CN113621536B - Paenibacillus polymyxa SP1 and application thereof - Google Patents
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Abstract
The invention discloses paenibacillus polymyxa SP1 and application thereof. The Paenibacillus polymyxa SP1 is deposited at the microorganism strain collection of Guangdong province at 2021, 4 months and 16 days, and the deposit number is GDMCC No:61605. the Paenibacillus polymyxa SP1 can produce antibacterial peptide, and the supernatant and the sediment after fermentation both contain antibacterial substances; the antibacterial agent has broad-spectrum antibacterial effect, has inhibition effect on common livestock and poultry breeding pathogens, breeding insect pathogens and the like and common human skin pathogens, 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 development of human skin pathogens inhibition drugs and cosmetics. Meanwhile, the strain can also produce lipase, the activity of the lipase is 0.59U/mL, the problem of fat feed absorption of young bred livestock and poultry can be solved, the fat absorption is promoted, the feed utilization rate is improved, the livestock and poultry production performance and intestinal health are improved, and a foundation is provided for developing a novel feed additive.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to paenibacillus polymyxa and application thereof.
Background
Antibiotics are widely used in animal production as growth promoters and therapeutic drugs to improve resistance to infectious diseases. However, with the lack of effective antibiotics and the occurrence of problems such as drug-resistant bacteria caused by using a large amount of antibiotics, great harm is brought to human health, and in order to maintain animal-derived food safety and public health safety in China, livestock and poultry feed is forbidden to be comprehensively implemented in the beginning of 7 months of 2020, and development of antibiotic substitutes for non-toxic harmless sustainable production has become an urgent need for animal husbandry.
The antibacterial peptide is one of the most promising antibiotic substitutes, can replace antibiotics to play a broader antibacterial role, has no or low drug resistance, has the functions of killing cancer cells, inhibiting viruses and the like, and can play an important role in regulating the infection of the immune system of an organism as an immunomodulator. Therefore, the antibacterial peptide has good application prospect in the fields of livestock, medicine, food, cosmetics and the like.
Paenibacillus polymyxa (Paenibacillus polymyxa) is a G+ bacterium producing spores, is non-pathogenic to human beings, animals and plants, can produce various bioactive substances such as polypeptide antibiotics, is a type of 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-class strain free from safety identification by the agricultural department of China.
Chinese patent CN110484467B discloses a strain of bacillus polymyxa for producing antibacterial peptide, which has the molecular weight of 5kDa-3kDa (especially antibacterial peptide A3 with molecular weight of 4 kDa) and better stability. However, the bacteria have bacteriostasis spectrums of only a few of chicken escherichia coli, chicken salmonella typhi, pig escherichia coli, staphylococcus aureus, salmonella and bovine staphylococcus aureus; and the bacteria are common bacteria for livestock and poultry cultivation, and the application of the bacteria in inhibiting human skin pathogenic bacteria is avoided. In addition, microbial bacteria are also at risk of degradation, so that continuous enrichment of the beneficial bacteria library is of great importance.
On the other hand, the antibacterial peptide production activity and the antibacterial activity of bacteria are mainly focused in the prior art, and the problem of adverse effects of fat components in the culture feed is not considered. In livestock and poultry cultivation, especially for young animals, the young animals have different maturity degrees of digestive functions, and the problem of low survival rate of the young animals caused by insufficient digestion of fat is caused.
Disclosure of Invention
The invention aims to provide a 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.
The invention aims at providing paenibacillus polymyxa SP1.
Another object of the present invention is to provide the use of said Paenibacillus polymyxa SP1.
The invention aims at realizing the following technical scheme:
the Paenibacillus polymyxa for producing the antibacterial peptide is named as Paenibacillus polymyxa SP1, wherein the Paenibacillus polymyxa SP1 is deposited in the microorganism strain collection center of Guangdong province, the deposit unit address is the institute of microorganisms (building 5 No. 59 of the university of Mitsui 100 of Guangzhou City), the deposit date is 2021, the month 16 is the deposit number is GDMCC No:61605.
furthermore, the antibacterial peptide-producing Paenibacillus polymyxa SP1 is a gram-positive rod-shaped bacterium and has elliptic end-growing endophytic spores.
Furthermore, the antibacterial peptide-producing Paenibacillus polymyxa SP1 grows on an R2A flat plate for 2 days, and the bacterial colony is round, convex, smooth in surface, neat in edge, semitransparent and sticky.
