CN113621536A

CN113621536A - Paenibacillus polymyxa SP1 and application thereof

Info

Publication number: CN113621536A
Application number: CN202110820349.XA
Authority: CN
Inventors: 林碧敏; 钟杨生; 陈芳艳; 张莹; 严会超; 李家安; 林健荣
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-07-20
Filing date: 2021-07-20
Publication date: 2021-11-09
Anticipated expiration: 2041-07-20
Also published as: CN113621536B

Abstract

The invention discloses paenibacillus polymyxa SP1 and application thereof. The paenibacillus polymyxa SP1 is preserved in Guangdong province microorganism strain collection center in 2021, 4 months and 16 days, and the preservation number is GDMCC No: 61605. the paenibacillus polymyxa SP1 can produce antibacterial peptide, and the fermented supernatant and the precipitate both contain antibacterial substances; has broad-spectrum antibacterial effect, has inhibiting effect on pathogenic bacteria of common livestock and poultry breeding bacteria, breeding insects such as silkworms and the like and common human skin pathogenic bacteria, reduces diseases of breeding insects such as silkworms and the like, is expected to achieve the effect of replacing antibiotics, and can also be applied to the development of human skin pathogenic bacteria inhibiting medicaments and cosmetics. Meanwhile, the strain can also produce lipase, the activity of the lipase is 0.59U/mL, the problem of absorption of cultured livestock and poultry young animals on fat feeds can be solved, the absorption of fat is promoted, the feed utilization rate is improved, the production performance and intestinal health of the livestock and poultry are improved, and a foundation is provided for developing a novel feed additive.

Description

Paenibacillus polymyxa SP1 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to paenibacillus polymyxa and application thereof.

Background

Antibiotics are widely used in animal production as growth promoters and therapeutic drugs to improve resistance to infections. However, with the shortage of effective antibiotics and the occurrence of problems of drug-resistant bacteria generated by using a large amount of antibiotics, great harm is brought to human health, in order to maintain the safety of animal-derived food and public health in China, from 7/1/2020, the complete implementation of livestock and poultry feed is forbidden, and the development of nontoxic and harmless antibiotic substitutes for sustainable production is an urgent need in animal husbandry.

The antibacterial peptide is one of the most promising antibiotic substitutes, can be used as a novel bioactive peptide to replace antibiotics to play a wider antibacterial role, has no or low drug resistance, has the functions of killing cancer cells, inhibiting viruses and the like, and can also play an important role in regulating the infection of an immune system of an organism as an immunomodulator. Therefore, the antibacterial peptide has good application prospect in the fields of animal husbandry, medicine, food, cosmetics and the like.

Paenibacillus polymyxa (Paenibacillus polymyxa) is a G + bacterium for producing spores, is nonpathogenic to human, animals and plants, can produce various bioactive substances such as polypeptide antibiotics and the like, is a biocontrol bacterium with great development prospect and application value, can be widely applied to the fields of industry, medicine, agriculture, food and the like, and is classified as a first-level strain free of safety identification by the department of agriculture in China.

Chinese patent CN110484467B discloses a strain of Bacillus polymyxa for producing antibacterial peptide, the molecular weight of the antibacterial peptide produced by the strain is 5KDa-3KDa (especially antibacterial peptide A3 with the molecular weight of 4 KDa), and the antibacterial peptide has better stability. However, the bacterial inhibition spectrum of the strain is only a few of Escherichia coli, Salmonella gallinarum, Escherichia coli of pigs, Staphylococcus aureus, Salmonella and Staphylococcus aureus of cows; and the bacterial strains are all common bacteria for livestock and poultry breeding, and have no inhibiting application of human skin pathogenic bacteria. In addition, the microorganism bacteria also have the risk of degradation, so that the continuous enrichment of the beneficial bacteria library has important significance.

On the other hand, in the prior art, the antibacterial peptide production activity and the antibacterial activity of bacteria are concerned more, and the problem of adverse effects of fat components in the culture feed is not considered. In the cultivation of poultry and livestock, especially for young animals, the maturity of digestive functions of the young animals and adults are different, and the problem of low survival rate of the young animals of the poultry and the livestock caused by insufficient digestion of fat exists.

