CN109251879B - High-density fermentation method of Paenibacillus jimila - Google Patents
High-density fermentation method of Paenibacillus jimila Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses a high-density fermentation method of Paenibacillus jimila. The fermentation medium adopted by the invention comprises the following components: corn flour and soybean meal powderThe invention activates Paenibacillus jimila, and then the Paenibacillus jimila is prepared and cultured by first-stage and second-stage seed liquid, when more than 90% of thallus in the fermentation liquor is converted into spore, the fermentation is stopped, and a two-step fermentation technology is adopted in the fermentation process, the first step aims to ensure that the thallus is greatly proliferated in a short time, the second step builds an environment which is not suitable for the growth of the thallus, the conversion of live bacteria to spore is promoted, the spore generation rate is improved, and the effective number of live bacteria reaches 7.5 × 10 under the condition of 50L fermentation tank scale9More than cfu/mL, and the spore yield of the buds reaches more than 92.6 percent. The invention has simple fermentation process, cheap and easily obtained raw materials and low cost, and is suitable for industrialized production.
Description
Technical Field
The invention belongs to the field of microbial fermentation, and particularly relates to a Paenibacillus jimila high-density fermentation method.
Background
Plant diseases and insect pests are always the large enemies of agricultural production, seriously affect the growth of crops, restrict the sustainable development of agriculture and cause huge economic loss, so that the research on the control of plant diseases and insect pests is more and more emphasized. At present, chemical pesticide prevention and control are mainly used in China, and a large amount of chemical pesticide is applied for a long time to cause serious harm to the ecological environment and human beings. With the intensive research on biological control of plant diseases, the control method which is green, environment-friendly, harmless to human and livestock and capable of reducing the use of chemical pesticides gets more and more attention and recognition by society, becomes a hotspot for researching plant disease control in recent years, and antagonistic bacteria play an important role in biological control.
At present, bacillus is most researched in antagonistic bacteria, the bacillus has the advantages of various types, environmental friendliness, easiness in field planting on the surface of a plant rhizosphere and the like, various antibacterial active substances generated by the bacillus, such as lipopeptide antibiotics, antibacterial proteins or polypeptide compounds, show strong antibacterial activity on most plant pathogenic fungi, and the bacillus has strong endophytic spores, strong stress resistance and high propagation speed, and is beneficial to industrial production. The microorganism is used for preventing and treating plant diseases, and has wide application prospect.
Paenibacillus jimilae is a bacillus, and the Paenibacillus jimilae can secrete active substances such as antibacterial protein, antibiotics, enzyme or polypeptide, has a good inhibitory effect on plant pathogenic bacteria and fungi, remarkably improves the disease resistance of plants, can promote the growth of the plants, improves the yield of the plants, and is a good biocontrol growth-promoting bacterium. The applicant of the invention separates and obtains Paenibacillus jimiila (Paenibacillus jamila) ZDC-04 from deep sea sediments, and the preservation number is CGMCC No. 16231. Experiments prove that the strain has good control effect on anthracnose of fruit trees (citrus, apple and grape) caused by colletotrichum gloeosporioides and vegetable blight caused by fusarium oxysporum.
The microbial inoculum mostly acts as live bacteria, the effective number of the live bacteria is an important quality index of the microbial inoculum, and the microbial inoculum must contain a certain number of the live bacteria to achieve the effect. China has definite regulations on the number and the dosage of effective viable bacteria for various microbial inoculum approved for production. The number of live bacteria and the yield of spores are important indexes for measuring the Paenibacillus jimila microbial inoculum, but at present, the research on the high-density fermentation optimization of biocontrol Paenibacillus jimila at home and abroad is rare, most of the high-density fermentation process optimization only improves the number of live bacteria, the number of spore formation is not high, the activity and the quality of the microbial inoculum are unstable due to low number of spores, the storage time is greatly reduced, and the microbial inoculum is not suitable for practical use to prevent diseases. Therefore, the development of a microbial inoculum product which has the biocontrol function and high spore number has great significance for practical application.
