CN108570431B

CN108570431B - Paenibacillus mucilaginosus and application thereof

Info

Publication number: CN108570431B
Application number: CN201810432213.XA
Authority: CN
Inventors: 梁锏文; 廖美德; 杜景德; 李伟
Original assignee: Yunnan Bosar Biotechnology Co ltd; South China Agricultural University
Current assignee: Yunnan Bosar Biotechnology Co ltd; South China Agricultural University
Priority date: 2018-05-08
Filing date: 2018-05-08
Publication date: 2020-09-01
Anticipated expiration: 2038-05-08
Also published as: CN108570431A

Abstract

The invention discloses a paenibacillus mucilaginosus strain and application thereof. The Paenibacillus kribbensis YB326 provided by the invention has the preservation number: GDMCC NO: 60326. the strain can produce natural active substances for inhibiting fungi, extracellular active polysaccharide and oligosaccharide, and fermentation metabolites of the strain have strong inhibition effects on more than ten plant pathogens such as rice sheath blight, rice blast and the like; the active saccharide is levan and fructo-oligosaccharide. The fermentation metabolite can not only effectively prevent and treat rice sheath blight and rice blast diseases, but also improve the resistance of rice and the rice yield, has a great application prospect, and has important significance for reducing the use of a large amount of chemical fertilizers and pesticides in rice planting and reducing agricultural non-point source pollution; on the other hand, the extracted extracellular active polysaccharide and oligosaccharide can be added into the feed, so that the immunity of animals can be effectively enhanced, the intestinal health of the animals can be improved, and the production performance of the animals can be improved.

Description

Paenibacillus mucilaginosus and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to paenibacillus mucilaginosus and application thereof in rice planting and animal breeding.

Background

The rice is the most important grain crop in China, and the rice yield accounts for nearly 40% of the yield of the three grain crops as the main grain variety. There are many factors affecting rice production, and the problem of disease is one of the important obstacles restricting rice production. The types of rice diseases are many, and nearly hundreds of types are reported all over the world. More than 70 kinds of rice are recorded in China, the rice blast, the sheath blight and the bacterial leaf blight occur more commonly in production, more than 20 kinds of diseases causing important economic losses are caused, the rice blast, the sheath blight and the bacterial leaf blight occur most frequently, the damage degree is the highest, the three kinds of diseases are three important diseases in rice production, the diseases are rice diseases causing the most serious damage to rice growing areas in south and north China, the yield can be greatly reduced, and even no grain harvest can be caused when the diseases are serious.

For the prevention and treatment of rice diseases, cultivation management measures and chemical pesticides are mainly used for prevention and treatment at present. However, most of the rice diseases have the characteristics of long incubation period, long disease period and multi-point damage, and the damage can be well controlled only by carrying out multiple treatments in one growth period and applying chemical pesticides for multiple times in the prevention and treatment process. Has the problems of high prevention and treatment cost, long time consumption, environmental pollution and the like. Therefore, the search for new economic, efficient, green and environment-friendly prevention and treatment technologies and approaches becomes a problem to be urgently solved by the current rice planting industry.

As a feed additive, the active oligosaccharide is an oligosaccharide which is not degraded by digestive enzymes of organisms, can promote the proliferation of beneficial bacteria in animal intestinal tracts, inhibit the adhesion of pathogenic bacteria in the intestinal tracts, increase the immune response of intestinal mucosa and promote the recovery of calcium, magnesium, iron, zinc and other elements of animals, and the application research as a novel feed additive is one of the focuses of the current research. Compared with the currently commonly used feed additives such as antibiotics, viable bacteria preparations and the like, the active oligosaccharide has the advantages of small dosage, no toxic or side effect, no residue, good stability, strong drug resistance and the like, and has higher tolerance in the processes of granulation, puffing, oxidation, storage and transportation, so the active oligosaccharide has a wide prospect when being used as a novel green feed additive for development, and has fructooligosaccharide, chitosan oligosaccharide, mannan oligosaccharide, galactooligosaccharide and the like which are currently applied.