Disclosure of Invention

The invention aims to provide a bacterial strain which can produce antibacterial peptide, has a wider antibacterial spectrum and can produce lipase, is particularly suitable for producing livestock and insect breeding feed and is expected to be applied to cosmetics and medicines for inhibiting human skin pathogenic bacteria.

One of the objects of the present invention is to provide a Paenibacillus polymyxa SP 1.

The invention also aims to provide application of the paenibacillus polymyxa SP 1.

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

the paenibacillus polymyxa for producing antibacterial peptide is named as paenibacillus polymyxa SP1, the paenibacillus polymyxa SP1 is preserved in the Guangdong province microorganism strain preservation center, the preservation unit address is the Guangdong province academy of sciences microorganism research institute (No. 59 building 5 of Michelia Torula No. 100 McHeim, Guangzhou), the preservation date is 2021, 4 and 16 days, and the preservation number is GDMCC No: 61605.

further, the paenibacillus polymyxa SP1 producing the antibacterial peptide is a gram-positive rod-shaped bacterium and has oval terminal endospores.

Furthermore, the paenibacillus polymyxa SP1 producing the antibacterial peptide grows on the R2A flat plate for 2 days, and the bacterial colony is circular, convex, smooth in surface, neat in edge, semitransparent and sticky.

Further, the paenibacillus polymyxa SP1 for producing the antibacterial peptide has the 16s rDNA length of 1406bp, and the 16s rDNA sequence is shown as SEQ ID No. 1. The 16s rDNA sequence of the Paenibacillus polymyxa SP1 is shown in the sequence, the 16SrDNA sequence obtained by sequencing is 1406bp, and nucleotide homology comparison is carried out on the sequence and the registered sequence in Genebank by using Blast program, and the homology of the 16SrDNA sequence of the strain and the Paenibacillus polymyxa reaches 99.50 percent; the morphological characteristics and physiological and biochemical characteristics of the strain are most similar to those of Paenibacillus polymyxa.

Furthermore, the paenibacillus polymyxa SP1 producing the antibacterial peptide can grow on a YEPD culture medium, the optimal growth temperature is 30 ℃, and the pH value of the optimal growth environment is 6.0-6.5.

Further, the method comprisesThe test of the paenibacillus polymyxa SP1 and the paenibacillus polymyxa SP1 oxidase for producing the antibacterial peptide is negative, and H is₂The S production test is negative, the citrate utilization test is negative, the indole test is negative, and the urease test is negative. The anaerobic growth test is positive, the catalase test is positive, the glucose fermentation test is positive, the nitrate reduction test is positive, the arabinose fermentation test is positive, the mannitol fermentation test is positive, the xylose fermentation test is positive, the glycerol fermentation test is positive, the VP test is positive, the casein hydrolysis test is positive, and the starch hydrolysis test is positive.

The paenibacillus polymyxa SP1 can be fermented to generate antibacterial peptide, and provides a basis for replacing antibiotics.

Furthermore, the fermentation product of the paenibacillus polymyxa SP1 producing the antibacterial peptide has high broad-spectrum antibacterial action and has antibacterial and inhibitory effects on staphylococcus aureus, escherichia coli K12D31, escherichia coli O78, salmonella typhimurium, salmonella choleraesuis, bacillus thuringiensis, septicemia pleuropterus, klebsiella pneumoniae, propionibacterium acnes, staphylococcus epidermidis, gypseous sporophytes and sphagnum rubrum.

Furthermore, the fermentation product of the paenibacillus polymyxa SP1 for producing the antibacterial peptide has higher lipase activity, and the lipase activity is 0.59U/mL.

Furthermore, the paenibacillus polymyxa SP1 producing the antibacterial peptide and the fermentation product thereof have high thermal stability and can be applied to high-temperature fermentation in production.

Therefore, the following applications should be within the scope of the present invention:

the paenibacillus polymyxa SP1 producing the antibacterial peptide is applied to the production of the antibacterial peptide and/or lipase.