Disclosure of Invention
Aiming at the problems, the invention provides a Paenibacillus jimila high-density fermentation method. The invention uses cheap and easily obtained agricultural byproducts as carbon and nitrogen source liquid to ferment and produce Paenibacillus jimila, reduces the production cost, improves the viable count and spore yield in unit volume of fermentation liquid, and provides reference for industrial production and popularization of Paenibacillus jimila.
The invention firstly provides a fermentation medium of Paenibacillus jimila, which comprises the following components: corn flour, soybean meal, bran, sucrose, calcium carbonate, monopotassium phosphate and sodium chloride. The Paenibacillus jimiila used in the invention is Paenibacillus jimiila (Paenibacillus jamila) ZDC-04.
Preferably, the fermentation medium of the present invention consists of: 12-30g/L of corn flour, 5-17g/L of soybean meal, 5-14g/L of bran, 6-12g/L of cane sugar, 1-5g/L of calcium carbonate, 1-6g/L of potassium dihydrogen phosphate, 0-5g/L of sodium chloride and 6.4-7.4 of initial pH value.
More preferably, the fermentation medium of the present invention consists of: 15g/L of corn flour, 10g/L of soybean meal, 8g/L of bran, 6g/L of sucrose, 5g/L of calcium carbonate, 5g/L of monopotassium phosphate, 2g/L of sodium chloride and 6.4-7.4 of initial pH value.
The soybean meal is crushed soybean meal, the bran is crushed wheat bran, the sodium chloride is industrial sodium chloride, and the sucrose is primary white granulated sugar.
The invention provides an application of the fermentation medium in fermentation of Paenibacillus jimila.
The invention also provides a high-density fermentation method of Paenibacillus jimila, which is characterized in that the Paenibacillus jimila is activated, and then is subjected to primary and secondary seed liquid preparation and fermentation culture, and when more than 90% of thalli in fermentation liquid are converted into spores, the fermentation is stopped;
the method specifically comprises the following steps:
(1) preparing first-order seed liquid by shaking a flask: transferring the activated Paenibacillus jimila into a shake flask seed culture medium, and culturing for 16-30h at the temperature of 28-37 ℃ and the rotation speed of a shaking table of 160-;
the shake flask seed culture medium comprises the following components: 10 plus or minus 2g/L of peptone, 10 plus or minus 2g/L of yeast extract, 30 plus or minus 5g/L of sucrose and pH6.5 plus or minus 0.1.
(2) Preparing a secondary seed liquid in a seed tank: transferring the cultured shake flask seed solution into a secondary seed culture medium according to the inoculation amount of 1-5% (volume ratio), and culturing at 30 + -2 deg.C for 14-18 h;
the secondary seed culture medium comprises the following components: corn flour 20 +/-5 g/L, soybean meal 15 +/-3 g/L, cane sugar 30 +/-5 g/L, potassium dihydrogen phosphate 5 +/-1 g/L and pH6.5 +/-0.1.
(3) Inoculating the seed liquid obtained in the step (2) into a fermentation tank filled with the fermentation culture medium according to the inoculation amount of 3-10% (volume ratio), performing fermentation culture at 30 +/-2 ℃, and stopping fermentation when more than 90% of thalli in the fermentation liquid are converted into spores.
Preferably, the stirring speed in the step (2) is 300-400r/min, the liquid loading amount is 50-70%, and the ventilation amount is 0.4-1m3The pressure in the tank is 0.03-0.05 MPa.
When the fermentation culture in the step (3) is carried out, the fermentation conditions are as follows for 0-10 h: the stirring speed is 300-3H, the tank pressure is 0.03-0.05 Mpa; the fermentation culture conditions after 11h were: the stirring speed is 200-300r/min, and the air flow is 1-2.5m3The pressure in the tank is 0.03-0.05 MPa.