Furthermore, the length of 16s rDNA of the Paenibacillus polymyxa SP1 for producing the antibacterial peptide is 1406bp, and the sequence of 16s rDNA is shown as SEQ ID No. 1. The 16s rDNA sequence of the bacillus polymyxa (Paenibacillus polymyxa) SP1 is 1406bp, the nucleotide homology between the 16s rDNA sequence of the bacillus polymyxa and the registered sequence is compared in Genebank by using Blast program, and the homology between the 16s rDNA sequence of the bacillus polymyxa and Paenibacillus polymyxa (bacillus polymyxa) reaches 99.50%; the morphological and physiological characteristics are most similar to those of Paenibacillus polymyxa (Paenibacillus polymyxa).
Furthermore, the antibacterial peptide-producing 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 6.0-6.5.
Furthermore, the antibacterial peptide-producing paenibacillus polymyxa SP1, the paenibacillus polymyxa SP1 oxidase test is negative, H 2 The S-producing test was negative, the citrate utilizing test was negative, the indole test was negative, and the urease test was negative. The anaerobic growth test is positive, the contact enzyme 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 ferment to generate antibacterial peptide, and provides basis for replacing antibiotics.
Furthermore, the fermentation product of the paenibacillus polymyxa SP1 for producing the antibacterial peptide has a high broad-spectrum antibacterial effect, and has an antibacterial inhibition effect on staphylococcus aureus, escherichia coli K12D31, escherichia coli O78, salmonella typhimurium, salmonella choleraesuis, bacillus thuringiensis, septicemia nigricans, klebsiella pneumoniae, propionibacterium acnes, staphylococcus epidermidis, gypsum-like spore bacteria and trichoderma rubrum.
Furthermore, the fermentation product of the antibacterial peptide-producing paenibacillus polymyxa SP1 has higher lipase activity, and the lipase activity is 0.59U/mL.
Furthermore, the antibacterial peptide-producing Paenibacillus polymyxa SP1 and the fermentation product thereof have high thermal stability, and can be applied to high-temperature fermentation in production.
Thus, the following applications are within the scope of the present invention:
the Paenibacillus polymyxa SP1 for producing the antibacterial peptide is applied to the production of the antibacterial peptide and/or lipase.
The paenibacillus polymyxa SP1 is applied to the preparation of antibacterial peptide and/or lipase bacterial agents.
The paenibacillus polymyxa SP1 is applied to the preparation of a bacteriostat.
The paenibacillus polymyxa SP1 is applied to the production of feed. Optionally fermenting to produce antibacterial feed.
The paenibacillus polymyxa SP1 is applied to the production of skin antibacterial 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 SP1:
(1) The bacteria can produce antibacterial peptide; provides basis for replacing antibiotics.
(2) The strain has high and broad-spectrum antibacterial capability, has an inhibition effect on pathogenic bacteria of common livestock and poultry intestinal bacteria, and also has an inhibition effect on common pathogenic bacteria of breeding insects such as silkworms and the like; it also has inhibitory effect on pathogenic bacteria of common skin problems.
(3) The strain can produce lipase, and the activity of the lipase produced by fermentation is 0.59U/mL; can also reduce the addition of fat substances in the feed, save the cost and provide a basis for developing novel feed additives. Can solve the problem of absorbing fat feed by young livestock and poultry, reduce dyspepsia and diarrhea caused by too high addition amount of grease, promote fat absorption, improve feed utilization rate, improve livestock and poultry productivity, improve intestinal health, and provide a basis for developing novel feed additives.
(4) The bacterium and the fermentation product thereof have strong thermal stability and good application prospect.
(5) The bacteria can be cultured in liquid state and solid state, both culture modes can produce good antibacterial effect, and the supernatant and the sediment after fermentation contain antibacterial substances.
Drawings
FIG. 1 shows the colony morphology of Paenibacillus polymyxa SP1.
FIG. 2 is a phylogenetic tree constructed by homology alignment of Paenibacillus polymyxa SP1 and 16s rDNA sequences of similar strains.
Detailed Description
The present invention will be further described with reference to the drawings and specific examples in the following, but should not be construed as limiting the invention. Simple modifications and substitutions of the method, steps or conditions of the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention; the technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
EXAMPLE 1 isolation and screening of strains
1. Selecting a culture medium:
LB medium includes LB liquid medium and LB solid medium.
LB liquid medium (1000 mL): 10g/L peptone, 5g/L yeast extract and 10g/L sodium chloride, and sterilizing at 121deg.C for 20min under high pressure with distilled water to 1L pH of 6.0-7.0.