The application of the paenibacillus polymyxa SP1 in preparing antibacterial peptide and/or lipase inoculants.

The paenibacillus polymyxa SP1 is applied to the preparation of bacteriostatic agents.

The application of the paenibacillus polymyxa SP1 in producing feed. Optionally fermenting to produce bacteriostatic feed.

The paenibacillus polymyxa SP1 is applied to the production of skin bacteriostatic drugs.

The Paenibacillus polymyxa SP1 is applied to the production of acne-removing cosmetics.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a paenibacillus polymyxa SP 1:

(1) the bacterium can produce antibacterial peptide; provides basis for replacing antibiotics.

(2) The strain has high and broad-spectrum antibacterial ability, has inhibiting effect on common livestock and poultry intestinal bacteria pathogenic bacteria, and also has inhibiting effect on common breeding pathogenic bacteria of insects such as silkworms and the like; it also has inhibitory effect on pathogenic bacteria of common skin problems of human.

(3) The strain can produce lipase, and the activity of producing lipase by fermentation is 0.59U/mL; the addition of fat substances in the feed can be reduced, the cost is saved, and a foundation is provided for developing a novel feed additive. The additive can solve the problem of absorption of the bred young livestock and poultry to the fat feed, reduce indigestion and diarrhea caused by overhigh addition amount of the grease, promote fat absorption, improve the feed utilization rate, improve the production performance of the livestock and poultry, improve the intestinal health and provide a foundation for developing a novel feed additive.

(4) The strain and the fermentation product thereof have strong thermal stability and good application prospect.

(5) The strain can be cultured in liquid and solid states, and both culture modes can produce good antibacterial effect, and the fermented supernatant and precipitate both contain antibacterial substances.

Drawings

FIG. 1 shows the colony morphology of Paenibacillus polymyxa SP 1.

FIG. 2 shows a phylogenetic tree constructed by homology alignment of the 16s rDNA sequences of Paenibacillus polymyxa SP1 and a similar strain.

Detailed Description

The invention is further described in the following description with reference to the figures and specific examples, which should not be construed as limiting the invention. It is within the scope of the present invention to make simple modifications or alterations to the methods, procedures or conditions of the present invention without departing from the spirit and substance of the invention; unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

EXAMPLE 1 isolation and screening of the strains

1. Selecting a culture medium:

the LB medium includes LB liquid medium and LB solid medium.

LB liquid medium (1000mL): 10g/L of peptone, 5g/L of yeast extract and 10g/L of sodium chloride, diluting to 1L with distilled water, adjusting pH to 6.0-7.0, and autoclaving at 121 ℃ for 20 min.

LB solid medium (1000mL): 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride and 1.5% agar powder, wherein the volume is determined to be 1L by using distilled water, the pH value is 6.0-7.0, and the agar powder is sterilized at 121 ℃ for 20min under high pressure.

2. Strain isolation and screening:

the original source of the strain is separated from soil of a mulberry field of southern China agricultural university, an agar hole diffusion method is adopted, escherichia coli and staphylococcus aureus are used as indicator bacteria, a sterilized LB culture medium is placed at room temperature and cooled to 48-50 ℃, 100 mu L of indicator bacteria diluent is sucked and added into 10mL of LB solid culture medium which is dissolved and cooled to 40-50 ℃, the LB solid culture medium is immediately poured into a culture dish with the diameter of 90mm, the culture dish is quickly shaken up by shaking to uniformly disperse the bacteria, and after the bacteria are cooled and solidified, a hole puncher with the diameter of 2.7mm is used for punching for standby application. Adding 5uL of paenibacillus polymyxa with uncertain bacteriostatic activity into each hole, standing for 30min, culturing at 37 ℃ for 24h, observing and measuring the size of a bacteriostatic zone, and recording data. And obtaining a strain with the highest bacteriostatic activity by comparison and screening of bacteriostatic activity, and marking the strain as a strain SP 1.

