A kind of Paenibacillus pilorei and its metabolites and preparation method

Abstract

The present invention discloses a bacillus pilorei ( Paenibacillus peoriae R5‑3). The Paenibacillus pierrei can effectively produce lipopeptide dodecapeptide. The present invention also discloses a method for producing lipopeptide dodecapeptide using the Paenibacillus pierrei. The lipopeptide dodecapeptide produced by the present invention has high purity, stable structure under the gastrointestinal environment, and strong activity in inhibiting Gram-negative bacteria, and can be used for the development and utilization of leading veterinary drugs and feed additives for inhibiting diseases caused by various Gram-negative pathogens.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a novel Paenibacillus pierrei, its metabolite lipopeptide dodecapeptide and a preparation method thereof. Background Art

[0002] Paenibacillus pilorei Paenibacillus peoriae ) is a type of Gram-positive bacillus, a microorganism of the genus Paenibacillus, which can produce a variety of novel lipopeptide compounds with antibacterial and other active structures. It is one of the common strains for developing lipopeptides. Lipopeptides are secondary metabolites produced by microorganisms (such as certain Bacillus). They are a class of highly diverse antimicrobial peptides composed of two parts: hydrophilic cyclic or short linear oligopeptides and hydrophobic long-chain fatty acids. They are mainly synthesized through the bacterial non-ribosomal peptide and polyketide hybrid pathways. The lipopeptide structure contains both hydrophilic amino acid residues and hydrophobic fatty acid chains, so the entire lipopeptide has significant amphipathicity. Lipopeptides can exert their biological functions of antibacterial and anti-infection through membrane targeting, non-membrane targeting and immunomodulatory mechanisms. Therefore, lipopeptides have great potential as new feed additives in animal husbandry.

[0003] The long-term use of traditional antibiotics in animal husbandry has accelerated the development of microbial resistance, so it is urgent to develop new antimicrobial drugs to replace traditional antibiotics. Lipopeptides have great potential to replace antibiotics in animal husbandry, but there are currently no excellent strains for producing lipopeptides and lipopeptide products with stable performance. Compared with other lipopeptide production strains, Paenibacillus peoriae Demonstrated unique production advantages. For example, Bacillus amyloliquefaciens In contrast, although strains 1 / 6k and 4 / 9 from the Adriatic Sea produced cyclic lipopeptides such as surfactin, iturin A, and fengycin, Paenibacillus peoriae It can produce lipopeptides with higher yields. In addition, compared with Brevibacillus laterosporus and Brevibacillus sp. strains, such as GI9 and SKDU10, although these strains also showed resistance to a broad spectrum of microorganisms, Paenibacillus peoriae The strains showed better growth and production stability, and showed more efficient antibacterial and anti-biofilm formation abilities in terms of biological activity. This is particularly important for applications in agriculture and medicine. This comparison shows that although other strains have shown some potential in producing specific types of lipopeptides, Paenibacillus peoriae Due to its efficient lipopeptide production capacity and production stability, it has significant advantages in the commercial production of lipopeptides. These characteristics not only enhance its competitiveness in the existing market, but also provide a strong technical support foundation for future industrial applications. Summary of the invention

[0004] In view of the above problems, the present invention aims to provide a strain of Paenibacillus piloreus ( Paenibacillus peoriae R5-3), and the preparation method and application of the lipopeptide expressed by the strain.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The present invention provides a strain of Paenibacillus pilorei ( Paenibacillus peoriae R5-3), the strain was deposited in China Center for Type Culture Collection, the deposit date is September 13, 2023, the address is Wuhan University, Wuhan, China, and the deposit number is CCTCC No. M20231701.

[0007] The present invention provides the use of the above-mentioned Paenibacillus pierrei in the preparation of lipopeptide dodecapeptide.

[0008] The present invention also provides the above-mentioned Paenibacillus pierrei metabolite, a lipopeptide dodecapeptide, the structural formula of which is as follows:

[0009] .