Paenibacillus (Paenibacillus) is aerobic or facultative anaerobic bacillus-producing bacillus rod-shaped bacteria, widely exists in nature, has various physiological characteristics, can produce various bioactive substances such as various antibiotics, saccharides, enzymes, phytohormones, flocculating agents and the like, has good antagonistic activity on plant pathogenic fungi, and can be used as a feed additive to improve the intestinal health of animals and improve the production performance of the animals, wherein the metabolite active polysaccharides and oligosaccharides of the bacillus (Paenibacillus) can improve the stress resistance of the plants. Therefore, the development and application of Paenibacillus are of great significance.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide the paenibacillus mucilaginosus.

The invention also aims to provide application of the paenibacillus mucilaginosus.

The application has two main aspects: firstly, the application of rice planting is verified by experiments, and the biological control preparation prepared by adopting the Paenibacillus mucilaginosus kribbensis YB326 provided by the invention not only can effectively control rice sheath blight and rice blast and reduce plant diseases and insect pests, but also can promote the growth of rice, improve the quality of the rice and improve the yield; secondly, in the aspect of animal breeding, the active oligosaccharide in the fermentation product can be used as a feed additive, so that the intestinal health of animals is improved, and the production performance of the animals is improved.

The purpose of the invention is realized by the following technical scheme:

the invention provides a strain of Paenibacillus mucilaginosus named Paenibacillus mucilaginosus Kribbensis YB326, which is obtained by separating and screening from soil (the colony morphology is shown in figure 1, and the colony microscopic morphology is shown in figure 2).

The preservation information of the Paenibacillus kribbensis YB326 is as follows: the preservation unit: guangdong province microbial culture Collection (GDMCC), the preservation date is 2018, 2 and 6 months, and the preservation address is as follows: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, Guangdong province, of the Zhonglu-Jieli, Guangzhou city, the preservation number: GDMCC NO: 60326.

the Paenibacillus mucilaginosus Kribbensis YB326 provided by the invention is a spore-producing bacterium, and the strain can be preserved by any technical method for preserving bacillus provided by the published documents in the field.

The invention provides application of Paenibacillus mucilaginosus kribbensis YB326 in rice planting.

The invention provides application of Paenibacillus mucilaginosus Kribbensis YB326 in animal breeding.

The Paenibacillus mucilaginosus Paenibacillus YB326 can be metabolized to generate plant pathogenic fungus resistant active substances and active oligosaccharides, and can be used for two purposes: in the aspect of rice planting, the strain is used for preparing a biological control preparation for controlling rice sheath blight and rice blast, improving stress resistance of rice and increasing rice yield; and secondly, in the aspect of animal breeding, the polysaccharide and the active oligosaccharide in the strain fermentation product can be used as feed additives for improving the intestinal health of animals and improving the production performance of the animals.

The strain Paenibacillus kribbensis YB326 is utilized to prepare a biological control preparation.

The preparation method of the biological control preparation mainly comprises the following steps:

(1) preparing a seed solution: selecting a preserved strain Paenibacillus kribbensis YB326, inoculating the strain into a liquid PDA culture medium, and culturing at 35-37 ℃ for 16-30 h to obtain a seed solution;

(2) inoculating the seed solution prepared in the step (1) into a fermentation culture medium according to the inoculation amount of 2% -5%, ventilating at the temperature of 35-37 ℃ and the speed of 0.5-1.0 vvm, fermenting for 3-4 d at the stirring speed of 100-300 r/min, centrifuging to remove thalli, and collecting fermentation products to obtain a biological control preparation;

the fermentation medium comprises a carbon source, a nitrogen source, phosphate, magnesium salt and water. The carbon source is one or more of glucose, sucrose and starch hydrolysate, and the dosage is 20-40 g/L; the nitrogen source is one or more of beef extract, peptone, yeast extract and ammonium sulfate, and the dosage is 3-5 g/L; the phosphate is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate and disodium hydrogen phosphate, and the using amount of the phosphate is 1-3 g/L; the magnesium salt is magnesium sulfate and magnesium chloride, and the dosage of the magnesium salt is 0.2-1 g/L respectively; the balance being water.