Example 2 identification of Strain SP1 and measurement of physiological and biochemical indices

1. And (3) identifying colony morphology of the strain:

the strain SP1 is a gram-positive rod-shaped bacterium and has elliptic terminal endophyte spores. The colonies grow on the R2A plate for 2 days, and are round, convex, smooth in surface, neat in edge, translucent and sticky (as shown in FIG. 1).

2. Physiological and biochemical identification

The physiological and biochemical identification of the SP1 strain was carried out with reference to Bergey's Manual of bacteria identification (ninth edition) and Manual of identification of common bacteria systems (scientific Press, King of Dongxu bead): strain SP1 oxidase test is negative, H₂The S production test is negative, the citrate utilization test is negative, the indole test is negative, and the urease test is negative. The anaerobic growth test is positive, the catalase test is positive, the glucose fermentation test is positive, the nitrate reduction test is positive, the arabinose fermentation test is positive, the mannitol fermentation test is positive, the xylose fermentation test is positive, the glycerol fermentation test is positive, the VP test is positive, the casein hydrolysis test is positive, and the starch hydrolysis test is positive.

3. Molecular characterization- -sequencing of Strain 16S rDNA

The screened strain SP1 was subjected to molecular identification, and the 16S rDNA of the screened SP1 was sequenced by requesting the Guangdong provincial microbiological analysis and detection center.

The 16SrDNA sequence of the strain SP1 is 1406bp, the 16s rDNA sequence is shown in SEQ ID No.1, a Blast program is used for carrying out nucleotide homology comparison with registered sequences in Genebank to construct a phylogenetic tree (figure 2), and the 16SrDNA sequence of the strain has 99.50 percent of homology with Paenibacillus polymyxa; the morphological characteristics and physiological and biochemical characteristics of the strain are most similar to those of Paenibacillus polymyxa.

Therefore, in summary of the identification results, the identified strain SP1 belongs to Paenibacillus polymyxa (Paenibacillus polymyxa), and is deposited at 16.4.2021 in the collection center of microbial cultures in Guangdong province, where the collection unit address is the institute of microbiology of the academy of sciences in Guangdong province (building 5 of Middy 100 college 59, Guangzhou city), and the collection number is GDMCC No: 61605.

EXAMPLE 3 determination of optimum temperature and optimum pH of Strain SP1

The YEPD culture medium comprises a YEPD liquid culture medium and a YEPD liquid culture medium, and the formula of the YEPD culture medium is as follows:

YEPD liquid medium (1000mL) containing peptone 20g/L, yeast extract 10g/L, and glucose 20g/L, adding distilled water to a volume of 1L, adjusting pH to 6-7, and autoclaving at 115 deg.C for 20 min.

YEPD solid culture medium (1000mL) comprises peptone 20g/L, yeast extract 10g/L, glucose 20g/L, 1.5% agar powder, distilled water to 1L, pH 6-7, and autoclaving at 115 deg.C for 20 min.

Respectively taking 7 groups of YEPD liquid culture media, and setting the culture temperature gradients to be 25 ℃ respectively; 30 ℃; 35 ℃; culturing in a constant temperature shaking table at 180r/min at 40 ℃ for 24h to obtain a bacterial liquid. The optimal temperature of 30 ℃ is obtained by counting the density of bacteria in the bacteria liquid after fermentation

Respectively taking 8 groups of YEPD liquid culture media, regulating the alkalinity of cultured amino acid by using hydrochloric acid and sodium hydroxide, and setting the pH value gradient to be 2; pH 4; pH 6; pH 7; pH 8; pH 10; pH 12; and (3) culturing the mixture for 24 hours in a constant temperature shaking table at 180r/min at the temperature of 30 ℃ under the condition that the pH value is 14 to obtain a bacterial liquid. And counting the density of bacteria in the fermented bacteria liquid to obtain the optimal pH value of 6-7.

Therefore, the paenibacillus polymyxa SP1 can grow on a YEPD culture medium, the optimal growth temperature is 30 ℃, and the pH value of the optimal growth environment is 6-7.

