentation time is 120h, mannitol is used as a carbon source, and the inoculation amount is 5%, the pH values are respectively 7,8,9,10,11 and 12, and the influence of pH factors on the sugar content and the yield is carried out:
the method comprises the following steps:
pretreatment: centrifuging fermentation liquor 7000rpm obtained under fermentation conditions of different pH values of 7,8,9,10,11 and 12 for 10min, collecting supernatant, and concentrating to 1/3 with rotary evaporator;
alcohol precipitation: respectively concentrating the fermentation broth polysaccharide obtained by six different pH values of 7,8,9,10,11 and 12, adding glacial ethanol according to the volume ratio of 1; and (3) purification: dissolving the obtained precipitate with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1:3, stirring for 30min, standing for 30min in a separating funnel after stirring, taking the lower layer for repeating the experiment twice, dialyzing the deproteinized sample with a dialysis bag with the molecular weight cutoff MW3500Da for 48h, and freeze-drying the dialyzed sample to obtain the azotobacter As101 extracellular polysaccharide; and (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution by gradient to 0, 30, 60, 90, 120, 150 and 180 mu g/ml, preparing the obtained azotobacter As101 extracellular polysaccharide into 200 mu g/ml concentration, adding 50 mu L of the obtained azotobacter As101 extracellular polysaccharide into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, standing for 10min at 90 ℃, taking out enzyme labeling solution with 632nm to measure the absorption wavelength, drawing a standard curve, and calculating the sugar content of the crude polysaccharide.
Polysaccharide is extracted from fermentation liquor obtained after five different carbon sources are fermented:
under the conditions of 35 ℃ of fermentation temperature, 120h of fermentation time, 7 pH value and 5% of inoculation amount, five different carbon sources are selected as lactose, sucrose, mannitol, mannose and glucose to carry out the influence of the carbon sources on the sugar content and yield: the method comprises the following steps:
pretreatment: centrifuging at 7000rpm for 10min with fermented broth of lactose, sucrose, mannitol, mannose or glucose as different carbon sources, collecting supernatant, and concentrating to 1/3 with rotary evaporator;
alcohol precipitation: adding glacial ethanol into the obtained concentrated fermentation liquor according to the volume ratio of 1;
and (3) purification: dissolving the obtained precipitate with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1 to 3, stirring for 30min, standing for 30min in a separating funnel after stirring, taking the lower layer for repeating the experiment for 2 times, dialyzing the deproteinized sample with a dialysis bag with the molecular weight cutoff MW3500Da for 48h, and freeze-drying the dialyzed sample to obtain the azotobacter As101 extracellular polysaccharide; and (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution by gradient to 0, 30, 60, 90, 120, 150 and 180 mu g/ml, preparing the obtained azotobacter As101 extracellular polysaccharide into 200 mu g/ml, adding 50 mu L of the obtained azotobacter As101 extracellular polysaccharide into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, standing for 10min at 90 ℃, taking out enzyme labeling solution with 632nm to measure the absorption wavelength, drawing a standard curve, and calculating the sugar content of the crude polysaccharide.
Azotobacter As101 extracellular polysaccharide salt adsorption capacity experiment: naCl with different contents of 0mg/ml, 10 mg/ml, 20 mg/ml and 30mg/ml are respectively added into the azotobacter culture fermentation liquor, and the polysaccharide content is detected after the culture.
The preparation method of the saline-alkali soil Azotobacter polysaccharide obtains Azotobacter by separating and purifying soil, the strain is named As Azotobacter salinaris As101, and the preparation method has the advantages of reasonable design, simple process, convenience in operation, high purity, low preparation cost, capability of effectively extracting extracellular polysaccharide, easiness in large-scale production, popularization and application and the like. The obtained azotobacter As101 can be used in the fields of environmental management, medicines and the like. Provides technical support for the structural analysis of the microbial polysaccharide in the future.
The growth temperature range of the nitrogen-fixing strain provided by the invention is that the optimal growth temperature is 30-40 ℃; the optimal growth pH is 7; the invention provides a material basis for opening the adsorption of extracellular polysaccharide to heavy metals and the improvement and application of saline-alkali soil, and deeply understands the structure of extracellular polysaccharide from azotobacter and the optimal fermentation conditions of the azotobacter As101 and the sugar content, which are provided by the research on the activity of the azotobacter, namely 35 ℃ of fermentation temperature, 120 hours of fermentation time, 7 of pH value, 5 percent of inoculation amount and mannitol As a carbon source.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the yield of the exopolysaccharide is 6.34g/L and the polysaccharide content is 70.45% under the fermentation conditions (the fermentation temperature is 35 ℃, the fermentation time is 120h, the pH value is 7, the inoculation amount is 5%, and mannitol is a carbon source) confirmed by a single-factor experiment on the azotobacter As101 obtained by separating and purifying a saline-alkali soil sample. In order to further open the salt adsorption of the exopolysaccharide, the saline-alkali soil improvement application provides a material basis and is helpful for deeply understanding the structure and other activities of the obtained nitrogen-fixing bacteria source exopolysaccharide.
The invention discloses a nitrogen-fixing bacterium in a saline-alkali resistant land, which is an Azotobacter salinistris strain As101. The azotobacter As101 is obtained by separating and purifying soil of Wuluqi salt lake, xinjiang, measuring a partial sequence of 16S rDNA of a strain, determining the azotobacter As azotobacter through phylogenetic analysis, and naming the azotobacter As As101. The morphological characteristics of the strain are as follows: gram-negative; milky white; the viscosity is very high. After 3 to 5 days of incubation at 37 ℃, the colonies or the culture broth appeared as a milky white sticky mass.
The Azotobacter is obtained by separating and purifying soil, and the strain is named As Azotobacter salinaris As101; the GenBank registration number is OK445517. Acquisition time 2021 year, 6 months, 5 days, acquisition place: salt lake scenic spot of Wulu wood Qi city.