The fermentation product of the Paenibacillus mucilaginosus kribbensis YB326 is diluted by 150-200 times and is respectively sprayed in the early stage of rice transplanting (seedling field application) and in the early stage of flowering after tillering is finished, so that the Paenibacillus mucilaginosus is used for preventing and treating rice sheath blight and rice blast diseases, the stress resistance and disease resistance of rice are improved, and the growth and income increase of the rice can be effectively promoted.

In the aspect of animal breeding application, the strain Paenibacillus kribbensis YB326 is utilized to prepare active polysaccharide and oligosaccharide which are used as feed additives.

The preparation method of the immunopotentiator for the cultured animals mainly comprises the following steps:

(1) fermentation: selecting a preserved strain Paenibacillus kribbensis YB326, inoculating the strain into a liquid PDA culture medium, and culturing at 35-37 ℃ for 16-30 h to obtain a seed solution; inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 2-5%, ventilating at the temperature of 35-37 ℃ and the ventilation speed of 0.5-1.0 vvm, fermenting for 3-4 d at the stirring speed of 100-300 r/min, and collecting a fermentation product;

the fermentation medium comprises a carbon source, a nitrogen source, magnesium phosphate and water. The carbon source is one or more of glucose, sucrose and starch hydrolysate, and the dosage is 30-60 g/L; the nitrogen source is one or more of beef extract, peptone, yeast extract and ammonium sulfate, and the dosage is 2-6 g/L; the phosphate is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate and disodium hydrogen phosphate, and the using amount of the phosphate is 2-3 g/L; the magnesium salt is magnesium sulfate and magnesium chloride, and the dosage of the magnesium salt is 0.2-1 g/L respectively; the balance being water.

(2) Extraction: and (2) centrifuging the fermentation product obtained in the step (1) to remove thalli, adding 1-2 times of organic solvent to precipitate extracellular active polysaccharide and oligosaccharide, drying and dehydrating the supernatant to obtain the rest of active oligosaccharide, and combining the extracellular active polysaccharide and the active oligosaccharide to obtain the cultured animal immunopotentiator.

Preferably, the organic solvent in step (2) is absolute ethyl alcohol.

Preferably, the drying in step (2) is drying to a water content of less than 10%.

The Paenibacillus mucilaginosus Kribbensis YB326 is fermented, extracted and dried to prepare active polysaccharide and oligosaccharide, and the active polysaccharide and oligosaccharide are added according to 0.05-5% of the total weight of the feed and are applied to animal breeding. Can obviously improve the intestinal health of animals and improve the production performance of the animals.

Preferably, the feed additive is added according to 0.05-3% of the total weight of the feed; more preferably 1 to 2%.

Compared with the prior art, the invention has the following advantages and effects:

the Paenibacillus mucilaginosus kribbensis YB326 provided by the invention can generate natural active substances for inhibiting fungi, extracellular active polysaccharide and oligosaccharide, and fermentation metabolites of the Paenibacillus mucilaginosus have strong inhibition effect on more than ten plant pathogenic bacteria such as rice sheath blight, rice blast and the like; the active saccharide is levan and fructo-oligosaccharide. The biological control preparation prepared from the paenibacillus mucilaginosus can efficiently control rice sheath blight and rice blast diseases, can improve the resistance of rice and the rice yield, is a biological control preparation with a great application prospect, and has important significance for reducing the use of a large amount of fertilizers and pesticides in rice planting and reducing agricultural non-point source pollution; on the other hand, extracellular active polysaccharide and oligosaccharide extracted from the fermentation product of the strain can be added into feed, so that the immunity of animals can be effectively enhanced, the intestinal health of the animals can be improved, and the production performance of the animals can be improved.