EXAMPLE 4 determination of Lipase-producing Activity of Strain SP1

1. Purpose of the experiment:

and (3) detecting whether the metabolite of the paenibacillus polymyxa SP1 contains lipase or not so as to facilitate wider application in animal husbandry.

The lipase is added into the feed, so that the digestion utilization rate of the animal daily ration grease can be improved, the deficiency of the endogenous digestive enzyme activity and the secretion of young animals caused by the fact that the digestive function is not developed well is supplemented, the dyspepsia and diarrhea caused by the fact that the addition amount of the grease is too high are reduced, the grease dosage is reduced, and the feed cost is reduced. In addition, in insect breeding, the insects have high fat demand, and the additive rich in lipase can promote the absorption of the insects to the fat.

2. The experimental steps are as follows:

(1) activation of paenibacillus polymyxa SP 1: aseptically handling in a clean bench, a certain amount of Paenibacillus polymyxa SP1 was picked up with an inoculating loop and streaked onto YEPD solid medium. And (3) carrying out inversion culture at the constant temperature of 30 ℃ for 12-24 hours until a single colony visible to the naked eye exists.

(2) Preparation of a paenibacillus polymyxa SP1 bacterial liquid: inoculating the activated single colony of Paenibacillus polymyxa SP1 in 5-6 mL YEPD liquid culture medium, and culturing in a constant temperature shaking table at 30 ℃ and 180r/min for 24h to serve as seed bacterial liquid.

(3) Drawing an enzyme activity standard curve: 0.08346g of p-nitrophenol is weighed, dissolved by a small amount of 95% ethanol, and then is made to be 100mL by water until the concentration is 6mmol. Different amounts of p-nitrophenol solution and 50mmo/L Tris-HCl (pH8.0) buffer were added to the reaction mixture, respectively, in a total volume of 2 mL. The concentration of p-nitrophenol in the system was set to 0. mu. mol/L, 5. mu. mol/L, 10. mu. mol/L, 15. mu. mol/L, 20. mu. mol/L, 30. mu. mol/L, 40. mu. mol/L, 60. mu. mol/L, 80. mu. mol/L, 100. mu. mol/L. Then 0.5mL 10% trichloroacetic acid was added to each tube, followed by 0.5mL 10% Na2CO3 solution in a total volume of 3mL, and the light absorption was measured at 410 mm. Then, a standard curve of the enzyme activity was plotted.

(4) Substrate solution A, 90mg of p-nitrophenyl palmitate (p-NPP), 30mL of isopropanol, and buffer solution B, 50mmol/LTriscl (pH 8.0). Take 4 numbered tubes, 1 tube as blank control, and the other 3 tubes as reaction tubes. Adding the solution B1.8mL and the substrate solution A0.2 mL into four test tubes respectively, preserving the heat in a water bath at 37 ℃ for 5min, adding the YEPD culture medium 1mL into a control tube, adding the paenibacillus polymyxa SP1 bacterial liquid 1mL into a sample tube, immediately mixing uniformly, timing, adding the trichloroacetic acid solution with the concentration of 0.5mL 10% after accurately reacting in the water bath for 10min to terminate the reaction, and adding the Na solution with the concentration of 0.5mL 10% into the sample tube₂CO₃And the solution develops color. And measuring the absorption value of the p-nitrophenol generated by the enzyme catalysis under a 410nm spectrophotometer. Lipase 1 enzyme activity unit definition: the amount of enzyme that released 1. mu. mol of p-nitrophenol per minute at pH8.0 and 37 ℃ was defined as 1 lipase activity unit (U).

Experimental results show that the activity of the lipase produced by fermentation of the Paenibacillus polymyxa SP1 is 0.59U/mL, and the fermentation of the Paenibacillus polymyxa SP1 has higher lipase output and activity.

Example 5 bacteriostatic Effect assay

1. Selection of pathogens

Staphylococcus aureus: gram-positive bacteria are common food-borne pathogenic microorganisms, have wide distribution range, can generate enterotoxin and have different degrees of harm to livestock and poultry breeding and human beings.