Further, the method for activating Paenibacillus jimila strains comprises the following steps: streaking Paenibacillus jimila preserved in a sand soil pipe on an LB culture medium flat plate for culture, culturing for 16-48h at 28-37 ℃, then selecting a single colony, streaking and transferring to an LB slant culture medium, and culturing for 16-48h at 28-37 ℃ to obtain an activated strain; the LB plate culture medium and the LB slant culture medium comprise: 100g/L of peptone, 100g/L of sodium chloride, 50g/L of yeast extract and 15-20g/L, pH 6.5.5 +/-0.1 of agar.
According to the method, in the Paenibacillus jie-mirabilis high-density fermentation process, different culture media are respectively prepared according to different fermentation conditions of primary seeds, secondary seeds and a fermentation tank in a shake flask so as to improve the activity of the seeds and promote the high-density fermentation. In addition, a two-step fermentation technology is adopted in the fermentation culture process, the first step aims to ensure that the thalli are greatly proliferated in a short time, and the second step creates an environment which is not suitable for the growth of the thalli, promotes the transformation of the viable bacteria to spores, improves the sporulation rate, and further improves the viable bacteria number and the spore yield.
Compared with the prior art, the invention has the following beneficial effects:
1) the invention researches the high-density fermentation process of Paenibacillus jimila for the first time and provides reference for the industrial production and popularization of Paenibacillus jimila;
2) the raw materials of the fermentation medium mainly come from cheap and easily-obtained agricultural and sideline products of corn flour, soybean meal and bran, the cost is low, and the expanded production is easy to realize;
3) the inventionThe fermentation process is simple, the fermentation period is short (15h), the effective viable count and the spore yield are high, and the effective viable count reaches 7.5 × 10 under the condition of 50L fermentation tank scale9More than cfu/mL, and the spore yield of the buds reaches more than 92.6 percent.
Detailed Description
Example 1: separation and screening of Paenibacillus jimila ZDC-04
(1) Separation method of Paenibacillus jimila ZDC-04
The Paenibacillus jimirabilis ZDC-04 is separated from sediments (120.6 degrees E and 34 degrees N) in the sea bottom of the east China sea, is separated in a water depth of 17 meters by adopting a dilution coating method, and specifically comprises the following steps: weighing 10g of submarine sediment sample, dissolving in 90mL of sterile water, placing in a shaking table, and oscillating for 30min to fully mix the sample with water to obtain 10-1And (4) diluting the solution. Mixing 1ml of the above diluted solution with 9ml of sterile water, and shaking to obtain 10-2And (4) diluting the solution. The same procedure gave 10-3、10-4、10-5、10-6The diluent (2). Respectively take 10-4、10-5、10-6The diluted solution of (2) was poured into an LB plate (100. mu.L), and the diluted solution was uniformly applied by an applicator, and each concentration was repeated 3 times. Culturing in 30 deg.C incubator for 48h, selecting single colonies with different culture characteristics, plating, purifying, and numbering for storage.
(2) Screening of antagonistic bacillus of colletotrichum gloeosporioides and fusarium oxysporum
Firstly, primary screening: adopting a confronting plate method, taking colletotrichum gloeosporioides and fusarium oxysporum as targets, inoculating bacteria in the center of a PDA plate by streaking, inoculating pathogenic bacteria cakes at two ends 2.5cm away from the bacteria, culturing at a constant temperature of 25 ℃, and inoculating only the pathogenic bacteria cakes in a contrast manner. Each treatment is repeated for 3 times, when the contrast grows over 3/4 culture dishes, the width of the bacteriostatic band is measured, and bacterial strains with obvious antagonism to the pathogenic bacteria are screened out.