Drawings

FIG. 1 is a colony morphology map of strain YB326 according to the present invention.

FIG. 2 is a colony microscopic morphology of strain YB326 according to the present invention.

FIG. 3 is a phylogenetic tree of strain YB326 constructed based on the gene sequence of strain 16S rRNA.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The paenibacillus mucilaginosus YB326 strain related to the invention is separated from garden soil and is preserved in a strain preservation library of a microorganism laboratory of Yunnan Shibo ao biotechnology limited company and a microorganism strain preservation center of Guangdong province.

Example 1: paenibacillus mucilaginosus Kuibbensis YB326 separation and screening

Collecting samples: taking soil in a certain garden in Guangzhou city, Guangdong province, removing surface soil by using an aseptic shovel, taking soil with the depth of 5-10 cm below the ground surface by using an aseptic sampler, collecting 5 samples at each point, combining the samples, filling the samples into an aseptic bag, and taking the aseptic bag back to a laboratory for strain separation.

Preparing a soil diluent: weighing 10g of soil, putting the soil into a triangular flask filled with 90mL of sterile water, and fully oscillating to prepare the soil with the dilution degree of 10^-1The soil dilution of (1). Then, the mixture was diluted in a gradient to prepare a dilution of 10^-2、10^-3、10^-4、10^-5And 10^-6The soil solution is ready for use.

And (3) bacteria separation: get 10^-4、10^-5And 10^-60.1mL of each of the three diluted soil suspensions was dropped into LB medium, the suspensions were spread evenly using a sterile spreading rod, and inverted for culture at 37 ℃ with three replicates per dilution sample. 30 strains of bacteria were isolated as described above.

Screening: respectively inoculating 30 isolated strains into 500mL conical flasks filled with 100mL liquid Chashi culture medium, placing the conical flasks in a shaking table for 180r/min to oscillate, culturing at 37 ℃ for 72h, centrifuging to remove thalli, collecting fermentation supernatant, placing oxford cups on PDA plates just coated with Rhizoctonia solani and Pyricularia oryzae, adding 200 mu L of fermentation supernatant into each oxford cup, culturing at 30 ℃ for 3-5 d, measuring and recording the diameter of a bacterial colony when the bacterial colony on a control culture medium grows to be more than 90% of the whole culture dish, and calculating the inhibition rate.

And (4) screening results: the 1 bacterial strain with strong antagonistic effect is obtained through the test and named as YB326, the inhibition rate of the bacterial strain on rhizoctonia solani and pyricularia oryzae is more than 85 percent, and substances such as exopolysaccharides and the like are found to be generated in the metabolic process.

Example 2: identification of Paenibacillus mucilaginosus Kuibbecensis YB326

The colony morphology characteristics of strain YB326 are shown in figure 1: the growth in the Chachi culture medium is good, the surface of a colony is smooth, the colony is milky white, is in a mucus shape, and has a protrusion in the middle. The microscopic morphological characteristics of the thalli YB326 are shown in figure 2, the thalli YB326 is rod-shaped and has capsules, and the thalli is 0.8-1.2 mu m.

Sequencing the rDNA gene of Paenibacillus mucilaginosus Kribbon-brensis YB 32616S, performing Blast comparison analysis on the sequence, finding that the strain YB326 belongs to the Paenibacillus (Paenibacillus), the similarity with the typical strain Paenibacillus Kribbon-brensis of the genus reaches 99%, and judging that the strain YB326 is identified as the Paenibacillus mucilaginosus (Paenibacillus Kribbon-brensis) by combining morphological characteristics and the like of the strain, wherein the phylogenetic tree of the strain is shown in figure 3.

Therefore, the strain is named as Paenibacillus mucilaginosus Kuibbensis YB326, and the preservation information is preservation unit: guangdong province microbial culture Collection (GDMCC), the preservation date is 2018, 2 and 6 months, and the preservation address is as follows: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, Guangdong province, of the Zhonglu-Jieli, Guangzhou city, the preservation number: GDMCC NO: 60326.

the determination result of the 16S rDNA gene sequence of the Paenibacillus mucilaginosus kribbensis YB326 is shown as SEQ ID NO: 1 is shown.