Drawings
FIG. 1 is the effect of fermentation time on the yield and content of exopolysaccharides of azotobacter As101 in the present invention;
FIG. 2 is the influence of pH value on the yield and content of exopolysaccharide of azotobacter As101 in the invention;
FIG. 3 shows the effect of carbon source on the yield and content of exopolysaccharide of azotobacter As101 in the present invention
FIG. 4 is the monosaccharide composition analysis of the As101 exopolysaccharide of azotobacter.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
a. Separation and purification: taking 10g soil sample, adding into 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Uniformly coating 100 mu L of diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a thermostat with the temperature of 37 ℃ and the humidity of 55% for culturing for 5 days, selecting single bacterial colonies which grow faster and have obviously different bacterial colony forms, continuously scribing and purifying to obtain single bacterial colonies with consistent forms, and using the single bacterial colonies with consistent forms after being obtained by continuous scribing and purifying for classification and identification; wherein the Ashby culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate and 1000mL of distilled water, and 1 multiplied by 105Pa for sterilization for 30min;
identification of azotobacter As 101:
extracting DNA of each strain by using a TIANGEN genome DNA extraction kit (Lot # W9104; cat. # DP 302-02), detecting the DNA, adjusting the DNA to a proper concentration, using the DNA as a template, amplifying a target fragment by using a 16S rRNA universal primer 27F/1492R, wherein the DNA comprises the following steps: AGAGTTTGATCCTGGCTCAG;1492R: ggttactctgttacgactt; the PCR amplification system is as follows: 1.5 mu L of template, 1 mu L of each of the upstream primer and the downstream primer, 25 mu L of 2 Taq PCR Green Mix and sterile water to make up the total volume of 50 mu L; the PCR amplification condition is that the temperature is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ and 7min, and PCR amplification products are sent to Shanghai workers for sequencing; after sequencing the 16S rRNA gene sequence of the strain, carrying out similarity comparison on an NCBI website, and submitting to a GenBank database to obtain a strain sequence registration number;
b. and (3) fermentation: b, picking the azotobacter separated in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium which uses carbon source mannitol, culturing for 48 hours at the pH value of 7, rotating the shaking table at 170rpm, transferring the azotobacter culture medium with the inoculation amount of 5 percent into 1L of azotobacter culture medium which uses carbon source mannitol, culturing for 24 hours at the pH value of 7 and the culture temperature of 35 ℃, and obtaining fermentation liquor, wherein the azotobacter culture medium is prepared by the steps of: 20.0g of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernate, and concentrating the supernate to 1/3 by using a rotary evaporator;
d. c, adding glacial ethanol into the concentrated fermentation liquor obtained in the step c according to the volume ratio of 1;
e. d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1:3, stirring for 30min, standing for 30min in a separating funnel after stirring, taking the lower layer, repeating the experiment twice, dialyzing the sample obtained after deproteinizing with a dialysis bag with the molecular weight cutoff MW3500Da for 48h, and freeze-drying the sample after dialysis to obtain azotobacter As101 extracellular polysaccharide;
and (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution by gradient to 0, 30, 60, 90, 120, 150 and 180 mu g/ml, preparing the obtained azotobacter As101 extracellular polysaccharide into 200 mu g/ml concentration, adding 50 mu L of samples into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, placing at 90 ℃ for standing for 10min, taking out enzyme labeling solution with 632nm to measure absorption wavelength, drawing a standard curve, calculating the sugar content of the crude polysaccharide, wherein the final polysaccharide yield is 0.845g/L and the polysaccharide content is 1.06%.
Example 2
a. Separation and purification: taking 10g soil sample, adding into 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Uniformly coating 100 mu L of diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a thermostat with the temperature of 37 ℃ and the humidity of 55% for culturing for 6d, selecting single bacterial colonies which grow faster and have obviously different bacterial colony forms, continuously scribing and purifying to obtain single bacterial colonies with consistent forms, and using the single bacterial colonies with consistent forms after the single bacterial colonies with consistent forms are obtained by continuous scribing and purifying for classification and identification, wherein the Ashby solid culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate and 1000mL of distilled water, and sterilizing at 1 × 105Pa for 30min;
identification of azotobacter As 101:
DNA of each strain was extracted using a TIANGEN genomic DNA extraction kit (Lot # W9104; cat. # DP 302-02). After the DNA is detected and adjusted to proper concentration, the DNA is used as a template, a target fragment is amplified by adopting a 16S rRNA universal primer 27F/1492R, and the DNA sequence of the DNA is shown in the specification, wherein the sequence is shown in the specification, and the sequence is shown in the specification, wherein the sequence is shown in the specification: AGAGTTTGATCCTGGCTCAG;1492R: ggttactctgttacgactt; the PCR amplification system is as follows: 1.5 muL of template, 1 muL of upstream primer and downstream primer respectively, 25 muL of 2 taq PCR Green Mix, and supplementing sterile water to the total volume of 50 muL; the PCR amplification condition is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ and 7min, and PCR amplification products are sent to Shanghai workers for sequencing; after sequencing the 16S rRNA gene sequence of the strain, carrying out similarity comparison on an NCBI website, and submitting the result to a GenBank database to obtain a strain sequence registration number;
b. and (3) fermentation: b, picking the azotobacter obtained by separating in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium using carbon source mannitol, culturing for 48 hours at the pH value of 7, rotating the shaking table at 170rpm, inoculating 5 percent of azotobacter to 1L of azotobacter culture medium using carbon source mannitol, culturing for 48 hours at the pH value of 7 and the culture temperature of 35 ℃, and obtaining fermentation liquor, wherein the azotobacter culture medium: 20.0g of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water at 1 x 105Pa for sterilization for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernate, and concentrating the supernate to 1/3 by using a rotary evaporator;
d. c, adding glacial ethanol into the concentrated fermentation liquor obtained in the step c according to the volume ratio of 1;
e. d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1:3, stirring for 30min, standing for 30min in a separating funnel after stirring, taking the lower layer, repeating the experiment twice, dialyzing the sample obtained after deproteinizing with a dialysis bag with the molecular weight cutoff MW3500Da for 48h, and freeze-drying the sample after dialysis to obtain azotobacter As101 extracellular polysaccharide;
and (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution by gradient to 0, 30, 60, 90, 120, 150 and 180 mu g/ml, preparing the obtained azotobacter As101 exopolysaccharide into 200 mu g/ml concentration, adding 50 mu L of samples into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, standing for 10min at 90 ℃, taking out enzyme labeling solution with 632nm to measure absorption wavelength, drawing a standard curve, calculating the sugar content of crude polysaccharide, wherein the final polysaccharide yield is 0.93g/L and the polysaccharide content is 1.54%.