LB solid medium (1000 mL): 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 1.5% agar powder, and distilled water to 1L, pH 6.0-7.0, and sterilizing at 121deg.C for 20min.
2. Strain isolation and screening:
the original source of the strain is separated from soil of a mulberry field of agricultural university of south China, 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, when the LB culture medium is cooled to 48-50 ℃, 100 mu L of indicator bacteria diluent is sucked, the indicator bacteria diluent is added into 10mL of LB solid culture medium which is dissolved and cooled to 40-50 ℃, the indicator bacteria diluent is immediately poured into a culture dish with the diameter of 90mm, and the culture dish is rapidly vibrated and uniformly shaken to uniformly disperse thalli, and after the indicator bacteria are cooled and solidified, a puncher with the diameter of 2.7mm is used for punching for standby. 5uL of Paenibacillus polymyxa with uncertain antibacterial activity is added into each hole, the mixture is kept stand for 30min, then is cultured for 24h at 37 ℃, and the size of a bacteriostasis zone is observed and measured, and data are recorded. The strain with the highest antibacterial activity is obtained through comparative screening of antibacterial activity and is marked as strain SP1.
Example 2 identification of Strain SP1 and determination of physiological and Biochemical indicators
1. Identification of bacterial colony morphology:
the strain SP1 is a gram-positive rod-shaped bacterium with oval end-growing endophytic spores. The colony was round, convex, smooth in surface, clean in edge, translucent and viscous (as in FIG. 1) and grown on the R2A plate for 2 days.
2. Physiological and biochemical identification
Physiological and biochemical identification of SP1 strain was performed with reference to the ninth edition of the berjie bacteria identification handbook and the common bacteria systems identification handbook (scientific press of the main code of eastern Xiuzhu): strain SP1 oxidase test negative, H 2 The S-producing test was negative, the citrate utilizing test was negative, the indole test was negative, and the urease test was negative. The anaerobic growth test is positive, the contact enzyme 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
Molecular identification was performed on the strain SP1 obtained by screening, and 16S rDNA of the strain SP1 obtained by screening was sequenced by entrusting the microorganism analysis and detection center in guangdong province.
The 16SrDNA sequence of the strain SP1 is 1406bp, the 16s rDNA sequence is shown as SEQ ID No.1, nucleotide homology comparison is carried out on the sequence in Genebank by using Blast program, a phylogenetic tree (figure 2) is constructed, and the homology of the 16SrDNA sequence of the strain with Paenibacillus polymyxa (Paenibacillus polymyxa) reaches 99.50%; the morphological and physiological characteristics are most similar to those of Paenibacillus polymyxa (Paenibacillus polymyxa).
Thus, in summary of the identification results, the identified strain SP1 belongs to paenibacillus polymyxa (Paenibacillus polymyxa) and was deposited at the collection of microorganism strains in the cantonese province on 4 months 16 days 2021, with the deposit unit address being the institute of microorganisms (building 5, 100. Mouth. 100. University, pioneer, guangzhou city) with the deposit number GDMCC No:61605.
example 3 determination of optimal temperature and optimal pH for Strain SP1
The YEPD medium comprises a YEPD liquid medium and a YEPD liquid medium, and the formula is as follows:
YEPD liquid culture medium (1000 mL) comprising peptone 20g/L, yeast extract 10g/L, glucose 20g/L, distilled water to 1L, pH=6-7, and autoclaving at 115℃for 20min.
YEPD solid culture medium (1000 mL) comprising 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℃for 20min.
Respectively taking 7 groups of YEPD liquid culture mediums, and setting the culture temperature gradients to be 25 ℃;30 ℃;35 ℃; culturing in a shaking table at a constant temperature of 180r/min at 40 ℃ for 24 hours to obtain bacterial liquid. Counting the bacterial density in the bacterial liquid after fermentation to obtain the optimal temperature of 30 DEG C
Respectively taking 8 groups of YEPD liquid culture mediums, regulating the pH value of the culture mediums by using hydrochloric acid and sodium hydroxide, and setting the pH value gradient to be pH=2; ph=4; ph=6; ph=7; ph=8; ph=10; ph=12; pH=14, and culturing in a shaking table at a constant temperature of 180r/min at 30 ℃ for 24 hours to obtain bacterial liquid. And counting the bacterial density in the bacterial liquid after fermentation to obtain the optimal pH value of 6-7.
Therefore, paenibacillus polymyxa SP1 can grow on a YEPD medium, and the optimal growth temperature is 30 ℃, and the pH value of the optimal growth environment is 6-7.