Example 3: shake flask fermentation

Seed culture medium: PDA culture medium

Fermentation medium: 30g/L of cane sugar, 3g/L of beef extract and K₂HPO₄·7H₂O 2g/L，MgSO₄·7H₂O0.5g/L, initial pH 7.2, solvent is water.

Inoculating activated Paenibacillus mucilaginosus Kuibbensis YB326 to a seed culture medium for culturing for 24h to prepare a seed solution, then inoculating the seed solution into a 250mL triangular flask filled with 50mL of a fermentation culture medium, wherein the inoculation amount is 5% (v/v), the temperature is 37 ℃, the shaking table fermentation culture is carried out for 72h at 250r/min, 2 times of volume of absolute alcohol is added to precipitate polysaccharide, the content is 10g/L, and the supernatant is evaporated to dryness to obtain oligosaccharide, wherein the content is 8 g/L.

Example 4: preparation of biological control agent

Seed culture medium: PDA culture medium

Fermentation medium: 30g/L of cane sugar, 2g/L of beef extract and K₂HPO₄·7H₂O 2g/L，MgSO₄·7H₂O0.5g/L, potassium chloride 0.5g/L, initial pH 7.5, solvent is water.

Inoculating activated Paenibacillus mucilaginosus YB326 to a seed culture medium for culturing at 37 ℃ and 200r/min for 24h to prepare a seed solution, then inoculating the seed solution to a fermentation culture medium, wherein the fermentation volume of a 100L fermentation tank is 70L, the inoculation amount is 3% (v/v), the temperature is 35 ℃, the ventilation is 0.8vvm, the stirring speed is 150r/min, and the fermentation is 68h, so that the biological rice control preparation is obtained.

Example 5: test for prevention and control of rice

In order to determine the control effect of the fermentation product of the Paenibacillus mucilaginosus on Rhizoctonia solani and Magnaporthe oryzae, the method provides a basis for popularization and application of the fermentation product. In 2017, in 3-10 months, microbial agent YB326 rice field application effect tests are respectively carried out in places such as Yongshang town of Hainan province, Qianxi county of Guizhou province and Cinnamomum camphora city of Jiangxi province, 1 mu of test field is prepared in each area, 1 mu of test field is averagely divided into 4 small test fields, and rice of the same variety is planted in the test fields in the same area. The effect test of the application of the microbial agent YB326 in the paddy field was carried out as follows.

Treatment 1: conventional fertilization and planting (0 treatment) of rice;

and (3) treatment 2: treating with rice microbial agent YB 326.

And (3) test results:

after microbial agent YB326 is applied to the experimental fields for planting three kinds of permanently grown rice in Guizhou province, camphor tree city (rice sheath blight disease) and Hainan province, the test results are shown in tables 1-3, wherein the main diseases in Guizhou province are rice blast, the main diseases in Jiangxi province are rice sheath blight, the disease incidence is obviously reduced, the acre yields of the three kinds of permanently grown rice are respectively increased by 10.81%, 7.13% and 15.22%, and the yield and the efficiency are obviously increased.

TABLE 1 Guizhou Rice test results

Note: the rice variety is big grain fragrant; purchased from the research center of rice engineering technology in Guizhou province.

TABLE 2 Rice test results for Jiangxi Cinnamomum camphora

Note: the rice variety is 35 in the morning, and the number is examined and determined: rice 2010005 was examined in county.

TABLE 3 Hainan perpetual-growth rice test results

Note: the rice variety is rice super-excellent 458, and the number is examined and determined: jojoba 2010015.

Example 6: preparation of active polysaccharides and oligosaccharides

Seed culture medium: PDA culture medium

Fermentation medium: 50g/L of cane sugar, 4g/L of beef extract and Na₂HPO₄·7H₂O 2g/L，MgSO₄·7H₂O0.2 g/L, potassium chloride 0.5g/L, initial pH 7.5, solvent is water.