Escherichia coli K12D31, Escherichia coli O78: common intestinal gram-negative bacteria of livestock and poultry infect the intestinal tracts of the livestock and poultry, can cause acute diarrhea and is not beneficial to the health of the livestock and poultry.

Salmonella typhimurium: it is present in the intestinal tract of various animals such as poultry, livestock and rats, and is one of the main pathogenic bacteria causing acute gastroenteritis and typhoid fever.

Salmonella choleraesuis: is a main pathogenic bacterium causing paratyphoid of piglets and causes great harm to the pig industry.

Bacillus thuringiensis: the most widespread microbial insecticides. The principle of the bacillus thuringiensis for preventing insects is that the strain can produce two toxins of endotoxin (parasporal crystal) and exotoxin to stop pests from eating, the pests die due to hunger, blood destruction and nerve poisoning, and the bacillus thuringiensis has high toxicity to various silkworms and can cause the damping-off disease of the silkworms.

B, bacteriostasis of septicemia melanothorax: bacillus bombycis (Bb) is a pathogenic bacterium of silkworm (Bombyx mori), has high toxicity to various silkworms, and can cause septicemia of the silkworms.

Klebsiella pneumoniae: klebsiella pneumoniae is present in the upper respiratory tract and intestinal tract of a human body, causing serious infections in humans, including pneumonia, urinary tract infections and blood infections.

Propionibacterium acnes: it is usually inhabited in the hair follicles and sebaceous glands of the skin and is the main bacterium causing acne.

Staphylococcus epidermidis: parasitizing on the skin, vagina, etc. of human body can cause diseases such as chronic inflammatory dermatosis and urinary system infection, etc.

Gypsum-like spore bacteria: belongs to the genus microsporo and can cause a series of fungal infection diseases such as skin tinea of human and animals.

Sphagnum rubrum: is a human-derived dermatophyte, and causes common skin superficial mycosis, such as tinea manuum, tinea pedis, tinea capitis and the like.

2. Experiment implementation:

(1) and detecting the size of the inhibition zone by an agar hole diffusion method. The method is briefly described as follows: placing the sterilized LB culture medium at room temperature, cooling to 48-50 ℃, sucking 100 mu L of indicator bacterium diluent, and respectively taking the indicator bacterium: staphylococcus aureus, Escherichia coli K12D31, Escherichia coli O78, Salmonella typhimurium, Salmonella choleraesuis, Bacillus thuringiensis and Patrinia scabiosa.

Adding the indicator bacterium diluent into 10mL of LB solid culture medium which is dissolved and cooled to 40-50 ℃, immediately pouring the indicator bacterium diluent into a culture dish with the diameter of 90mm, quickly shaking and shaking up to uniformly disperse the thalli, and punching a hole with a hole puncher with the diameter of 2.7mm after the indicator bacterium diluent is cooled and solidified for later use. 5uL of paenibacillus polymyxa SP1 is added into each hole, after standing for 30min, the mixture is cultured for 24h at 37 ℃, the size of a bacteriostatic circle is observed and measured, and data are recorded.

(2) A filter paper sheet method: taking a certain amount of propionibacterium acnes, adding a proper amount of sterile water for dilution, fully and uniformly mixing, sucking 100 mu L of the mixture, and uniformly coating. Placing a sterile filter paper sheet with the diameter of 5mm in the center of a culture dish, dropwise adding 5 mu L of Paenibacillus polymyxa SP1, standing for 15min, inversely culturing for 48h in an anaerobic environment at 37 ℃, observing and measuring the size of a bacteriostatic zone, and recording data.

Adding Staphylococcus epidermidis and Klebsiella pneumoniae serving as indicator bacteria into an LB solid culture medium, cooling and solidifying, and then carrying out the steps.