Example 4 measurement of Lipase Activity produced by Strain SP1
1. The purpose of the experiment is as follows:
and detecting whether the metabolite of the paenibacillus polymyxa SP1 contains lipase or not so as to be widely applied to livestock.
The lipase is added into the feed, so that the digestion utilization rate of animal daily ration grease can be improved, the deficiencies of endogenous digestive enzyme activity and secretion amount caused by the undeveloped digestive function of young animals are supplemented, dyspepsia and diarrhea caused by the excessive addition of the grease are reduced, the grease consumption is reduced, and the feed cost is lowered. In addition, in insect culture, the requirement of insects on fat is high, and the lipase-rich additive can promote the absorption of the insects on the fat.
2. The experimental steps are as follows:
(1) Activation of Paenibacillus polymyxa SP1: sterile manipulation was performed in an ultra clean bench with inoculating loop to pick up a quantity of Paenibacillus polymyxa SP1 streaked on YEPD solid medium. Culturing at 30 deg.c for 12-24 hr until there is macroscopic single colony.
(2) Preparation of Paenibacillus polymyxa SP1 bacterial liquid: inoculating the activated paenibacillus polymyxa SP1 single colony into 5-6 mL of YEPD liquid culture medium, and culturing in a constant temperature shaking table at 30 ℃ and 180r/min for 24 hours to obtain seed bacterial liquid.
(3) Drawing an enzyme activity standard curve: 0.08346g of p-nitrophenol are weighed, dissolved with a small amount of 95% ethanol, and then fixed to a volume of 100mL with water, the concentration being 6mmol. Different amounts of p-nitrophenol solution and 50mmo/L Tris-HCl (pH 8.0) buffer were added, respectively, in a total volume of 2mL. The concentration of p-nitrophenol in the system was 0, 5, 10, 15, 20, 30, 40, 60, 80, 100. Then, 0.5mL of 10% trichloroacetic acid was added to each tube, and then 0.5mL of 10% Na2CO3 solution was added thereto, and the total volume was 3mL and 410mm, to measure the light absorption. Then, an enzyme activity standard curve was drawn.
(4) Substrate solution A90 mg p-palm eleostearic acid nitrobenzeneEster (p-NPP) solution was butyl 30mL of isopropanol, buffer solution B:50mmol/LTriscl (pH 8.0). 4 numbered test tubes were taken, of which 1 tube served as a blank and the other 3 tubes served as reaction tubes. Adding solution B1.8mL and substrate solution A0.2 mL into four test tubes, maintaining in water bath at 37deg.C for 5min, adding YEPD culture medium 1mL into control tube, adding Paenibacillus polymyxa SP1 bacterial liquid 1mL into sample tube, immediately mixing, timing, accurately reacting in water bath for 10min, adding trichloroacetic acid solution 0.5mL10%, stopping reaction, and adding Na 0.5mL10% 2 CO 3 The solution developed. The absorbance of the enzymatically produced p-nitrophenol was measured with 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 at 37℃was defined as 1 lipase activity unit (U).
Experimental results show that the activity of 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 determination of bacteriostatic action
1. Selection of pathogens
Staphylococcus aureus: gram positive bacteria are common food-borne pathogenic microorganisms, have wide distribution range, can produce enterotoxin, and have different degrees of harm to livestock and poultry cultivation and human beings.
Coli K12D31, e.coli O78: common intestinal gram-negative bacteria of livestock and poultry can cause infection to the intestinal tracts of livestock and poultry, can cause acute diarrhea, and is unfavorable for the health of livestock and poultry.
Salmonella typhimurium: the bacterial strain is one of main pathogenic bacteria causing acute gastroenteritis and typhoid fever, and exists in intestinal tracts of various animals such as poultry, livestock, mice and the like.
Salmonella choleraesuis: is a main pathogenic bacterium causing paratyphoid of piglets and causes serious harm to pig industry.
Bacillus thuringiensis: the most widespread microbial pesticides. The principle of insect prevention of bacillus thuringiensis is that its strain can produce endotoxin (companion crystal) and exotoxin, so that the pests stop feeding, and the pests are dead due to hunger, blood spoilage and neurotoxicity, and have high toxicity to various silkworms, so that the damping-off of silkworms can be resulted.
Bacteriostasis of black-chest-septicemia: bacillus myrtillus (Bacillus bombyseptieus, bb) is a pathogenic bacterium of silkworm (Bombyx mori), has high toxicity to various silkworms, and can cause sepsis of silkworms.