Secondly, re-screening: preparing seed solution from the primarily screened bacterial strains, inoculating 2mL of the seed solution into 100mL of LB culture medium, and carrying out constant temperature shaking culture at 180r/min and 30 ℃ for 48 h. And (3) uniformly mixing the fermentation liquor and the melted and cooled PDA culture medium (45-50 ℃) by adopting a hypha growth rate method, pouring the mixture into a flat plate, taking the PDA culture medium added with the LB liquid culture medium with the same volume as the PDA culture medium as a control, and inoculating a pathogenic bacteria cake with the diameter of 5mm in the center of the flat plate. Each treatment is repeated for 3 times, when the control grows over 3/4 culture dishes, the colony diameters of the control group and the treatment group are measured by a cross method, the bacteriostasis rate is calculated, and the bacterial strains with the optimal antagonistic effect on colletotrichum gloeosporioides and fusarium oxysporum are screened out.
Inhibition (%) - (control colony radius (mm) -treatment colony radius (mm))/control colony radius (mm) × 100
(3) Results and analysis: 20 strains of bacteria are separated from seabed sediment by using a dilution plate method, 7 antagonistic strains with obvious inhibition effect on the pathogenic bacteria are screened by using a counter plate method, and account for 35 percent of the separated strains, wherein the antagonistic effect of the strain ZDC-04 is the best, and the widths of the bacteriostatic belts on colletotrichum gloeosporioides and fusarium oxysporum are respectively 17.00mm and 15.50 mm. The fermentation liquor of the strain is selected to be diluted by 10 times in a PDA culture medium for hypha growth rate method test, and the result shows that the inhibition rates of the fermentation liquor of the strain on the hypha growth of colletotrichum gloeosporioides and fusarium oxysporum are 100% and 96.5% respectively.
The invention is also proved by field experiments that: the control effect of the fermentation liquor of Paenibacillus jimila ZDC-04 on the leaves and fruits of citrus anthracnose is 81.53% and 81.82% respectively.
Example 2: identification of Paenibacillus jimila ZDC-04
(1) Strain morphology: the bacteria are long rod-shaped, the size is (0.5-1.1) Mumx (3.2-5.2) Mum, spores are produced, and gram staining is positive. The bacterial colony is white and round and has raised edges after being cultured for 48 hours at 30 ℃ on an LB culture medium.
(2) Results of molecular identification
The 16S rRNA gene sequence determination result (SEQ-1) of the strain is as follows:
AAGCTTGCTTCTAATTAACCTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCCACAAGACAGGGATAACTACCGGAAACGGTAGCTAATACCCGATACATCCTTTTCCTGCATGGGAGAAGGAGGAAAGGCGGAGCAATCTGTCACTTGTGGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGCCAGGGAAGAACGCTTGATAGAGTAACTGCTCTTGAAGTGACGGTACCTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCTCTTTAAGTCTGGTGTTTAATCCCGAGGCTCAACTTCGGGTCGCACTGGAAACTGGGGAGCTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGGCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTAGGGGTTTCGATACCCTTGGTGCCGAAGTTAACACATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCTCTGACCGGTCTAGAGATAGATCTTTCCTTCGGGACAGAGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGCTTAGTTGCCAGCAGGTCAAGCTGGGCACTCTAAGCAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCCGGTACAACGGGAAGCGAAGGAGCGATCTGGAGCCAATCCTAGAAAAGCCGGTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTACAACACCCGAAGTCGGTGAGGTAACC
according to the sequence homology comparison of Gen-Bank, the similarity of the strain ZDC-04 and the Paenibacillus jamilae is 100 percent, so that the strain ZDC-04 is identified as Paenibacillus jamilae (Paenibacillus jamilae). The strain is preserved in China general microbiological culture Collection center (CGMCC for short, No. 3 of West Lu No.1 of Beijing, Tokyo Yang district, China academy of sciences) at 8 months and 8 days in 2018, and the preservation number is CGMCC No. 16231.