Inoculating activated Paenibacillus mucilaginosus YB326 to a seed culture medium for culturing at 37 ℃ and 250r/min for 18h to prepare a seed solution, then inoculating the seed solution to a fermentation culture medium, wherein the fermentation volume of a 100L fermentation tank is 60L, the inoculation amount is 2% (v/v), the temperature is 35 ℃, ventilation is 0.8vvm, the stirring speed is 250r/min, fermentation is carried out for 72h, bacteria are removed by centrifugation, 2 times of volume of absolute ethyl alcohol is added, stirring and precipitation are carried out, precipitates are obtained by filtration, drying is carried out at 80 ℃ to obtain 1500g of polysaccharide and oligosaccharide, the extracting solution is heated and evaporated to dryness (recycling alcohol) to obtain 950g of oligosaccharide, the solid content is 2450g, and the extracting solution is crushed and sieved by a 80-mesh sieve to obtain active polysaccharide and oligosaccharide.

Example 7: effect of active polysaccharides and oligosaccharides on growth Performance of Tilapia

Tilapia (commercially available) of substantially uniform size was randomly placed into 9 aquaria, averaging 20 fish per aquarium, and the 9 aquaria were divided into groups of 3 replicates each, group 1: basal feed was fed, group 2: feed with 1% of the active polysaccharide and oligosaccharide feed prepared in example 6, group 3: 2% of the feed containing the active polysaccharides and oligosaccharides prepared in example 6 was added to the feed and the feed was fed 2 times a day (the feed level was about 2% of the weight of the fish/time). The test time is 4 weeks, feeding is stopped one day before the test is finished, the test fish is weighed, and the weight gain rate and the bait coefficient are calculated. From the test results shown in table 4, it can be seen that the weight gain rate of the feed added with active oligosaccharide is significantly higher than that of the control group (P <0.05), the addition amount is 2% and is slightly higher than 1%, but the weight gain is not significant.

The weight gain (%) (average weight of fish at end of test-average weight of fish at initial test)/average weight of fish at initial test ] × 100

The bait coefficient is the total weight of ingested feed/(average weight of test end fish-average weight of test initial fish)

TABLE 4 Effect of active polysaccharides and oligosaccharides on growth Performance of Tilapia mossambica

Test group	Initial average weight/g	End average weight/g	Weight gainPercentage (%)	Coefficient of bait
					Group 1 (control)	28.65±1.24	59.63±6.24	108.13^a	2.06±0.14
Group 2 (1%)	28.45±1.34	65.63±7.76	130.69^b	1.72±0.26
					Group 3 (2%)	28.60±1.27	66.46±8.24	132.38^b	1.68±0.18

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> Yunan Boshiao Biotechnology Ltd

SOUTH CHINA AGRICULTURAL University

<120> Paenibacillus mucilaginosus and application thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>1494

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 16S rDNA gene sequence of Paenibacillus kribbensis YB326