Example 3
a. Separation and purification: taking 10g soil sample, adding into 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Uniformly coating 100 mu L of diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a thermostat with the temperature of 37 ℃ and the humidity of 55% for culture for 7d, selecting single bacterial colonies which grow faster and have obviously different bacterial colony forms, continuously scribing and purifying to obtain single bacterial colonies with consistent forms, and using the single bacterial colonies with consistent forms after the single bacterial colonies with consistent forms are obtained by continuous scribing and purifying for classification and identification, wherein the Ashby solid culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate and 1000mL of distilled water, and sterilizing at 1 × 105Pa for 30min;
identification of azotobacter As 101:
DNA of each strain was extracted by using a TIANGEN genomic DNA extraction kit (Lot # W9104; cat. # DP 302-02). After the DNA is detected and adjusted to a proper concentration, the DNA is used as a template, a target fragment is amplified by adopting a 16S rRNA universal primer 27F/1492R, and the sequence of the DNA is shown in the specification, wherein the sequence is shown in the specification, and is shown in the specification: AGAGTTTGATCCTGGCTCAG;1492R: ggttactctgttacgactt; the PCR amplification system is as follows: 1.5 mu L of template, 1 mu L of each of the upstream primer and the downstream primer, 25 mu L of 2 Taq PCR Green Mix and sterile water to make up the total volume of 50 mu L; the PCR amplification condition is that the temperature is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ for 7min, and PCR amplification products are sent to Shanghai worker for sequencing; after sequencing the 16S rRNA gene sequence of the strain, carrying out similarity comparison on an NCBI website, and submitting the result to a GenBank database to obtain a strain sequence registration number;
b. and (3) fermentation: b, picking the azotobacter separated in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium which uses carbon source mannitol, culturing for 48 hours at the pH value of 7, rotating the shaking table at 170rpm, transferring the azotobacter culture medium with the inoculation amount of 5 percent into 1L of azotobacter culture medium which uses carbon source mannitol, culturing for 72 hours at the pH value of 7 and the culture temperature of 35 ℃, and obtaining fermentation liquor, wherein the azotobacter culture medium: 20.0g of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernatant, and concentrating the supernatant to 1/3 by using a rotary evaporator;
d. c, adding glacial ethanol into the concentrated fermentation liquor obtained in the step c according to the volume ratio of 1;
e. d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1;
and (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution by gradient to 0, 30, 60, 90, 120, 150 and 180 mu g/ml, preparing the obtained azotobacter As101 extracellular polysaccharide into 200 mu g/ml concentration, adding 50 mu L of samples into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, placing the samples into a temperature of 90 ℃, standing for 10min, taking out enzyme labeling solution with 632nm to measure absorption wavelength, drawing a standard curve, calculating the sugar content of the crude polysaccharide, and finally obtaining the polysaccharide with the yield of 1.485g/L and the polysaccharide content of 18.27%.
Example 4
a. Separation and purification: taking 10g soil sample, adding into 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Uniformly coating 100 mu L of diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a thermostat with the temperature of 37 ℃ and the humidity of 55% for culturing for 5-7d, selecting single bacterial colonies which grow faster and have obviously different bacterial colony forms, continuously scribing and purifying to obtain the single bacterial colonies with the consistent forms, and using the single bacterial colonies with the consistent forms obtained by continuous scribing and purifying for classification and identification, wherein the Ashby solid culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate and 1000mL of distilled water, and sterilizing at 1 × 105Pa for 30min;
identification of azotobacter As 101:
DNA of each strain was extracted using a TIANGEN genomic DNA extraction kit (Lot # W9104; cat. # DP 302-02). After the DNA is detected and adjusted to proper concentration, the DNA is used as a template, a target fragment is amplified by adopting a 16S rRNA universal primer 27F/1492R, and the DNA sequence of the DNA is shown in the specification, wherein the sequence is shown in the specification, and the sequence is shown in the specification, wherein the sequence is shown in the specification: AGAGTTTGATCCTGGCTCAG;1492R: ggttactctgttacgactt; the PCR amplification system is as follows: 1.5 muL of template, 1 muL of upstream primer and downstream primer respectively, 25 muL of 2 taq PCR Green Mix, and supplementing sterile water to the total volume of 50 muL; the PCR amplification condition is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ and 7min, and PCR amplification products are sent to Shanghai workers for sequencing; after sequencing the 16S rRNA gene sequence of the strain, carrying out similarity comparison on an NCBI website, and submitting to a GenBank database to obtain a strain sequence registration number;
b. fermentation: b, picking the azotobacter obtained by separating in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium using carbon source mannitol, culturing for 48 hours at the pH value of 7, rotating the shaking table at 170rpm, inoculating 5 percent of azotobacter to 1L of azotobacter culture medium using carbon source mannitol, culturing for 96 hours at the pH value of 7 and the culture temperature of 35 ℃, and obtaining fermentation liquor, wherein the azotobacter culture medium: 20.0g of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernatant, and concentrating the supernatant to 1/3 by using a rotary evaporator;
d. c, adding glacial ethanol into the concentrated fermentation liquor obtained in the step c according to the volume ratio of 1;
e. d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent with the proportion of 1;
and (3) determination of polysaccharide content: glucose is used As standard solution, the mother solution is prepared into 200 mu g/ml, the mother solution is diluted by 0, 30, 60, 90, 120, 150 and 180 mu g/ml in a gradient manner, the obtained azotobacter As101 exopolysaccharide is prepared into 200 mu g/ml concentration, 50 mu L of samples are added into a 96-well plate, 150 mu L of anthrone sulfuric acid is added into each sample, the mixture is uniformly mixed, the temperature is kept still for 10min at 90 ℃, 632nm of enzyme labeling solution is taken out to measure the absorption wavelength, a standard curve is drawn, the sugar content of crude polysaccharide is calculated, the final polysaccharide yield is 2.681g/L, and the polysaccharide content is 23.12%.
Example 5
a. Separation and purification: taking 10g soil sample, adding into 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Uniformly coating 100 mu L of diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a constant temperature box with the temperature of 37 ℃ and the humidity of 55 percent for culture for 7 days, selecting single bacterial colonies which grow faster and have obviously different bacterial colony forms, continuously scribing and purifying to obtain the single bacterial colonies with consistent forms,and (3) performing continuous streak purification to obtain single strains with consistent morphology for classification and identification, wherein the ratio of the Ashby culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate and 1000mL of distilled water, and sterilizing at 1 × 105Pa for 30min;
identification of azotobacter As 101:
extracting DNA of each strain by using a TIANGEN genome DNA extraction kit (Lot # W9104; cat. # DP 302-02); after the DNA is detected and adjusted to a proper concentration, the DNA is used as a template, a target fragment is amplified by adopting a 16S rRNA universal primer 27F/1492R, and the sequence of the DNA is shown in the specification, wherein the sequence is shown in the specification, and is shown in the specification: AGAGTTTGATCCTGGCTCAG;1492R: ggttactctgttacgactt; the PCR amplification system is as follows: 1.5 mu L of template, 1 mu L of each of the upstream primer and the downstream primer, 25 mu L of 2 Taq PCR Green Mix and sterile water to make up the total volume of 50 mu L; the PCR amplification condition is that the temperature is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ and 7min, and PCR amplification products are sent to Shanghai workers for sequencing; after sequencing the 16S rRNA gene sequence of the strain, carrying out similarity comparison on an NCBI website, and submitting to a GenBank database to obtain a strain sequence registration number;
b. fermentation: b, picking the azotobacter separated in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium which uses carbon source mannitol, culturing for 48 hours at the pH value of 7, rotating the shaking table at 170rpm, transferring the azotobacter culture medium with the inoculation amount of 5 percent into 1L of azotobacter culture medium which uses carbon source mannitol, culturing for 120 hours at the pH value of 7 and the culture temperature of 35 ℃, and obtaining fermentation liquor, wherein the azotobacter culture medium is prepared by the steps of: 20.0g of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernatant, and concentrating the supernatant to 1/3 by using a rotary evaporator;
d. c, adding glacial ethanol into the concentrated fermentation liquor obtained in the step c according to the volume ratio of 1;
e. d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1;
and (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution by gradient to 0, 30, 60, 90, 120, 150 and 180 mu g/ml, preparing the obtained azotobacter As101 extracellular polysaccharide into 200 mu g/ml concentration, adding 50 mu L of samples into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, placing at 90 ℃ for standing for 10min, taking out enzyme labeling solution with 632nm to measure absorption wavelength, drawing a standard curve, calculating the sugar content of the crude polysaccharide, and finally obtaining the polysaccharide with the yield of 6.34g/L and the polysaccharide content of 70.45 percent.