[0010] The present invention also provides a method for preparing the above-mentioned lipopeptide dodecapeptide, comprising the following steps:

[0011] Step 1: Bacillus pilorei with a deposit number of CCTCC No. M20231701 ( Paenibacillus peoriae R5-3) was inoculated in NB slant medium for activation, cultured at 33°C and 220 rpm for 12 h, inoculated into a sterilized NB medium shake flask, cultured at 33°C and 220 rpm for 15 h to obtain seed liquid, and the seed liquid was inoculated into an optimized homemade liquid medium at a volume ratio of 1% inoculation amount, and cultured at 33°C and 220 rpm for 18 h to obtain a lipopeptide dodecapeptide fermentation product. The fermentation broth was centrifuged to obtain the supernatant, which was freeze-dried to obtain lyophilized powder.

[0012] Step 2: The obtained lyophilized powder was dissolved in ultrapure water and extracted with an equal volume of n-butanol. Ether was added to the n-butanol extract and allowed to stand at 4°C for 2 h to precipitate the lipopeptide dodecapeptide.

[0013] Step 3: The obtained precipitate is dissolved with the initial mobile phase, and separated and purified by semi-preparative reversed-phase high performance liquid chromatography. The obtained fraction is lyophilized after removing acetonitrile by rotary evaporation to obtain the pure product of lipopeptide dodecapeptide.

[0014] Step 4: Purity test by analytical reverse phase HPLC.

[0015] Preferably, the homemade liquid culture medium described in step 1 is prepared as follows: 3.0 g soluble starch, 10 g yeast extract powder, 10 g ammonium sulfate, and 3 g anhydrous magnesium sulfate are dissolved in 1 L ultrapure water, and the pH is adjusted to 6.5 with hydrochloric acid after dissolution. The culture medium is sterilized at 121°C for 20 min, the inoculation amount is 1%, the fermentation temperature is 33°C, the fermentation time is 14-18 h, and the fermentation broth is centrifuged to obtain the supernatant, which is freeze-dried to obtain freeze-dried powder.

[0016] Preferably, the extraction process in step 2 is as follows: the lyophilized powder obtained in step 1 is dissolved with ultrapure water, the ratio of lyophilized powder to ultrapure water is 1:1~1:5 (W / V), after dissolution, an equal volume of n-butanol is added and extracted 1~3 times, the n-butanol extract is transferred to a new conical flask and an equal volume of ether is added to precipitate the lipopeptide dodecapeptide.

[0017] Preferably, the reverse phase high performance liquid chromatography preparation process in step 3 is: preparative liquid chromatography column, Agilent Zorbax 300SB-C18 PrepHT; mobile phase contains 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B);

[0018] The elution modes are shown in the table below:

[0019]

[0020] The flow rate was 5 mL / min, the column temperature was 30 °C, and the detection wavelength was 220 nm.

[0021] The present invention has the following advantages:

[0022] 1. The present invention provides a strain of Paenibacillus piloreus ( Paenibacillus peoriae R5-3), which can effectively produce lipopeptide dodecapeptide;

[0023] 2. The present invention prepares a high-purity lipopeptide dodecapeptide finished product through liquid seed preparation, fermentation liquid preparation, fermentation liquid pretreatment, lipopeptide dodecapeptide extraction and purification. Compared with the initial extraction by filter paper filtration, the use of n-butanol for initial extraction improves the purification efficiency, and the purity of the lipopeptide dodecapeptide is as high as 97.71%.

[0024] 3. The present invention utilizes extraction precipitation combined with preparative reverse phase high performance liquid chromatography to improve the preparation efficiency of lipopeptide dodecapeptide.