Example 3: screening of fermentation Medium
Activation of bacterial species
Inoculating Paenibacillus jimila ZDC-04 stored in a sand soil tube on an LB culture medium flat plate for culture by streaking, culturing for 48h at 30 ℃, then picking up for microscopic examination, transferring a typical single colony streaking to an LB test tube slant culture medium, and culturing for 48h at 30 ℃ to obtain an activated strain for later use;
the composition of the LB medium was: 100g/L peptone, 100g/L sodium chloride, 50g/L yeast extract, 15-20g/L, pH 6.5.5 + -0.1 agar, and high pressure steam sterilizing at 121 deg.C for 30 min.
Second, determination of fermentation medium
The fermentation medium is mainly obtained through the following steps:
1. screening of Paenibacillus jimila fermentation medium
The Paenibacillus jimila is researched for the first time, and the invention utilizes NCBI and national knowledge network to look up related documents at home and abroad to find that no Paenibacillus jimila fermentation culture medium is reported, so that 11 species can be found only by referring to the fermentation culture medium of the strain which has the closest genetic relationship with Paenibacillus jimila, and then the 11 species of fermentation culture media are screened by combining strain growth conditions, fermentation principles, spore production conditions and the like, and finally 3 species of bacillus fermentation culture media are determined for subsequent experiments.
2. Determination of ingredients of Paenibacillus jimila ZDC-04 fermentation medium
Analyzing the principle and spore production condition of the fermentation medium, combining with the actual raw material purchasing approach, cost and the like, and combining with the single-factor test method to screen the components of the medium, and adopting a dilution plate counting method for result evaluation. Finally, the main culture medium components are determined to be corn flour, soybean meal, bran and sucrose.
3. Determination of content of ingredients in Paenibacillus jimila ZDC-04 fermentation medium
And (3) finally determining the components and the content of the culture medium through an orthogonal test method for the nutrient components of the fermentation culture medium: 15g/L of corn flour, 10g/L of soybean meal, 8g/L of bran, 6g/L of sucrose, 5g/L of calcium carbonate, 5g/L of monopotassium phosphate and 2g/L of sodium chloride.
4. Determination of fermentation temperature
After the fermentation medium is inoculated, the fermentation temperature is set to be 25, 28, 30, 35 and 37 ℃ respectively, the rotating speed of a shaking table is 200r/min, the viable bacteria amount and the spore rate are determined, and the test is repeated for three times. The optimum culture temperature was finally determined to be 30 ℃.
5. Determination of fermentation pH
Setting the initial pH values of the culture medium as follows: 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2 and 7.4, after inoculation, the culture temperature is 37 ℃, the rotating speed of a shaking table is 200r/min, the viable bacteria amount and the spore rate are determined, and the test is repeated three times. Finally, the optimal pH value is determined to be 6.5 +/-0.1.
Example 4: 50L fermentation tank high-density fermentation Paenibacillus jimila ZDC-04
Shake flask seed culture medium: 10g/L of peptone, 10g/L of yeast extract and 30g/L, pH 6.5.5 +/-0.1 of sucrose;
secondary seed culture medium: 20g/L of corn flour, 15g/L of soybean meal, 30g/L of cane sugar, 5g/L of monopotassium phosphate and pH6.5 +/-0.1;
fermentation medium: 15g/L of corn flour, 10g/L of soybean meal, 8g/L of bran, 6g/L of sucrose, 5g/L of calcium carbonate, 5g/L of monopotassium phosphate, 2g/L of sodium chloride and initial pH of 6.5 +/-0.1.
(1) Preparing first-order seed liquid by shaking a flask: the shaking flask seed culture medium is filled into 1 500mL triangular flask, the flask is sterilized by high-pressure steam at 121 ℃ for 30min, the activated Paenibacillus jimila ZDC-04 in the example 1 is inoculated into the shaking flask seed culture medium by an inoculating loop in a clean bench, and the culture conditions are as follows: the temperature is 30 ℃, the rotating speed of a shaking table is 200r/min, and the culture time is 24 h.