(3) Growth rate assay: adding 100 μ L of Paenibacillus polymyxa SP1 on the surface of PDA plate, coating uniformly, inoculating fungal stipe with diameter of 5mm in the center of the plate after absorption, wherein the indicator bacteria are Gypsum Fibrosum-like spore bacteria and Gypsum Fibrosum-like spore bacteria, and using equal amount of sterile deionized water as control. And (3) carrying out inverted culture for 5-7 days in a constant temperature incubator at 25-28 ℃, wherein the growth condition is observed every 24 hours, and the size of the colony is observed and recorded. The inhibitory effects of paenibacillus polymyxa SP1 on the hyphae of two fungi were compared, and the inhibition ratio (Ir) was calculated.

Ir ═ [ (CK bacterial disc average diameter-treated bacterial disc average diameter)/CK bacterial disc average diameter ] × 100%

3. Results of the experiment

The experimental results show (as in table 1): the fermentation product of the paenibacillus polymyxa SP1 has a high broad-spectrum antibacterial effect, has an inhibiting effect on common livestock and poultry intestinal bacteria pathogenic bacteria, has an inhibiting effect on common insect breeding pathogenic bacteria, and has an inhibiting effect on common skin problem pathogenic bacteria.

TABLE 1 Paenibacillus polymyxa SP1 solid Medium bacteriostasis test

Pathogenic bacteria	Diameter of inhibition zone (inhibition rate)
		Staphylococcus aureus	16.45mm
Escherichia coli K12D31	9.28mm
		Escherichia coli O78	10.73mm
Salmonella typhimurium	9.83mm
		Salmonella choleraesuis	9.30mm
Bacillus thuringiensis	11.5mm
		Septicemia latrine (Fr.) Kuntze	9.00mm
Klebsiella pneumoniae	8.15mm
		Propionibacterium acnes	7.98mm
Staphylococcus epidermidis	18.41mm
		Gypsum spore bacteria	90.38％
Trichophyton rubrum	72％

EXAMPLE 6 Strain SP1 thermostability assay

And (3) placing the sterilized LB culture medium at room temperature, cooling to 48-50 ℃, sucking 100 mu L of indicator bacterium diluent, and taking staphylococcus aureus and escherichia coli K12D31 as indicator bacteria. Adding the indicator bacterium diluent into 10mL of LB solid culture medium which is dissolved and cooled to 40-50 ℃, immediately pouring the indicator bacterium diluent into a culture dish with the diameter of 90mm, quickly shaking and shaking up to uniformly disperse the thalli, and punching a hole with a hole puncher with the diameter of 2.7mm after the indicator bacterium diluent is cooled and solidified for later use. Heating Paenibacillus polymyxa SP1 in water bath at 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C, and 100 deg.C for 10 min. And (3) taking the bacteria liquid which is not subjected to heating treatment as a blank control, adding 5uL of paenibacillus polymyxa SP1 into each hole, standing for 30min, culturing at 37 ℃ for 24h, observing and measuring the size of a bacteriostatic circle, and recording data.

The diameter of the zone of inhibition for staphylococcus aureus is shown in table 2:

TABLE 2 bacteriostatic test of strain SP1 on Staphylococcus aureus and Escherichia coli K12D31 at different temperatures

Therefore, the fermentation product of the paenibacillus polymyxa SP1 has high thermal stability. The test result shows that: the paenibacillus polymyxa SP1 is strong in heat stability.

EXAMPLE 7 determination of the thermal stability of the supernatant and of the precipitate

And (3) centrifuging the fermentation liquor of the paenibacillus polymyxa SP1, heating the supernatant and the thallus precipitate in water baths at 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ and 100 ℃ for 10min respectively, taking the unheated bacteria liquid as a blank control, adopting an agar hole diffusion method and the experiment six, observing and measuring the size of the inhibition zone, and recording data.