[0025] 4. The present invention provides the use of lipopeptide dodecapeptide in the preparation of anti-Gram-negative bacteria feed additives or anti-infective drugs. Through the screening of antibacterial activity, it was found that the lipopeptide dodecapeptide has a strong activity of inhibiting clinical drug-resistant strains of Escherichia coli, Shigella dysenteriae, Salmonella, and Pseudomonas aeruginosa, and can be used for the development and utilization of feed additives or anti-infective drugs that inhibit related diseases and infections caused by various Gram-negative pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the HPLC preparation diagram of the lipopeptide dodecapeptide in Example 1 of the present invention;

[0027] Figure 2 This is a HPLC chart of purity analysis of the lipopeptide dodecapeptide in Example 1 of the present invention;

[0028] Figure 3 The mass spectrum of the lipopeptide dodecapeptide in Example 1 of the present invention;

[0029] Figure 4 The stability of the lipopeptide dodecapeptide in Example 3 of the present invention in aqueous solutions of different pH values

[0030] Figure 5 The inhibition zone of the lipopeptide dodecapeptide in Example 4 of the present invention after being treated in artificial gastric juice

[0031] Figure 6 The stability test of the lipopeptide dodecapeptide in Example 4 of the present invention in artificial gastric juice (left: degradation retention rate; right: inhibition zone diameter)

[0032] Figure 7 The inhibition zone of the lipopeptide dodecapeptide in Example 5 of the present invention after being treated in artificial intestinal fluid

[0033] Figure 8 The stability test of the lipopeptide dodecapeptide in the artificial intestinal fluid in Example 5 of the present invention (left: degradation retention rate; right: inhibition zone diameter)

[0034] Fig. 9 Hemolytic activity of lipopeptide dodecapeptide in Example 6 of the present invention DETAILED DESCRIPTION

[0035] The strain used in the present invention is Paenibacillus pilorei isolated from the soil of Rongchang District, Chongqing. Paenibacillus peoriae R5-3), which was deposited in the China Center for Type Culture Collection on September 13, 2023. The deposit number is CCTCC No. M20231701.

[0036] The strain isolated by the present invention has been identified, and its morphological characteristics are: the colony is white, the surface is shiny, and there is no wrinkled edge; the bacteria are rod-shaped, 1.0-3.5 μm long, 0.4-0.8 μm wide, the bacteria have flagella, can form spores, and are Gram-positive. The 16S rRNA gene of the strain was amplified by PCR, and sequencing and phylogenetic analysis were performed to identify it as Paenibacillus pierreiu, and it was named Paenibacillus pierreiu ( Paenibacillus peoriae R5-3).

[0037] Example 1

[0038] The method for separating and preparing lipopeptide dodecapeptide specifically comprises the following steps:

[0039] (1) Fermentation

[0040] The pilori bacillus with the deposit number of CCTCC No. M20231701 ( Paenibacillus peoriae R5-3) was inoculated in a NB solid culture dish for activation and cultured at 33°C and 220 rpm for 12 h, then inoculated into a sterilized NB culture medium shake flask, cultured at 33°C and 220 rpm for 15 h to obtain a seed solution, and the seed solution was inoculated into an optimized homemade liquid culture medium at a volume ratio of 1% inoculation amount, and cultured at 33°C and 220 rpm for 18 h to obtain a lipopeptide dodecapeptide fermentation product. The fermentation broth was centrifuged to obtain a supernatant, which was freeze-dried to obtain a lyophilized powder.

[0041] (2) Extraction, separation and purification

[0042] The obtained lyophilized powder was dissolved in ultrapure water and extracted with an equal volume of n-butanol. An equal volume of ether was added to the n-butanol extract and allowed to stand at 4°C for 1.5 h to precipitate the lipopeptide dodecapeptide. The precipitate was dissolved in the initial mobile phase and then subjected to the following steps: Figure 1 As shown, the fraction corresponding to the chromatographic peak No. 2 is lipopeptide dodecapeptide. The chromatographic column used in the preparation process is an Agilent Zorbax300SB-C18 PrepHT preparative liquid chromatography column, and the mobile phase is water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% trifluoroacetic acid (B);

[0043] The elution modes are shown in the table below:

[0044]

[0045] The flow rate was 5 mL / min, the column temperature was 30 °C, and the detection wavelength was 220 nm.