Example 6
a. Separation and purification: taking 10g soil sample, adding 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Uniformly coating 100 mu L of diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a thermostat with the temperature of 37 ℃ and the humidity of 55% for culturing for 5 days, selecting single bacterial colonies which grow faster and have obviously different bacterial colony forms, continuously scribing and purifying to obtain single bacterial colonies with consistent forms, and using the single bacterial colonies with consistent forms after the single bacterial colonies with consistent forms are obtained by continuous scribing and purifying for classification and identification, wherein the Ashby solid culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate and 1000mL of distilled water, and sterilizing at 1 × 105Pa for 30min;
identification of azotobacter As 101:
DNA of each strain was extracted by using a TIANGEN genomic DNA extraction kit (Lot # W9104; cat. # DP 302-02). After the DNA is detected and adjusted to proper concentration, the DNA is used as a template, a target fragment is amplified by adopting a 16S rRNA universal primer 27F/1492R, and the DNA sequence of the DNA is shown in the specification, wherein the sequence is shown in the specification, and the sequence is shown in the specification, wherein the sequence is shown in the specification: AGAGTTTGATCCTGGCTCAG;1492R: ggttactctgttacgactt; the PCR amplification system is as follows: 1.5 muL of template, 1 muL of upstream primer and downstream primer respectively, 25 muL of 2 taq PCR Green Mix, and supplementing sterile water to the total volume of 50 muL; the PCR amplification condition is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ and 7min, and PCR amplification products are sent to Shanghai workers for sequencing; after sequencing the 16S rRNA gene sequence of the strain, carrying out similarity comparison on an NCBI website, and submitting the result to a GenBank database to obtain a strain sequence registration number;
b. fermentation: b, picking the azotobacter obtained by separating in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium using carbon source mannitol, culturing for 48 hours at the pH value of 7 and at the shaking table rotating speed of 170rpm, transferring the azotobacter culture medium with the inoculation amount of 5 percent to 1L of azotobacter culture medium using carbon source mannitol, culturing for 120 hours at the pH value of 8 and the culture temperature of 35 ℃, and obtaining fermentation liquor, wherein the azotobacter culture medium: 20.0g of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernate, and concentrating the supernate to 1/3 by using a rotary evaporator;
d. c, adding glacial ethanol into the concentrated fermentation liquor obtained in the step c according to the volume ratio of 1;
e. d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1:3, stirring for 30min, standing for 30min in a separating funnel after stirring, taking the lower layer, repeating the experiment twice, dialyzing the sample obtained after deproteinizing with a dialysis bag with the molecular weight cutoff MW3500Da for 48h, and freeze-drying the sample after dialysis to obtain azotobacter As101 extracellular polysaccharide;
and (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution by gradient to 0, 30, 60, 90, 120, 150 and 180 mu g/ml, preparing the obtained azotobacter As101 exopolysaccharide into 200 mu g/ml concentration, adding 50 mu L of samples into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, standing for 10min at 90 ℃, taking out enzyme labeling solution with 632nm to measure absorption wavelength, drawing a standard curve, calculating the sugar content of crude polysaccharide, wherein the final polysaccharide yield is 1.36g/L, and the polysaccharide content is 20.08%.
Example 7
a. Separation and purification: taking 10g soil sample, adding 90mlShaking in sterile water for 30min, and gradient diluting the supernatant for 10min -1 、10 -2 、10 -3 、10 -4 、10 -5 Taking 100 mu L of diluted bacterial liquid, uniformly coating the diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a thermostat with the temperature of 37 ℃ and the humidity of 55% for culture for 7d, selecting single bacterial colonies with faster growth and obviously different bacterial colony forms, continuously streaking and purifying to obtain the single bacterial colonies with the consistent forms, and using the single bacterial colonies with the consistent forms after the single bacterial colonies are obtained by continuous streaking and purifying for classification and identification, wherein the Ashby solid culture medium comprises the following components: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate and 1000mL of distilled water, and sterilizing at 1 × 105Pa for 30min;
identification of azotobacter As 101:
DNA of each strain was extracted using a TIANGEN genomic DNA extraction kit (Lot # W9104; cat. # DP 302-02). After the DNA is detected and adjusted to proper concentration, the DNA is used as a template, a target fragment is amplified by adopting a 16S rRNA universal primer 27F/1492R, and the DNA sequence of the DNA is shown in the specification, wherein the sequence is shown in the specification, and the sequence is shown in the specification, wherein the sequence is shown in the specification: AGAGTTTGATCCTGGCTCAG;1492R: ggttactctgttacgactt; the PCR amplification system is as follows: 1.5 muL of template, 1 muL of upstream primer and downstream primer respectively, 25 muL of 2 taq PCR Green Mix, and supplementing sterile water to the total volume of 50 muL; the PCR amplification condition is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ and 7min, and PCR amplification products are sent to Shanghai workers for sequencing; after sequencing the 16S rRNA gene sequence of the strain, carrying out similarity comparison on an NCBI website, and submitting the result to a GenBank database to obtain a strain sequence registration number;
b. fermentation: b, picking the azotobacter separated in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium which uses carbon source mannitol, culturing for 48 hours at the pH value of 7 at the shaking table rotating speed of 170rpm with the inoculation amount of 5 percent, transferring the azotobacter culture medium to 1L of azotobacter culture medium which uses carbon source mannitol, culturing for 120 hours at the pH value of 9 and the culture temperature of 35 ℃, and obtaining fermentation liquor, wherein the azotobacter culture medium is prepared by the steps of: 20.0g of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernate, and concentrating the supernate to 1/3 by using a rotary evaporator;
d. c, adding glacial ethanol into the concentrated fermentation liquor obtained in the step c according to the volume ratio of 1;
e. and d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent with the proportion of 1. And (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution by gradient to 0, 30, 60, 90, 120, 150 and 180 mu g/ml, preparing the obtained azotobacter As101 exopolysaccharide into 200 mu g/ml concentration, adding 50 mu L of samples into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, standing for 10min at 90 ℃, taking out enzyme labeling solution with 632nm to measure absorption wavelength, drawing a standard curve, calculating the sugar content of crude polysaccharide, wherein the final polysaccharide yield is 0.6g/L, and the polysaccharide content is 17.42%.