<400>1

agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60

ggggttaatt agaagcttgc ttctaattaa cctagcggcg gacgggtgag taacacgtag 120

gcaacctgcc cacaagacag ggataactac cggaaacggt agctaatacc cgatacatcc 180

ttttcctgca tgggagaagg aggaaagacg gagcaatctg tcacttgtgg atgggcctgc 240

ggcgcattag ctagttggtg gggtaaaggc ctaccaaggc gacgatgcgt agccgacctg 300

agagggtgat cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag 360

tagggaatct tccgcaatgg gcgaaagcct gacggagcaa cgccgcgtga gtgatgaagg 420

ttttcggatc gtaaagctct gttgccaggg aagaatgctt gggagagtaa ctgctctcaa 480

ggtgacggta cctgagaaga aagccccggc taactacgtg ccagcagccg cggtagtacg 540

tagggggcaa gcgttgtccg gaattattgg gcgtaaagcg cgcgcaggcg gctctttaag 600

tctggtgttt aatcccgagg ctcaacttcg ggtcgcactg gaaactggag agcttgagtg 660

cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgaagagatg tggaggaaca 720

ccagtggcga aagcgactct ctgggctgta actgacgctg aggcgcgaaa gcgtggggag 780

caaacaggat tagataccct ggtagtccac gccgtaacga tgaatgctag tgttagggtt 840

tcgataccct tggtgccgaa gttacacatt aagcattccg cctggggagt acggtcgcaa 900

gactgaaact caaaggaatt gacggggacc cgcacaagca gtggagtatg tggtttaatt 960

cgaagcaacg cgaagaacct taccaggtct tgacatccct ctgatcggtc tagagataga 1020

tctttccttc gggacagagg agacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag 1080

atgttgggtt aagtcccgca acgagcgcaa cccttatgct tagttgccag caggtgaagc 1140

tgggcactct aagcagactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc 1200

atcatgcccc ttatgacctg ggctacacac gtactacaat ggccggtaca acgggaagcg 1260

aaaccgcgag gtggagcgaa tcctagaaaa gccggtctca gttcggattg taggctgcaa 1320

ctcgcctaca tgaagtcgga attgctagta atcgcggatc agcatgccgc ggtgaatacg 1380

ttcccgggtc ttgtacgcac cgcccgtcac accacgagag tttacaacac ccgaagtcgg 1440

tggggtaacc cgcaagggag ccagccgccg aaagtggggt agatgattgg ggtg 1494

Claims

1. A strain of Paenibacillus mucilaginosus is characterized in that: name isPaenibacillus kribbensisYB326, deposited in No. 59 building No. 5 building Guangdong province microorganism research institute of Mie Zhonglu No. 100 college in Guangzhou city in 2018, 2.6.8, with the deposit number: GDMCC NO: 60326.

2. the use of Paenibacillus mucilaginosus as defined in claim 1 for promoting weight gain of tilapia mossambica.

3. A tilapia mossambica weight increasing agent is characterized in that: fermenting the Paenibacillus mucilaginosus of claim 1, extracting, drying and collecting solid content to obtain the tilapia mossambica weight increasing agent.

4. The method for preparing tilapia mossambica weighting agent according to claim 3, characterized in that it mainly comprises the following steps:

(1) fermentation: selecting strainsPaenibacillus kribbensisInoculating YB326 into a liquid PDA culture medium, and culturing for 16-30 h at 35-37 ℃ to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 2-5%, ventilating at the temperature of 35-37 ℃ and the ventilation speed of 0.5-1.0 vvm, fermenting for 3-4 d at the stirring speed of 100-300 r/min, and collecting a fermentation product;

(2) extraction: and (2) centrifuging the fermentation product obtained in the step (1) to remove thalli, adding 1-2 times of organic solvent in volume to precipitate extracellular active polysaccharide and oligosaccharide, drying and dehydrating the supernatant to obtain the remaining active oligosaccharide, and combining the extracellular active polysaccharide and the active oligosaccharide to obtain the tilapia mossambica weight gain agent.

5. The method for preparing tilapia mossambica weighting agent according to claim 4, characterized in that:

the fermentation medium comprises a carbon source, a nitrogen source, magnesium phosphate and water; the carbon source is one or more of glucose, sucrose and starch hydrolysate, and the dosage is 30-60 g/L; the nitrogen source is one or more of beef extract, peptone, yeast extract and ammonium sulfate, and the dosage is 2-6 g/L; the phosphate is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate and disodium hydrogen phosphate, and the using amount of the phosphate is 2-3 g/L; the magnesium salt is magnesium sulfate and magnesium chloride, and the dosage of the magnesium salt is 0.2-1 g/L respectively; the balance of water;

and (3) drying in the step (2) until the water content is less than 10%.

6. The use of a tilapia weighting agent according to claim 3, characterized in that:

the tilapia weight increasing agent is added according to 1-2% of the total weight of the feed, and is applied to tilapia culture.