[0046] The obtained fractions were subjected to rotary evaporation to remove acetonitrile and then freeze-dried to obtain pure lipopeptide dodecapeptide. The purity was tested by analytical reverse phase high performance liquid chromatography. The results were as follows: Figure 2 As shown. The purity of the lipopeptide dodecapeptide sample prepared in the present invention can be as high as 97.71%. The molecular structure was identified by mass spectrometry, and the results are as follows Figure 3 As shown. After ionization, the lipopeptide dodecapeptide molecule can form two charging modes. The mass-to-charge ratio (m / z) in the spectrum: 507.2747 and 760.4046 are the molecular ion peaks of the lipopeptide dodecapeptide with three charges and two charges, respectively. The accurate molecular weight of the lipopeptide dodecapeptide calculated by the mass-to-charge ratio is: 1518.8092.

[0047] Example 2

[0048] Antibacterial activity of lipopeptide dodecapeptide

[0049] According to the National Committee for Clinical Laboratory Standards, MH medium was used to serially dilute the lipopeptide dodecapeptide to 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / mL. 50 μL of the diluted lipopeptide dodecapeptide was added to each well of the 96-well plate, and 50 μL of the lipopeptide resuspended in MH at 2-7×10 5 CFU / mL indicator bacteria liquid. Shake and culture for at least 18 h, read the OD value at 600 nm with a microplate reader to obtain the minimum inhibitory concentration of the lipopeptide dodecapeptide. The indicator bacteria selected were: Escherichia coli ATCC 25922, Escherichia coli resistant strains, Shigella dysenteriae CGMCC 1.1869, Salmonella CVCC 534, Pseudomonas aeruginosa ATCC 27853, Streptococcus dysgalactiae ATCC 35666, Staphylococcus aureus ATCC43300, Staphylococcus aureus ATCC 29213, Listeria monocytogenes CVCC 3746, Bacillus cereus CVCC 4101, and the results are shown in Table 2:

[0050]

[0051] Among them, the lipopeptide dodecapeptide has good anti-Gram-negative bacteria activity, with MIC values ​​ranging from 4 to 8 μg / mL. The anti-Gram-positive bacteria activity is weak, with MIC values ​​ranging from 32 to 256 μg / mL. The results show that the lipopeptide dodecapeptide has the research and development value of a special lead drug for anti-Gram-negative bacteria and an antimicrobial peptide.

[0052] Example 3

[0053] Stability of lipopeptide dodecapeptide in aqueous solutions at different pH values

[0054] The pure lipopeptide dodecapeptide was prepared into a solution with a concentration of 1.0 mg / mL using ultrapure water, and the pH was adjusted to 1.0, 3.0, 5.0, 7.0 and 9.0 using hydrochloric acid and sodium hydroxide solutions, respectively. Samples were taken at 0, 0.5, 1, 1.5 and 2 h for HPLC and inhibition zone detection. The stability of the lipopeptide dodecapeptide in aqueous solutions of different pH values ​​was evaluated by the degradation retention rate obtained by HPLC detection. The results are shown in Figure 4 As shown, there was no difference in the degradation retention rate of lipopeptide dodecapeptide at pH 1.0~9.0 (p>0.05).

[0055] Example 4

[0056] Evaluation of the stability of lipopeptide dodecapeptide in artificial gastric juice

[0057] The stability of lipopeptide dodecapeptide in gastric protein solution was determined by HPLC and inhibition zone method. According to the Veterinary Pharmacopoeia of the People's Republic of China (2020 edition), 16.4 mL of dilute hydrochloric acid was taken, about 800 mL of water was added, and 10 g of pepsin was shaken, then transferred to a 1000 mL volumetric flask and diluted to the mark with ultrapure water to obtain artificial gastric juice. The lipopeptide dodecapeptide was dissolved and diluted with artificial gastric juice to a concentration of 200 μg / mL, placed in a 37 ℃ water bath, and samples were taken at 0, 2, 4, and 6 h. After the sample was inactivated by 85 ℃ water bath for 5 min, it was centrifuged at 9500 rpm and 4 ℃ for 15 min, and the chromatographic peak area of ​​lipopeptide dodecapeptide at different time points was detected, and the degradation retention rate of lipopeptide after treatment with artificial gastric juice was calculated. 100 μL of solution was taken to detect the antibacterial activity of lipopeptide dodecapeptide by the inhibition zone method. One blank control group and four experimental groups (samples taken at 0, 2, 4, and 6 h) were set up. The results are as follows: Figure 5 As shown. After 0-6 h in a 37℃ water bath, the degradation retention rate of lipopeptide dodecapeptide in artificial gastric juice was above 95%, and the size of the inhibition zone for indicator bacteria Escherichia coli ATCC 25922 was in the range of 1.50-1.75 cm ( Figure 6 ). This indicates that the lipopeptide dodecapeptide can maintain the stability of its structure and antibacterial activity in artificial gastric juice.