Example 8
a. Separation and purification: taking 10g soil sample, adding into 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Uniformly coating 100 mu L of diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a thermostat with the temperature of 37 ℃ and the humidity of 55% for culture for 7d, selecting single bacterial colonies which grow faster and have obviously different bacterial colony forms, continuously scribing and purifying to obtain the single bacterial colonies with the same form, and using the single bacterial colonies with the same form after the single bacterial colonies with the same form are obtained by continuous scribing and purifying for classification and identification, wherein the Ashby solid culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate and 1000mL of distilled water, and 1 multiplied by 105Pa for sterilization for 30min;
identification of azotobacter As 101:
DNA of each strain was extracted using a TIANGEN genomic DNA extraction kit (Lot # W9104; cat. # DP 302-02). After the DNA is detected and adjusted to proper concentration, the DNA is used as a template, a target fragment is amplified by adopting a 16S rRNA universal primer 27F/1492R, and the DNA sequence of the DNA is shown in the specification, wherein the sequence is shown in the specification, and the sequence is shown in the specification, wherein the sequence is shown in the specification: AGAGTTTGATCCTGGCTCAG;1492R: ggttactctgttacgactt; the PCR amplification system is as follows: 1.5 muL of template, 1 muL of upstream primer and downstream primer respectively, 25 muL of 2 taq PCR Green Mix, and supplementing sterile water to the total volume of 50 muL; the PCR amplification condition is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ and 7min, and PCR amplification products are sent to Shanghai workers for sequencing; after sequencing the 16S rRNA gene sequence of the strain, carrying out similarity comparison on an NCBI website, and submitting the result to a GenBank database to obtain a strain sequence registration number;
b. fermentation: b, picking the azotobacter separated in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium which uses carbon source mannitol, culturing for 48 hours at the pH value of 7 at the shaking table rotating speed of 170rpm with the inoculation amount of 5 percent, transferring the azotobacter culture medium to 1L of azotobacter culture medium which uses carbon source mannitol, culturing for 120 hours at the pH value of 10 and the culture temperature of 35 ℃, and obtaining fermentation liquor, wherein the azotobacter culture medium comprises the following components: 20.0g of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernatant, and concentrating the supernatant to 1/3 by using a rotary evaporator;
d. c, adding glacial ethanol into the concentrated fermentation liquor obtained in the step c according to the volume ratio of 1;
e. d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1;
and (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution in a gradient way (0, 30, 60, 90, 120, 150 and 180) mu g/ml, preparing the obtained azotobacter As101 exopolysaccharide into 200 mu g/ml concentration, adding 50 mu L of samples into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, standing for 10min at 90 ℃, taking out enzyme labeling solution with 632nm to measure the absorption wavelength, drawing a standard curve, calculating the sugar content of crude polysaccharide, wherein the final polysaccharide yield is 0.72g/L, and the polysaccharide content is 18.09%.
Example 9
a. Taking 10g soil sample, adding into 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Uniformly coating 100 mu L of diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a constant temperature box with the temperature of 37 ℃ and the humidity of 55 percent for culturing for 5-7d, selecting single bacterial colonies which grow faster and have obviously different bacterial colony forms, and continuously scribing and purifying to obtain single bacterial colonies with consistent forms for classification and identification; wherein the Ashby culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate and 1000mL of distilled water, and sterilizing at 1 × 105Pa for 30min;
identification of azotobacter As 101:
DNA of each strain was extracted using a TIANGEN genomic DNA extraction kit (Lot # W9104; cat. # DP 302-02). After the DNA is detected and adjusted to proper concentration, the DNA is used as a template, a target fragment is amplified by adopting a 16S rRNA universal primer 27F/1492R, and the DNA sequence of the DNA is shown in the specification, wherein the sequence is shown in the specification, and the sequence is shown in the specification, wherein the sequence is shown in the specification: AGAGTTTGATCCTGGCTCAG;1492R: ggttactctgttacgactt; the PCR amplification system is as follows: 1.5 muL of template, 1 muL of upstream primer and downstream primer respectively, 25 muL of 2 taq PCR Green Mix, and supplementing sterile water to the total volume of 50 muL; the PCR amplification condition is that the temperature is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ and 7min, and PCR amplification products are sent to Shanghai workers for sequencing; after sequencing the 16S rRNA gene sequence of the strain, carrying out similarity comparison on an NCBI website, and submitting the result to a GenBank database to obtain a strain sequence registration number;
b. fermentation: b, picking the azotobacter obtained by separating in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium using carbon source mannitol, culturing for 48 hours at the pH value of 7 and at the shaking table rotating speed of 170rpm, transferring the azotobacter culture medium with the inoculation amount of 5 percent into 1L of azotobacter culture medium using carbon source mannitol, culturing for 120 hours at the pH value of 11 and the culture temperature of 35 ℃, and obtaining fermentation liquor, wherein the azotobacter culture medium: 20.0g of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernatant, and concentrating the supernatant to 1/3 by using a rotary evaporator;
d. c, adding glacial ethanol into the concentrated fermentation liquor obtained in the step c according to the volume ratio of 1;
e. d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1:3, stirring for 30min, standing for 30min in a separating funnel after stirring, taking the lower layer, repeating the experiment twice, dialyzing the sample obtained after deproteinizing with a dialysis bag with the molecular weight cutoff MW3500Da for 48h, and freeze-drying the sample after dialysis to obtain azotobacter As101 extracellular polysaccharide;
and (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution by gradient to 0, 30, 60, 90, 120, 150 and 180 mu g/ml, preparing the obtained azotobacter As101 exopolysaccharide into 200 mu g/ml concentration, adding 50 mu L of samples into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, standing for 10min at 90 ℃, taking out enzyme labeling solution with 632nm to measure absorption wavelength, drawing a standard curve, calculating the sugar content of crude polysaccharide, wherein the final polysaccharide yield is 0.54g/L, and the polysaccharide content is 13.72%.
