CN114317317B - Bacillus belicus strain capable of resisting salt and producing lipopeptid at high yield and application thereof - Google Patents

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides bacillus bailii (Bacillus velezensis) which is salt-tolerant and high in lipopeptide yield and application. The bacillus bailii sieve is selected from northeast traditional naturally fermented bean paste, and the preservation number is CGMCC No.23402. The strain can produce lipopeptid at high yield, can effectively inhibit common pathogenic bacteria in foods such as staphylococcus aureus, listeria monocytogenes, penicillium, escherichia coli, salmonella and the like, and can be widely applied to the field of food production.

Description

Bacillus belicus strain capable of resisting salt and producing lipopeptid at high yield and application thereof

Technical Field

The invention relates to the technical field of functional microorganism screening and application, in particular to novel salt-tolerant bacillus belicus with high lipopeptid yield and application thereof.

Background

Lipopeptides are secondary metabolites of bacillus and are synthesized by a non-ribosomal polypeptide synthetase system formed by combining certain functional modules. The lipopeptid has the characteristics of low toxicity, high isoelectric point, stable physicochemical property, good heat resistance, good acid-base property, safety, no toxicity, high stability and the like. The lipopeptides have strong biological effects, such as plant disease control, anti-tumor and antiviral activities and a wide antibacterial spectrum, so that the lipopeptides are widely applied in the fields of foods, biological medicines, agriculture, processes and the like. Lipopeptides can be classified into different types according to their amino acid composition and fatty acid chain length, and their main types are surface active agents (Surfactin), iturin (Iturin) and fenugreek (fenycin). The Surfactin type lipopeptid is a substance with stronger biological surface activity, can reduce the surface tension of plant roots, can be used as a detergent for biological membranes, and has the effects of resisting ulcers, reducing cholesterol and resisting inflammation. The Iturin type lipopeptide has good inhibition effect on plant pathogenic bacteria, and has bacteriostasis effect on most fungi and a small part of bacteria, and is safe and nontoxic and does not destroy ecological environment, so that the type lipopeptide can be used as a component for producing agriculture in agriculture, and can replace the traditional chemical pesticide to ensure that agricultural products are safer. The Fengycin type lipopeptide has strong hemolytic activity and fungus inhibition effect, so that the Fengycin type lipopeptide has good biocontrol value.

The lipopeptid has the most remarkable characteristics and advantages that the lipopeptid has good antibacterial property and has a certain inhibition effect on gram-positive bacteria, gram-negative bacteria, fungi, viruses and mycoplasma. Food-borne microbial contamination often occurs in the food processing process, and people can cause food poisoning after eating by mistake, so that the human health is endangered. In all food safety events, staphylococcus aureus and listeria monocytogenes are the two major food-borne pathogenic microorganisms responsible for food-borne diseases. At present, most methods for killing two food-borne pathogenic microorganisms adopt antibiotics for treatment, but various drug-resistant bacteria appear along with abuse of antibiotics, and the health of human beings is endangered continuously. Studies have shown that the antibacterial mechanism of lipopeptides is completely different from that of antibiotics, and lipopeptides destroy cell wall structures or change the permeability of cell membranes through antagonism to produce antibacterial effects. In addition, in journal of nature in 2018, the fengyins lipopeptides are published to be similar to the AIP signaling molecular structure of the staphylococcus aureus Agr colony induction pathway, and can inhibit the synthesis of staphylococcus aureus toxin and intestinal tract colonisation by competitively binding with the Agr C receptor protein of staphylococcus aureus. Therefore, the application of the lipopeptid can be expected to replace the treatment of the traditional antibiotics with high efficiency, and the lipopeptid has an antibacterial effect on staphylococcus aureus with drug resistance, so that the problem of pollution of food-borne diseases is effectively inhibited, and the food safety is ensured.

Currently, the lipopeptid-producing microorganisms mainly include Bacillus (Bacillus), paenibacillus (Paenibacillus), lactobacillus (Lactobacillus), streptomyces (Streptomyces), pseudomonas (Pseudomonas), serratia (Serratia), burkholderia (Burkholderia) and the like. Among them, bacillus is considered to be the main genus for producing lipopeptides, and the amount and kind of lipopeptides produced are more than those of other microorganisms. And bacillus is safe and low in toxicity, has no pathogenicity, is nontoxic and harmless to human beings and domestic animals, does not pollute the environment, and can produce lipopeptides with strong bacteriostasis, so that bacillus is considered as a safe probiotic microorganism and has been widely applied to foods. Among the bacillus bacteria that produce lipopeptides in the food processing field are mainly bacillus subtilis (b. Subtilis), bacillus licheniformis (Bacillus licheniformis), bacillus methylotrophicus (b. Methyltrophius), bacillus amyloliquefaciens (b. Amyloliquefaciens), and the like. The lipopeptid produced by the bacillus has low yield, narrow bacteriostasis spectrum and insignificant bacteriostasis effect, so that the application range and the application effect of the lipopeptid in food production are greatly limited. Therefore, screening bacillus which can produce lipopeptid with high yield and good antibacterial effect is a research hotspot in the current food processing field.