Klebsiella pneumoniae: klebsiella pneumoniae is present in the upper respiratory tract and intestinal tract of humans, causing serious infections in humans, including pneumonia, urinary tract infection and blood infection.
Propionibacterium acnes: is generally lodged in hair follicles and sebaceous glands of the skin and is the main bacterium responsible for acne.
Staphylococcus epidermidis: parasitic on the skin, vagina and other parts of human body can cause chronic inflammatory dermatosis, urinary system infection and other diseases.
Spore bacteria of gypsum sample: belongs to microsporidian and can cause a series of fungal infection diseases such as tinea cutanea and the like of human beings and animals.
Physcomitrella rubra: is a human dermatophyte, which causes common superficial dermatomycosis such as tinea manuum, tinea pedis, tinea capitis, etc.
2. Experimental implementation:
(1) The size of the inhibition zone is detected by an agar pore diffusion method. The method is briefly described as follows: placing the sterilized LB culture medium at room temperature, and when the LB culture medium is cooled to 48-50 ℃, sucking 100 mu L of indicator bacteria diluent, and respectively taking the indicator bacteria: staphylococcus aureus, escherichia coli K12D31, escherichia coli O78, salmonella typhimurium, salmonella choleraesuis, bacillus thuringiensis, and septicemia.
Adding the indicator bacteria diluent into 10mL of LB solid medium which is dissolved and cooled to 40-50 ℃, immediately pouring the diluent into a culture dish with the diameter of 90mm, shaking the diluent rapidly and uniformly to uniformly disperse the thalli, and punching the thalli by a puncher with the diameter of 2.7mm after the thalli is cooled and solidified for later use. 5uL of Paenibacillus polymyxa SP1 was added to each well, and after standing for 30min, the culture was performed at 37℃for 24 hours, and the size of the inhibition zone was observed and measured, and the data were recorded.
(2) Filter paper sheet process: a certain amount of propionibacterium acnes is taken, diluted by a proper amount of sterile water, fully and uniformly mixed, and 100 mu L of the mixture is absorbed for uniform coating. A sterile filter paper sheet with the diameter of 5mm is placed in the center of a culture dish, 5 mu L of Paenibacillus polymyxa SP1 is dripped, the culture is carried out for 15min, the culture is carried out in an anaerobic environment at 37 ℃ for 48h in an inverted mode, the size of a bacteriostasis area is observed and measured, and data are recorded.
Staphylococcus epidermidis and klebsiella pneumoniae are used as indicator bacteria and added into LB solid culture medium, and after cooling and solidification, the steps are the same.
(3) Growth rate assay: 100 mu L of Paenibacillus polymyxa SP1 is added on the surface of a PDA flat plate, the flat plate is uniformly coated, a fungus stipe with the diameter of 5mm is inoculated in the center of the flat plate after the flat plate is absorbed, the indicator bacteria are gypsilosis and gypsilosis, and the equal amount of sterile deionized water is used as a control. Culturing in a constant temperature incubator at 25-28 deg.c for 5-7 days in reverse, observing the growth condition every 24 hr, and recording colony size. The inhibition of the hyphae of the two fungi by Paenibacillus mucilaginosus SP1 was compared and the inhibition rate (Ir) was calculated.
Ir= [ (average diameter of CK bacterial disc-average diameter of treated bacterial disc)/average diameter of CK bacterial disc ]. Times.100%
3. Experimental results
The experimental results showed (as in table 1): the Paenibacillus polymyxa SP1 fermentation product has a relatively high broad-spectrum antibacterial effect, has a function of inhibiting common pathogenic bacteria of intestinal bacteria of livestock and poultry, has a function of inhibiting common pathogenic bacteria of insect breeding, and has a function of inhibiting pathogenic bacteria of common skin problems.