Example 10
a. Taking 10g soil sample, adding into 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Uniformly coating 100 mu L of diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a thermostat with the temperature of 37 ℃ and the humidity of 55% for culturing for 5d, selecting single bacterial colonies which grow faster and have obviously different bacterial colony forms, and continuously scribing and purifying to obtain single bacterial colonies with consistent forms for classification and identification; wherein the Ashby culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate and 5.0g of calcium carbonate, and steamingDistilled water is 1000mL, sterilized at 1 × 105Pa for 30min;
identification of azotobacter As 101:
DNA of each strain was extracted using a TIANGEN genomic DNA extraction kit (Lot # W9104; cat. # DP 302-02). After the DNA is detected and adjusted to proper concentration, the DNA is used as a template, a target fragment is amplified by adopting a 16S rRNA universal primer 27F/1492R, and the DNA sequence of the DNA is shown in the specification, wherein the sequence is shown in the specification, and the sequence is shown in the specification, wherein the sequence is shown in the specification: AGAGTTTGATCCTGGCTCAG;1492R: ggttactctgttacgactt; the PCR amplification system is as follows: 1.5 muL of template, 1 muL of upstream primer and downstream primer respectively, 25 muL of 2 taq PCR Green Mix, and supplementing sterile water to the total volume of 50 muL; the PCR amplification condition is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ and 7min, and PCR amplification products are sent to Shanghai workers for sequencing; after sequencing the 16S rRNA gene sequence of the strain, carrying out similarity comparison on an NCBI website, and submitting the result to a GenBank database to obtain a strain sequence registration number;
b. fermentation: b, picking the azotobacter obtained by separating in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium using carbon source mannitol, culturing for 48 hours at the pH value of 7 and at the shaking table rotating speed of 170rpm, transferring the azotobacter culture medium with the inoculation amount of 5 percent into 1L of azotobacter culture medium using carbon source mannitol, culturing for 120 hours at the pH value of 12 and the culture temperature of 35 ℃, and obtaining fermentation liquor, wherein the azotobacter culture medium: 20.0g of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernate, and concentrating the supernate to 1/3 by using a rotary evaporator;
d. c, adding glacial ethanol into the concentrated fermentation liquor obtained in the step c according to the volume ratio of 1;
e. d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1:3, stirring for 30min, standing for 30min in a separating funnel after stirring, taking the lower layer, repeating the experiment twice, dialyzing the sample obtained after deproteinizing with a dialysis bag with the molecular weight cutoff MW3500Da for 48h, and freeze-drying the sample after dialysis to obtain azotobacter As101 extracellular polysaccharide;
and (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution by 0, 30, 60, 90, 120, 150 and 180 mu g/ml in a gradient manner, and preparing the obtained azotobacter As101 exopolysaccharide into 200 mu g/ml. Adding 50 μ L of the above samples into 96-well plate, adding 150 μ L of anthrone sulfuric acid into each sample, mixing, and standing at 90 deg.C for 10min. And taking out the enzyme labeling solution at 632nm to measure the absorption wavelength. The sugar content of the crude polysaccharide was calculated by drawing a standard curve. The final polysaccharide yield was 0.85g/L, with a polysaccharide content of 7.21%.
Example 11
a. Taking 10g soil sample, adding into 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Uniformly coating 100 mu L of diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a constant temperature box with the temperature of 37 ℃ and the humidity of 55 percent for culture for 6 days, selecting single bacterial colonies which grow faster and have obviously different bacterial colony forms, and continuously scribing and purifying to obtain single bacterial colonies with consistent forms for classification and identification; wherein the Ashby culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate and 1000mL of distilled water, and sterilizing at 1 × 105Pa for 30min;
identification of azotobacter As 101:
DNA of each strain was extracted using a TIANGEN genomic DNA extraction kit (Lot # W9104; cat. # DP 302-02) as follows: after the DNA is detected and adjusted to proper concentration, the DNA is used as a template, a target fragment is amplified by adopting a 16S rRNA universal primer 27F/1492R, and the DNA sequence of the DNA is shown in the specification, wherein the sequence is shown in the specification, and the sequence is shown in the specification, wherein the sequence is shown in the specification: AGAGTTTGATCCTGGCTCAG;1492R: ggttactctgttacgactt; the PCR amplification system is as follows: 1.5 muL of template, 1 muL of upstream primer and downstream primer respectively, 25 muL of 2 taq PCR Green Mix, and supplementing sterile water to the total volume of 50 muL; the PCR amplification condition is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ and 7min, and PCR amplification products are sent to Shanghai workers for sequencing; after sequencing the 16S rRNA gene sequence of the strain, carrying out similarity comparison on an NCBI website, and submitting the result to a GenBank database to obtain a strain sequence registration number;
b. and (3) fermentation: b, picking the azotobacter separated in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium which uses carbon source glucose, culturing for 48 hours at the pH value of 7, rotating the shaking table at 170rpm, transferring the azotobacter culture medium with the inoculation amount of 5 percent into 1L of azotobacter culture medium which uses carbon source glucose, culturing for 120 hours at the pH value of 7 and the culture temperature of 35 ℃, and obtaining fermentation liquor, wherein the azotobacter culture medium is prepared by the steps of: 20.0g of glucose, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernatant, and concentrating the supernatant to 1/3 by using a rotary evaporator;
d. c, adding glacial ethanol into the concentrated fermentation liquor obtained in the step c according to the volume ratio of 1;
e. d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1;
and (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution by gradient to 0, 30, 60, 90, 120, 150 and 180 mu g/ml, preparing the obtained azotobacter As101 extracellular polysaccharide into 200 mu g/ml concentration, adding 50 mu L of the sample into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, placing at 90 ℃ for standing for 10min, taking out enzyme labeling solution with 632nm to measure absorption wavelength, drawing a standard curve, calculating the sugar content of the crude polysaccharide, and finally obtaining the polysaccharide with the yield of 3.2g/L and the polysaccharide content of 48.44 percent.