Disclosure of Invention

The invention provides bacillus belicus (Bacillus velezensis) which is salt-tolerant and can produce lipopeptid in high yield and application thereof, and aims to solve the problems in the prior art. The bacillus bailii sieve is selected from the soybean paste of northeast traditional natural fermentation, can produce lipopeptid with high yield, can effectively inhibit common pathogenic bacteria in foods such as staphylococcus aureus, listeria monocytogenes, penicillium, escherichia coli, salmonella and the like, and can be widely applied to the field of food production.

The invention provides bacillus beleiensis, which is named as bacillus beleiensis SNBV-20 (Bacillus velezensis SNBV-20), and is preserved in 2021, 09 and 13 days to China general microbiological culture Collection center (China Committee for culture Collection), wherein the preservation address is CGMCC No.23402, and the preservation number is CGMCC No.23402.

The 16s rDNA sequence of the bacillus bailii SNBV-20 is SEQ ID NO:1.

in one aspect, the invention provides an application of bacillus bailii SNBV-20 in preparing a food starter.

In one aspect, the invention provides an application of bacillus bailii SNBV-20 in preparing an antibacterial composition.

The invention also provides an application of bacillus belicus SNBV-20 in lipopeptide production.

The invention also provides a production method of the lipopeptid, which comprises the following steps:

(1) Inoculating the activated bacillus belicus SNBV-20 into an LB liquid culture medium, and culturing at a constant temperature of 33 ℃ for 24 hours to obtain a fermentation broth;

(2) Centrifuging Bacillus bailii SNBV-20 fermentation broth at 4deg.C and 10000rpm for 10min, and keeping supernatant;

(3) Adjusting pH value of the fermentation supernatant with hydrochloric acid to 2.0,4 ℃ and placing in a refrigerator for 12 hours;

(4) Centrifuging the fermentation broth after acid precipitation at 4deg.C and 10000rpm for 10min, removing supernatant, and collecting precipitate;

(5) Extracting the precipitate with methanol for 3 times repeatedly, mixing the extractive solutions, and adjusting pH to neutrality with sodium hydroxide;

(6) Filtering the methanol extracting solution by using a 0.22 mu m needle type filter membrane filter to obtain a lipopeptide crude extracting solution;

(3) And (3) carrying out reduced pressure suspension evaporation on the methanol solution by a reduced pressure rotary evaporator at the temperature of 45-55 ℃ to obtain a brown yellow solid substance, namely the lipopeptide.

The lipopeptides comprise surface active agents (surfactans), fencines (fenycins) and Iturin (Iturin), and have remarkable inhibition effects on staphylococcus aureus.

The invention also provides an antibacterial composition comprising the lipopeptid.

The invention also provides application of the antibacterial composition in preparation of foods or medicines.

The invention has the beneficial effects that:

the bacillus belicus SNBV-20 provided by the invention is screened from northeast natural fermented soybean paste, has strong salt tolerance and good growth in a 10% concentration high-salt environment. The strain has remarkable inhibition effect on common pathogenic bacteria in five foods of staphylococcus aureus, listeria monocytogenes, penicillium, escherichia coli and salmonella, wherein the inhibition effect on staphylococcus aureus, listeria monocytogenes and penicillium is strongest, and the average inhibition zone diameter is more than 15mm.

Bacillus bailii SNBV-20 can ferment high-yield lipopeptid, and the extraction amount of the lipopeptid in fermentation liquor reaches 0.135g/L, namely, the extraction amount of the lipopeptid per g of thallus is about 0.11g, so that unexpected technical effects are achieved. The components with antibacterial effect in the bacillus belicus SNBV-20 lipopeptide are surfactant (Surfactin), fencine (fengycins) and Iturin (Iturin). The three types of lipopeptides have remarkable inhibitory effects on staphylococcus aureus. The observation under a scanning electron microscope shows that the lipopeptide can damage part of cell walls of staphylococcus aureus to form holes, even break cells, and has remarkable antibacterial effect. The bacillus belicus SNBV-20 can be widely applied to the production of natural lipopeptid, has short production period and high yield, is safe and free from toxic and side effects, and meets the requirements of the food industry.

Bacillus bailii SNBV-20, its fermentation product or extracted lipopeptides can be used alone or in combination for the production of antibacterial compositions, and are widely used for the preparation of foods or medicines.

Drawings

FIG. 1 is a gram-stain microscopy image;

FIG. 2 is a diagram showing the electrophoresis detection of PCR products;

FIG. 3 is a colony morphology;

FIG. 4 is a phylogenetic tree;

FIG. 5 shows the result of thin layer chromatography of lipopeptides produced by Bacillus bailii SNBV-20;

FIG. 6 is a MALDI-TOF-MS and analysis table of lipopeptides produced by Bacillus bailii SNBV-20;

FIG. 7 is a scanning electron micrograph of the inhibitory effect of Bacillus belicus SNBV-20 lipopeptide on Staphylococcus aureus.