[0058] Example 5

[0059] Evaluation of the stability of lipopeptide dodecapeptide in artificial intestinal fluid

[0060] The stability of lipopeptide dodecapeptide in pancreatic protein solution was determined by high performance liquid chromatography and inhibition zone method. According to the Veterinary Pharmacopoeia of the People's Republic of China (2020 edition), 250 mL of 0.2 mol / L potassium dihydrogen phosphate solution was taken, 118 mL of 0.2 mol / L sodium hydroxide solution was added, and 10 g of pancreatin was taken, and an appropriate amount of water was added to dissolve. After the two liquids were mixed, they were transferred to a 1000 mL volumetric flask and diluted to the mark with water to obtain a pancreatic protein solution. The lipopeptide dodecapeptide was dissolved and diluted with trypsin solution to a concentration of 200 μg / mL, placed in a 37 ℃ water bath, and samples were taken at 0, 2, 4, and 6 h. After the sample was inactivated by water bathing at 85 ℃ for 5 min, it was centrifuged at 9500 rpm and 4 ℃ for 15 min, and the chromatographic peak area of ​​lipopeptide dodecapeptide at different time points was detected, and the degradation retention rate was calculated. 100 μL of the solution was taken to detect the antibacterial activity of the lipopeptide dodecapeptide using the inhibition zone method. One blank control group and four experimental groups (samples taken at 0, 2, 4, and 6 h) were set up. The results are shown in the figure. Figure 7As shown. After 0-6 h in a 37 ℃ water bath, the degradation retention rate of lipopeptide dodecapeptide in artificial intestinal fluid was greater than 85%, and the size of the inhibition zone for indicator bacteria Escherichia coli ATCC 25922 was in the range of 1.7-1.9 cm ( Figure 8 ). The antibacterial properties of lipopeptide dodecapeptide are stable in artificial intestinal fluid environment.

[0061] Example 6

[0062] Evaluation of the hemolytic activity of lipopeptide dodecapeptide

[0063] Whole blood from pigs was collected intravenously using a blood collection tube containing sodium heparin, and red blood cells were obtained after centrifugation. The blood cells were washed with 10 mM PBS (pH7.3) until the supernatant had no obvious red color. The blood cells were prepared into a 4% suspension with PBS, and 100 μL of 4% blood cell suspension and 100 μL of a dilution of lipopeptide dodecapeptide solution with a concentration range of 0~512 μg / mL were added to the wells of a 96-well plate, incubated in a 37°C incubator for 1 h, centrifuged at 1500 rpm for 5 min, and the supernatant was transferred to a new 96-well plate. The OD value was detected at 540 nm using an enzyme reader. 10 mM PBS and 0.1% Triton X-100 were used as negative and positive controls, respectively. The hemolyticity of the lipopeptide dodecapeptide was calculated. The calculation formula is as follows:

[0064]

[0065] in, A 脂肽十二肽 The OD value of different concentrations of lipopeptide dodecapeptide added to the blood cell well; A PBS A is the OD value of adding PBS into the blood cell well; 0.1% Triton X-100 The OD value of 0.1% Triton X-100 added to the blood cell well. Fig. 9 As shown, the hemolytic rate of lipopeptide dodecapeptide was 0.23-0.75% in the concentration range of 256-0.5 μg / mL, and its variation was within 0.5%. The results showed that lipopeptide dodecapeptide had no obvious hemolytic activity.