Example 12
a. Taking 10g soil sample, adding into 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Uniformly coating 100 mu L of diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a thermostat with the temperature of 37 ℃ and the humidity of 55 percent for culturing for 5-7 days, selecting the bacteria which grow faster,Single colonies with obviously different colony morphologies are obtained by continuous streak purification and used for classification and identification after single colonies with consistent morphology are obtained; wherein the Ashby culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate and 1000mL of distilled water, and sterilizing at 1 × 105Pa for 30min;
identification of azotobacter As 101:
DNA of each strain was extracted by using a TIANGEN genomic DNA extraction kit (Lot # W9104; cat. # DP 302-02). After the DNA is detected and adjusted to a proper concentration, the DNA is used as a template, a target fragment is amplified by adopting a 16S rRNA universal primer 27F/1492R, and the sequence of the DNA is shown in the specification, wherein the sequence is shown in the specification, and is shown in the specification: AGAGTTTGATCCTGGCTCAG;1492R: ggttactctgttacgactt; the PCR amplification system is as follows: 1.5 muL of template, 1 muL of upstream primer and downstream primer respectively, 25 muL of 2 taq PCR Green Mix, and supplementing sterile water to the total volume of 50 muL; the PCR amplification condition is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ and 7min, and PCR amplification products are sent to Shanghai workers for sequencing; after sequencing the 16S rRNA gene sequence of the strain, carrying out similarity comparison on an NCBI website, and submitting to a GenBank database to obtain a strain sequence registration number;
b. and (3) fermentation: b, picking the azotobacter separated in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium which uses carbon source cane sugar, culturing for 48 hours at a pH value of 7 at a shaking table rotating speed of 170rpm with an inoculation amount of 5 percent, transferring the azotobacter culture medium to 1L of azotobacter culture medium which uses carbon source cane sugar, culturing for 120 hours at a pH value of 7 at a culture temperature of 35 ℃, and obtaining a fermentation liquid, wherein the azotobacter culture medium comprises the following components: 20.0g of cane sugar, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min; obtaining a fermentation liquor, wherein the nitrogen fixation culture medium: 20.0g of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernatant, and concentrating the supernatant to 1/3 by using a rotary evaporator;
d. c, adding glacial ethanol into the concentrated fermentation liquor obtained in the step c according to the volume ratio of 1;
e. d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1;
and (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution by gradient to 0, 30, 60, 90, 120, 150 and 180 mu g/ml, preparing the obtained azotobacter As101 exopolysaccharide into 200 mu g/ml concentration, adding 50 mu L of the samples into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, and standing at 90 ℃ for 10min. And (5) taking out the enzyme labeling solution with the wavelength of 632nm to measure the absorption wavelength, drawing a standard curve, and calculating the sugar content of the crude polysaccharide. The final polysaccharide yield was 3.54g/L, with a polysaccharide content of 51.48%.
Example 13
a. Taking 10g soil sample, adding into 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Uniformly coating 100 mu L of diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a constant temperature box with the temperature of 37 ℃ and the humidity of 55 percent for culturing for 5 days, selecting single bacterial colonies which grow faster and have obviously different bacterial colony forms, and continuously scribing and purifying to obtain single bacterial colonies with consistent forms for classification and identification; wherein the Ashby culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate and 1000mL of distilled water, and 1 multiplied by 105Pa for sterilization for 30min;
identification of azotobacter As 101:
DNA of each strain was extracted using a TIANGEN genomic DNA extraction kit (Lot # W9104; cat. # DP 302-02). After the DNA is detected and adjusted to proper concentration, the DNA is used as a template, a target fragment is amplified by adopting a 16S rRNA universal primer 27F/1492R, and the DNA sequence of the DNA is shown in the specification, wherein the sequence is shown in the specification, and the sequence is shown in the specification, wherein the sequence is shown in the specification: AGAGTTTGATCCTGGCTCAG;1492R: ggttactctgttacgactt; the PCR amplification system is as follows: 1.5 muL of template, 1 muL of upstream primer and downstream primer respectively, 25 muL of 2 taq PCR Green Mix, and supplementing sterile water to the total volume of 50 muL; the PCR amplification condition is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ for 7min, and PCR amplification products are sent to Shanghai worker for sequencing; after sequencing the 16S rRNA gene sequence of the strain, carrying out similarity comparison on an NCBI website, and submitting the result to a GenBank database to obtain a strain sequence registration number;
b. fermentation: b, picking the azotobacter separated in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium which uses carbon source lactose, culturing for 48 hours at the pH value of 7, rotating the shaking table at 170rpm, transferring the azotobacter culture medium with the inoculation amount of 5 percent into 1L of azotobacter culture medium which uses carbon source lactose, culturing for 120 hours at the pH value of 7 and the culture temperature of 35 ℃, and obtaining fermentation liquor, wherein the azotobacter culture medium comprises the following components: 20.0g of lactose, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min; obtaining a fermentation liquor, wherein the nitrogen fixation culture medium: 20.0g of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernatant, and concentrating the supernatant to 1/3 by using a rotary evaporator;
d. c, adding glacial ethanol into the concentrated fermentation liquor obtained in the step c according to the volume ratio of 1;
e. d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1;
and (3) determination of polysaccharide content: taking glucose As standard solution, preparing the mother solution into 200 mu g/ml, diluting the mother solution by gradient to 0, 30, 60, 90, 120, 150 and 180 mu g/ml, preparing the obtained azotobacter As101 exopolysaccharide into 200 mu g/ml concentration, adding 50 mu L of samples into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, standing for 10min at 90 ℃, taking out enzyme labeling solution with 632nm to measure absorption wavelength, drawing a standard curve, calculating the sugar content of crude polysaccharide, wherein the final polysaccharide yield is 5.13g/L, and the polysaccharide content is 53.99%.