TABLE 3 bacteriostatic test of supernatant of strain SP1 on Staphylococcus aureus and Escherichia coli K12D31 at different temperatures

TABLE 4 bacteriostasis test of bacterial precipitation of strain SP1 on Staphylococcus aureus and Escherichia coli K12D31 at different temperatures

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> southern China university of agriculture

<120> Paenibacillus polymyxa SP1 and application thereof

<130>

<160> 1

<170> PatentIn version 3.3

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<211> 1406

<212> DNA

<213> rDNA sequence of Paenibacillus polymyxa SP1

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ttcatgtagg cgagttgcag cctacaatcc gaactgagac cggcttttct aggattggct 180

ccacctcgct ccttcgcttc ccgttgtacc ggccattgta gtacgtgtgt agcccaggtc 240

ataaggggca tgatgatttg acgtcatccc caccttcctc cggtttgtca ccggcagtct 300

gcttagagtg cccagcttga cctgctggca actaagcata agggttgcgc tcgttgcggg 360

acttaaccca acatctcacg acacgagctg acgacaacca tgcaccacct gtctcctctg 420

tcccgaagga aagatctatc tctagaccgg tcaaagggat gtcaagacct ggtaaggttc 480

ttcgcgttgc ttcgaattaa accacatact ccactgcttg tgcgggtccc cgtcaattcc 540

tttgagtttc agtcttgcga ccgtactccc caggcggaat gcttaatgtg ttaacttcgg 600

caccaagggt atcgaaaccc ctaacaccta gcattcatcg tttacggcgt ggactaccag 660

ggtatctaat cctgtttgct ccccacgctt tcgcgcctca gcgtcagtta cagcccagag 720

agtcgccttc gccactggtg ttcctccaca tctctacgca tttcaccgct acacgtggaa 780

ttccactctc ctcttctgca ctcaagctcc ccagtttcca gtgcgacccg aagttgagcc 840

tcgggattaa acaccagact taaagagccg cctgcgcgcg ctttacgccc aataattccg 900

gacaacgctt gccccctacg tattaccgcg gctgctggca cgtagttagc cggggctttc 960

ttctcaggta ccgtcacttc aagagcagtt actctaccaa gcgttcttcc ctggcaacag 1020

agctttacga tccgaaaacc ttcatcactc acgcggcgtt gctccgtcag gctttcgccc 1080

attgcggaag attccctact gctgcctccc gtaggagtct gggccgtgtc tcagtcccag 1140

tgtggccgat caccctctca ggtcggctac gcatcgtcgc cttggtaggc ctttacccca 1200

ccaactagct aatgcgccgc aggcccatcc acaagtgaca gattgctccg cctttcctcc 1260

ttctcccatg caggaaaagg atgtatcggg tattagctac cgtttccggt agttatccct 1320

gtcttgtggg caggttgcct acgtgttact cacccgtccg ccgctaggtt agttagaagc 1380

aagcttctaa ttaaccccgc tcgact 1406

Claims

1. Paenibacillus polymyxa (Paenibacillus polymyxa) SP1, wherein the Paenibacillus polymyxa SP1 is deposited at the Guangdong province culture Collection center at 2021, 4 and 16 days, and the deposit number is GDMCC No: 61605.

2. the use of Paenibacillus polymyxa SP1 as claimed in claim 1 for bacteriostasis.

3. Use of the paenibacillus polymyxa SP1 of claim 1 for the preparation of a bacteriostatic agent.

4. The use according to claim 2 or 3, wherein the bacterial inhibition profile comprises one or more of Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Salmonella choleraesuis, Bacillus thuringiensis, Patrinia scabiosa, Klebsiella pneumoniae, Propionibacterium acnes, Staphylococcus epidermidis, Sphaerotheca gypseum, or Trichophyton rubrum.

5. Use of paenibacillus polymyxa SP1 according to claim 1 for the production of antimicrobial peptides and/or lipases.

6. Use of the Paenibacillus polymyxa SP1 as claimed in claim 1 in the preparation of a microbial inoculum for producing antimicrobial peptides and/or lipases.

7. Use of the Paenibacillus polymyxa SP1 of claim 1 for the preparation of feed.

8. Use of paenibacillus polymyxa SP1 according to claim 1 in the fermentative production of bacteriostatic feeds.

9. Use of the paenibacillus polymyxa SP1 of claim 1 in the preparation of a skin bacteriostatic cosmetic additive or in the preparation of a skin bacteriostatic cosmetic.

10. Use of the paenibacillus polymyxa SP1 of claim 1 in the preparation of a skin bacteriostatic medicament.