TABLE 1 Paenibacillus polymyxa SP1 solid Medium antibacterial test
| Germ (germ) | Diameter of inhibition zone (inhibition rate) |
| Staphylococcus aureus | 16.45mm |
| Coli K12D31 | 9.28mm |
| Coli O78 | 10.73mm |
| Salmonella typhimurium | 9.83mm |
| Salmonella choleraesuis (Fr.) karst | 9.30mm |
| Bacillus thuringiensis | 11.5mm |
| Brevibacterium melanosis | 9.00mm |
| Klebsiella pneumoniae | 8.15mm |
| Propionibacterium acnes | 7.98mm |
| Staphylococcus epidermidis | 18.41mm |
| Spore fungus gypsum | 90.38％ |
| Trichophyton rubrum (Fr.) |
72％ |
Example 6 measurement of Heat stability of Strain SP1
And (3) placing the sterilized LB culture medium at room temperature, and sucking 100 mu L of indicator bacteria diluent when the LB culture medium is cooled to 48-50 ℃, wherein the indicator bacteria are staphylococcus aureus and escherichia coli K12D31. Adding the indicator bacteria diluent into 10mL of LB solid medium which is dissolved and cooled to 40-50 ℃, immediately pouring the diluent into a culture dish with the diameter of 90mm, shaking the diluent rapidly and uniformly to uniformly disperse the thalli, and punching the thalli by a puncher with the diameter of 2.7mm after the thalli is cooled and solidified for later use. Heating Paenibacillus polymyxa SP1 in water bath at 50deg.C, 60deg.C, 70deg.C, 80deg.C, 90deg.C, and 100deg.C for 10min. Taking unheated bacterial liquid 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 bacteriostasis zone, and recording data.
The diameter of the zone of inhibition against staphylococcus aureus is shown in table 2:
TABLE 2 antibacterial test of Strain SP1 against Staphylococcus aureus and E.coli K12D31 at different temperatures
Therefore, the Paenibacillus polymyxa SP1 fermentation product has high thermal stability. The test results show that: paenibacillus polymyxa SP1 is strong in heat stability.
Example 7 determination of the thermal stability of the supernatant and the precipitate
Centrifuging Paenibacillus polymyxa SP1 fermentation broth, heating supernatant and thallus precipitate in water baths at 50deg.C, 60deg.C, 70deg.C, 80deg.C, 90deg.C and 100deg.C for 10min, taking unheated bacterial liquid as blank, observing and measuring bacteriostasis zone size by agar pore diffusion method, and recording data.
TABLE 3 antibacterial test of the supernatants of Strain SP1 against Staphylococcus aureus and Escherichia coli K12D31 at different temperatures
TABLE 4 antibacterial test of bacterial precipitation of Strain SP1 cells against Staphylococcus aureus and Escherichia coli K12D31 at different temperatures
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
SEQUENCE LISTING
<110> agricultural university of south China
<120> Paenibacillus polymyxa SP1 and application thereof
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1406
<212> DNA
<213> rDNA sequence of Paenibacillus polymyxa SP1
<400> 1
ttgcggttac ctcaccgact tcgggtgttg taaactctcg tggtgtgacg ggcggtgtgt 60
acaagacccg ggaacgtatt caccgcggca tgctgatccg cgattactag caattccgac 120
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 (8)
1. Paenibacillus polymyxa @Paenibacillus polymyxa) SP1, wherein said paenibacillus polymyxa SP1 is deposited with the collection of microorganism strains in the cantonese province at 2021, 4 months and 16 days, under the deposit number GDMCC No:61605.
2. the application of the paenibacillus polymyxa SP1 in preparing a bacteriostatic agent, which is characterized in that the bacteriostasis spectrum is one or more of staphylococcus aureus, escherichia coli, salmonella typhimurium, salmonella choleraesuis, bacillus thuringiensis, septicemia, klebsiella pneumoniae, propionibacterium acnes, staphylococcus epidermidis, gypsum-like spore bacteria or trichoderma rubrum.
3. Use of paenibacillus polymyxa SP1 according to claim 1 for the production of antibacterial peptides and/or lipases.
4. Use of paenibacillus polymyxa SP1 according to claim 1 for the preparation of a bacterial agent for producing an antibacterial peptide and/or a lipase.
5. The use of Paenibacillus polymyxa SP1 according to claim 1 for the preparation of a feed.
6. The use of paenibacillus polymyxa SP1 according to claim 1 for the fermentative production of a bacteriostatic feed, wherein the bacteriostasis is one or more of staphylococcus aureus, escherichia coli, salmonella typhimurium, salmonella choleraesuis, bacillus thuringiensis, septicemia and klebsiella pneumoniae.
7. The use of paenibacillus polymyxa SP1 according to claim 1 for the preparation of a cosmetic additive for skin bacteriostasis, wherein the bacteriostasis is one or more of propionibacterium acnes, staphylococcus epidermidis, sporophore gypseum or trichophyton rubrum.
8. The use of paenibacillus polymyxa SP1 according to claim 1 for the preparation of a skin antibacterial agent, wherein the antibacterial agent is one or more of propionibacterium acnes, staphylococcus epidermidis, gypsum-like spore bacteria or trichoderma rubrum.
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