(2) Preparing a secondary seed liquid in a seed tank: inoculating the seed liquid obtained in the step (1) into a 10L secondary seed tank according to the inoculum size of 2% after microscopic examination without mixed bacteria pollution, and culturing the seed liquid under the conditions that: the temperature is 30 ℃, the stirring speed is 300r/min, and the ventilation volume is 0.6m3Culture under 0.04MPa for 14 hr.
(3) High-density fermentation culture in a 50L fermentation tank: inoculating the seed liquid obtained in the step (2) into a 50L fermentation tank according to the inoculation amount of 5% after microscopic examination of the seed liquid without impurity bacterial pollution, wherein the liquid filling amount of the fermentation tank is 30L. Fermenting for 0-10h, wherein the culture conditions are as follows: the temperature is 30 +/-0.1 ℃, the stirring speed is 400r/min, and the air flow is 3m3H, the tank pressure is 0.03-0.05 Mpa; fermentation culture conditions after 11 h: the temperature is 30 +/-0.1 ℃, the stirring speed is 300r/min, and the air flow is 1m3H, the tank pressure is 0.05 Mpa. And (5) stopping fermentation when the microscopic examination of the fermentation liquor shows that more than 90% of the thallus in the visual field is spores, wherein the fermentation period is 15h, and the fermentation liquor is reserved.
(4) Calculation of the number of cells and spore yield
1) Measurement of the total cell count: counting by a dilution plate coating method, and counting the unit cfu/mL;
2) determination of the total number of spores: heating the bacterial liquid in 80 deg.C water bath for 15min to kill vegetative somatic cells, and coating with gradient dilution plate;
3) spore rate calculation formula: the total viable bacteria count and the number of spores are respectively measured by calculating the total viable bacteria count and calculating the spores after inactivation according to the following calculation formula:
the ratio of spores (number of spores/total number of viable bacteria) × 100%
(5) The experimental results are as follows:
under the condition of 50L fermentation tank scale, the fermentation period is 15h, and the effective viable count reaches 7.5 × 109More than cfu/mL, and the spore yield of the buds reaches more than 92.6 percent.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
SEQUENCE LISTING
<110> agriculture technology Limited in Qingdao
<120> high-density fermentation method of Paenibacillus jimila
<130>0
<160>1
<170>PatentIn version 3.3
<210>1
<211>1382
<212>DNA
<213> 16S rRNA gene sequence of Paenibacillus jimiera ZDC-04
<400>1
aagcttgctt ctaattaacc tagcggcgga cgggtgagta acacgtaggc aacctgccca 60
caagacaggg ataactaccg gaaacggtag ctaatacccg atacatcctt ttcctgcatg 120
ggagaaggag gaaaggcgga gcaatctgtc acttgtggat gggcctgcgg cgcattagct 180
agttggtggg gtaaaggcct accaaggcga cgatgcgtag ccgacctgag agggtgatcg 240
gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc 300
cgcaatgggc gaaagcctga cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt 360
aaagctctgt tgccagggaa gaacgcttga tagagtaact gctcttgaag tgacggtacc 420
tgagaagaaa gccccggcta actacgtgcc agcagccgcg gtaatacgta gggggcaagc 480
gttgtccgga attattgggc gtaaagcgcg cgcaggcggc tctttaagtc tggtgtttaa 540
tcccgaggct caacttcggg tcgcactgga aactggggag cttgagtgca gaagaggaga 600
gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag 660
gcgactctct gggctgtaac tgacgctgag gcgcgaaagc gtggggagca aacaggatta 720
gataccctgg tagtccacgc cgtaaacgat gaatgctagg tgttaggggt ttcgataccc 780
ttggtgccga agttaacaca ttaagcattc cgcctgggga gtacggtcgc aagactgaaa 840