Example 14
a. Taking 10g soil sample, adding into 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution for 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Uniformly coating 100 mu L of diluted bacterial liquid on an Ashby solid culture medium, placing the Ashby solid culture medium in a constant temperature box with the temperature of 37 ℃ and the humidity of 55 percent for culture for 7d, selecting single bacterial colonies which grow faster and have obviously different bacterial colony forms, and continuously scribing and purifying to obtain single bacterial colonies with consistent forms for classification and identification; wherein the Ashby culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate and 1000mL of distilled water, and sterilizing at 1 × 105Pa for 30min;
identification of azotobacter As 101:
DNA of each strain was extracted using a TIANGEN genomic DNA extraction kit (Lot # W9104; cat. # DP 302-02). After the DNA is detected and adjusted to proper concentration, the DNA is used as a template, a target fragment is amplified by adopting a 16S rRNA universal primer 27F/1492R, and the DNA sequence of the DNA is shown in the specification, wherein the sequence is shown in the specification, and the sequence is shown in the specification, wherein the sequence is shown in the specification: AGAGTTTGATCCTGGCTCAG;1492R: ggttactctgttacgactt; the PCR amplification system is as follows: 1.5 muL of template, 1 muL of upstream primer and downstream primer respectively, 25 muL of 2 taq PCR Green Mix, and supplementing sterile water to the total volume of 50 muL; the PCR amplification condition is 95 ℃ for 5min; the temperature is 94 ℃ for 50s; the temperature is 54 ℃ for 30s; the temperature is 72 ℃ for 30s for 30 cycles; the temperature is 72 ℃ and 7min, and PCR amplification products are sent to Shanghai workers for sequencing; after sequencing the 16S rRNA gene sequence of the strain, carrying out similarity comparison on an NCBI website, and submitting the result to a GenBank database to obtain a strain sequence registration number;
b. fermentation: b, picking the azotobacter obtained by separating in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium using carbon source mannose, culturing for 48 hours at the pH value of 7, rotating the shaking table at 170rpm, inoculating 5 percent of azotobacter to 1L of azotobacter culture medium using carbon source mannose, culturing for 120 hours at the pH value of 7 and the culture temperature of 35 ℃, and obtaining fermentation liquor, wherein the azotobacter culture medium: 20.0g of mannose, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min; obtaining a fermentation liquor, wherein the nitrogen fixation culture medium: 20.0g of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric chloride, trace sodium molybdate and 1000mL of distilled water are sterilized at 1 × 105Pa for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernatant, and concentrating the supernatant to 1/3 by using a rotary evaporator;
d. c, adding glacial ethanol into the concentrated fermentation liquor obtained in the step c according to the volume ratio of 1;
e. d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent with the volume ratio of 1;
and (3) determination of polysaccharide content: taking glucose As standard solution, preparing mother solution into 200 mu g/ml, diluting the mother solution by gradient to 0, 30, 60, 90, 120, 150 and 180 mu g/ml, preparing the obtained azotobacter As101 exopolysaccharide into 200 mu g/ml, adding 50 mu L of samples into a 96-well plate, adding 150 mu L of anthrone sulfuric acid into each sample, uniformly mixing, and standing at 90 ℃ for 10min. And (3) taking out the enzyme labeling solution with the wavelength of 632nm to measure the absorption wavelength, drawing a standard curve, and calculating the sugar content of the crude polysaccharide, wherein the final polysaccharide yield is 1.84g/L, and the polysaccharide content is 55.76%.
Example 15
Analysis of monosaccharide composition:
hydrolysis: taking 5mg of a purified azotobacter As101 exopolysaccharide sample, adding 4mL of 2mol/L trifluoroacetic acid into a headspace bottle, sealing, hydrolyzing at 110 ℃ for 6h at constant temperature, adding 2mL of methanol, reducing pressure, evaporating to dryness, and repeating for three times;
acetylated derivatives: adding 8mg of glycolic acid, 1mL of pyridine and 1mL of acetic anhydride into the hydrolysate, heating at 90 ℃ for 1h, cooling, drying N2, diluting the acetylated monosaccharide alcohol with chloroform, and performing gas chromatography-mass spectrometry (GS) analysis (figure 4), wherein GC analysis shows that the extracellular polysaccharide of azotobacter As101 consists of 0.644% of mannose, 74.542% of glucose and 24.794% of galactose.
Example 16
NaCl with different contents of 0, 10, 20 and 30mg/ml is added into the azotobacter culture fermentation liquor, and the polysaccharide content and the Na + content are detected after the azotobacter culture liquor is cultured for a certain time, so that the azotobacter has the highest sugar yield under the stress of no NaCl, the Na + adsorption capacity is enhanced along with the increase of the NaCl concentration, and the Na + removal rate is improved according to the table 1. The results show that: azotobacter salinistis (As 101) has sodium ion adsorption of 21.501 + -0.9023 mg/ml and sodium ion removal capacity of 71.67 + -0.9555%.
TABLE 1 adsorption of Na by azotobacteria + Capability of
The results show that: the pH value has great influence on the production of azotobacter As101, the yield and the polysaccharide content are highest under the condition of pH7, and the more alkaline the pH condition is, the more the generation of metabolites is inhibited. Thus, the optimum fermentation pH for azotobacter As101 bacteria is 7. It is shown that different carbon sources do not have great influence on the sugar yield and sugar content of azotobacter As101, wherein the yield and sugar content of azotobacter As101 are optimal after mannitol in the original formula of azotobacter medium is used As the carbon source. The carbon source is therefore mannitol as the optimal medium carbon source.
Claims (1)
1. The preparation method of the saline-alkali soil azotobacter polysaccharide is characterized in that azotobacter is obtained by separating and purifying soil, and the strain is named as azotobacterAzotobacter salinestris As101, the specific operation is carried out according to the following steps:
a. separation and purification: taking 10g soil sample, adding into 90ml sterile water, shaking for 30min, taking supernatant, and performing gradient dilution for 10 -1 、10 -2 、10 -3 、10 -4 And 10 -5 Taking 100 mu L of diluted bacterium liquid, uniformly coating the diluted bacterium liquid on an Ashby solid culture medium, placing the diluted bacterium liquid in a thermostat with the temperature of 37 ℃ and the humidity of 55% for culturing for 5-7d, selecting a single colony which grows faster and has an obvious colony morphology, and continuously carrying out scribing and purification to obtain the single colony with the consistent morphology, wherein the Ashby solid culture medium: 10.0g of glucose, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.2g of sodium chloride, 0.2g of calcium sulfate, 5.0g of calcium carbonate, 1000mL of distilled water and 1 multiplied by 105Pa for sterilization for 30min;
b. and (3) fermentation: b, picking the azotobacter obtained by separation in the step a by using an inoculating loop, inoculating the azotobacter into 100ml of azotobacter culture medium which uses carbon sources of lactose, sucrose, mannitol, mannose or glucose, culturing for 24-48 hours at a pH value of 7-12 at a rotating speed of a shaking table of 170rpm, transferring the azotobacter culture medium with the inoculation amount of 5 percent into 1L of azotobacter culture medium which uses carbon sources of lactose, sucrose, mannitol, mannose and glucose, culturing for 48-120 hours at a pH value of 7-12 at a culture temperature of 35 ℃, and obtaining fermentation liquor, wherein the azotobacter culture medium comprises the following steps: 20.0g of mannitol, mg of mannitol, 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.1g of calcium sulfate, 0.5g of yeast extract, trace ferric trichloride, trace sodium molybdate, 1000mL of distilled water and sterilization at 1 × 105Pa for 20min;
c. c, centrifuging the fermentation liquor obtained in the step b for 10min, taking supernate, and concentrating the supernate to 1/3 by using a rotary evaporator;
d. d, adding glacial ethanol into the concentrated solution obtained in the step c according to the volume ratio of 1;
e. and d, dissolving the precipitate obtained in the step d with water, deproteinizing, adding a Sevage reagent according to the volume ratio of 1.
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