[0066] In summary, in the present invention, based on Paenibacillus piloreus ( Paenibacillus peoriae The lipopeptide dodecapeptide produced by R5-3 has the advantages of resisting a variety of Gram-negative pathogens, has a stable structure, can maintain good activity in the complex gastrointestinal tract of animals or similar environments, and has a very broad application prospect.

[0067] The above are only embodiments of the present invention. The invention is not limited to the fields involved in this implementation case. The common sense such as the known specific structures and characteristics in the scheme is not described in detail here. The ordinary technicians in the relevant field know all the common technical knowledge in the technical field to which the invention belongs before the application date or priority date, can obtain all the existing technologies in the field, and have the ability to apply the conventional experimental means before that date. The ordinary technicians in the relevant field can improve and implement this scheme in combination with their own abilities under the enlightenment given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the relevant field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the present invention. The protection scope required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to explain the content of the claims.

Claims

1. A strain of Paenibacillus peoriae R5-3, characterized in that: The strain is deposited in the China Center for Type Culture Collection, the deposit date is September 13, 2023, the address is Wuhan University, Wuhan, China, and the deposit number is CCTCC No. M20231701.

2. Use of the Paenibacillus pilorioides according to claim 1 in the preparation of a lipopeptide dodecapeptide having a structural formula (I): 。 3. A method for preparing a lipopeptide dodecapeptide, characterized in that The steps include: (1) Cultivation and fermentation The Paenibacillus piloreus described in claim 1 is inoculated in a NB solid culture dish for culture activation, and after culturing in a constant temperature incubator at 33°C for 12 h, an activated single colony is picked and inoculated into a sterilized NB culture medium shake bottle, and cultured at 33°C and 200 rpm for 15 h to obtain a seed solution, and the seed solution is inoculated into an optimized self-made liquid culture medium at an inoculum volume ratio of 1%, and after culturing at 33°C and 200 rpm for 18 h, the fermentation liquid is centrifuged to obtain a supernatant, and the supernatant is freeze-dried to obtain a lipopeptide dodecapeptide fermentation liquid freeze-dried powder; (2) Crude extract extraction Add ultrapure water to the lyophilized powder obtained in step (1) to dissolve it, extract it with an equal volume of n-butanol, add ether to the n-butanol extract and let it stand at 4°C for 1.5 hours to precipitate the lipopeptide dodecapeptide; (3) Separation and preparation of lipopeptide dodecapeptide The lipopeptide dodecapeptide precipitate obtained in step (2) was dissolved in the initial mobile phase and separated and purified by semi-preparative reverse phase high performance liquid chromatography. The preparation process was as follows: preparative liquid chromatography column, Agilent Zorbax 300SB-C18 PrepHT; mobile phases contained 0.1% trifluoroacetic acid in pure water A and 0.1% trifluoroacetic acid in acetonitrile B; elution pattern as shown in Table 1: The flow rate was 5 mL / min, the column temperature was 30°C, and the detection wavelength was 220 nm; the obtained fractions were lyophilized after removing acetonitrile by rotary evaporation, and the lyophilized powder was the purified lipopeptide dodecapeptide.

4. The preparation method according to claim 3, characterized in that: The homemade liquid culture medium described in step (1) is: 3.0 g soluble starch, 10 g yeast extract powder, 10 g ammonium sulfate, and 3 g anhydrous magnesium sulfate are dissolved in 1 L ultrapure water, and after dissolution, the pH is adjusted to 6.5 with hydrochloric acid.

5. The preparation method according to claim 3, characterized in that: The ratio of the dissolved lyophilized powder to ultrapure water in step (2) is 1:1~1:5 W / V. After dissolution, an equal volume of n-butanol is added and extracted 1~3 times. The n-butanol extract is transferred to a new conical flask and an equal volume of ether is added to precipitate the lipopeptide dodecapeptide.

6. Use of the Paenibacillus pierrei according to claim 1 in the preparation of an anti-Gram-negative bacteria feed additive or an anti-infective drug.

7. The use according to claim 6, further characterized in that: The Gram-negative bacteria are clinical drug-resistant strains of Escherichia coli, Shigella dysenteriae, Salmonella or Pseudomonas aeruginosa.

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