ctcaaaggaa ttgacgggga cccgcacaag cagtggagta tgtggtttaa ttcgaagcaa 900
cgcgaagaac cttaccaggt cttgacatcc ctctgaccgg tctagagata gatctttcct 960
tcgggacaga ggagacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg 1020
ttaagtcccg caacgagcgc aacccttatg cttagttgcc agcaggtcaa gctgggcact 1080
ctaagcagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc 1140
ccttatgacc tgggctacac acgtactaca atggccggta caacgggaag cgaaggagcg 1200
atctggagcc aatcctagaa aagccggtct cagttcggat tgtaggctgc aactcgccta 1260
catgaagtcg gaattgctag taatcgcgga tcagcatgcc gcggtgaata cgttcccggg 1320
tcttgtacac accgcccgtc acaccacgag agtttacaac acccgaagtc ggtgaggtaa 1380
cc 1382
Claims (3)
1. A Paenibacillus jimila high-density fermentation method is characterized in that after Paenibacillus jimila is activated, the Paenibacillus jimila is subjected to shake flask primary seed liquid preparation, seed tank secondary seed liquid preparation and fermentation culture, and when more than 90% of thalli in fermentation liquid are converted into spores, the fermentation is stopped;
the Paenibacillus jimila is Paenibacillus jimila ZDC-04, and the preservation number of the strain is CGMCCNo.16231;
the first-stage seed culture medium for shaking the bottle is prepared by the following steps: peptone 10 plus or minus 2g/L, yeast extract 10 plus or minus 2g/L, sucrose 30 plus or minus 5g/L, pH6.5 plus or minus 0.1;
the preparation of the seeding tank secondary seed liquid adopts a seeding tank secondary seed culture medium which is as follows: corn flour 20 +/-5 g/L, soybean meal 15 +/-3 g/L, cane sugar 30 +/-5 g/L, potassium dihydrogen phosphate 5 +/-1 g/L and pH6.5 +/-0.1;
the fermentation culture medium adopted by the fermentation culture is as follows: 12-30g/L of corn flour, 5-17g/L of soybean meal, 5-14g/L of bran, 6-12g/L of sucrose, 1-5g/L of calcium carbonate, 1-6g/L of potassium dihydrogen phosphate, 0-5g/L of sodium chloride and initial pH value of 6.4-7.4;
the culture conditions of the fermentation culture are as follows: inoculating the secondary seed liquid in the seed tank into a 50L fermentation tank according to the inoculation amount of 5%, wherein the liquid filling amount of the fermentation tank is 30L; fermenting for 0-10h under the culture conditions: the temperature is 30 +/-0.1 ℃, the stirring speed is 400r/min, and the air flow is 3m3H, the tank pressure is 0.03-0.05 Mpa; fermentation culture conditions after 11h of fermentation: the temperature is 30 +/-0.1 ℃, the stirring speed is 300r/min, and the air flow is 1m3H, the tank pressure is 0.05 Mpa.
2. The Paenibacillus jimii high-density fermentation method according to claim 1, wherein the fermentation medium is: 15g/L of corn flour, 10g/L of soybean meal, 8g/L of bran, 6g/L of sucrose, 5g/L of calcium carbonate, 5g/L of monopotassium phosphate, 2g/L of sodium chloride and 6.4-7.4 of initial pH value.
3. The Paenibacillus jimii high-density fermentation method according to claim 1, wherein the shake flask primary seed liquid preparation and the seed tank secondary seed liquid preparation are as follows:
(1) preparing first-order seed liquid by shaking a flask: transferring the activated Paenibacillus jimila into a first-level seed culture medium of a shaking flask, and performing shake culture at 28-37 ℃ for 16-30 h;
(2) preparing a secondary seed liquid in a seed tank: transferring the cultured primary seed liquid of the shake flask into a secondary seed culture medium of a seeding tank according to the inoculation amount of 1-5%, and culturing for 14-18h at the temperature of 30 +/-2 ℃.
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