Detailed Description

The screening method of the present invention is not limited to the examples, but known screening methods can be used to achieve the screening purpose, and the screening description of the examples is only illustrative of the present invention and is not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

The culture medium used in the embodiment of the invention comprises the following components:

LB solid medium: 10g of sodium chloride, 10g of tryptone, 5g of yeast extract powder, 1000mL of distilled water and pH value of 3.0-3.2;20g of agar;

LB liquid medium: 10g of sodium chloride, 10g of tryptone, 5g of yeast extract powder, 1000mL of distilled water and pH value of 3.0-3.2.

EXAMPLE 1 isolation and screening of lipopeptide-producing Strain

1. Sample source

A traditional fermented soybean paste is collected in Shenyang city of Liaoning province.

2. Bacillus primary screening

1g of soybean paste sample was taken and placed in a test tube containing 9ml of physiological saline with a concentration of 0.9%, and thoroughly mixed. In order to only retain bacillus, the test tube after being uniformly mixed is put into a water bath kettle with the temperature of 85 ℃ and is taken out after being heated in the water bath for 20-25 min. And taking out the test tube, sequentially carrying out 10 times of gradient dilution, wherein the diluent is sterilized 0.9% physiological saline, and carrying out continuous dilution for 6 times. From a dilution gradient of 10 ^-5 And 10 ^-6 100. Mu.L of each diluted sample was pipetted into LB solid plates and coated using sterilized coating bars. After 18-36h of culture, the plate is observed, part of single colony is picked and transferred into a liquid LB culture medium for culture, and meanwhile, the single colony of the same part is subjected to gram staining microscopic examination to observe the morphological characteristics.

Blue, slender and rod-shaped single or chain-shaped substances with parts similar to spores and 24 strains of bacteria with spores in the middle of the bacteria and circular shapes are selected during microscopic examination, and the microscopic examination result is shown in figure 1. And (3) continuously activating and culturing each strain for three generations until the strain is pure strain.

Extracting DNA of each strain by using a bacterial DNA extraction kit, measuring and calculating the DNA concentration of each thallus by using an ultra-trace nucleic acid protein quantitative instrument after extraction, and preparing a PCR system according to concentration calculation.

TABLE 1 PCR System

The volume of the added DNA extracted is measured according to an ultra-trace nucleic acid protein quantitative analyzer, and the total amount of the added DNA is required to be 100ng, but the added volume is 10ul at maximum. After the addition, the remaining extracted DNA is put into a refrigerator with the temperature of minus 20 ℃ for preservation, and is used for the subsequent rescreening of bacillus for producing lipopeptid.

PCR amplification was performed using bacterial universal upstream and downstream primers 23F and 1492R (synthesized by Biotechnology Co., ltd.) using the strain DNA as a template. The reaction procedure: pre-denaturation at 94 ℃ for 5min,30 cycles: 94℃30s,56℃30s,32℃1min,32℃10min.

23F：AGAGTTTGATCCTGGCTCAG；

1492R：TACGGCTACCTTGTTACGACTT。

The results of gel electrophoresis detection of the PCR amplification products are shown in FIG. 2.

The corresponding electrophoresis band of about 1500bp was sent to the Shanghai Co., ltd for sequencing. The sequencing results were logged into the NCBI website and BLAST analysis was performed. The sequence alignment shows 11 strains of Bacillus sp.

3. Rescreening of bacillus stearothermophilus

The related gene detection of bacillus cyclic lipopeptide synthesis is carried out by adopting a PCR technology, and 11 strains of bacillus obtained by primary screening are subjected to secondary screening, so that bacillus mainly producing the surfactant, the fencine and the iturin type lipopeptid is screened.

(1) Configuring PCR amplification reaction system

3 pairs of PCR primers fen B, sfp and itu A are designed and synthesized, and PCR amplification is carried out by taking genomic DNA of 11 bacillus strains obtained through preliminary screening as templates. Primers were synthesized by Shanghai Paenox Biomedicine technologies Co.

The primer sequences were as follows:

Sfp-F：GGCCGTATGATAGGATGGTT；

Sfp-R：GAAGTCGAGCGGCTGTTTCA；

fenB-F：CTATAGTTTGTTGACGGCTC；

fenB-R：CAGCACTGGTTCTTGTCGCA；

ituA-F：ATGTATACCAGTCAATTCC；

ituA-R：GATCCGAAGCTGACAATAG。

TABLE 2 PCR amplification reaction System (50. Mu.L)

TABLE 3 PCR amplification reaction procedure

(2) Recovery of PCR products:

the PCR product was recovered using AxyPrep DNA gel recovery kit (Boyao, ASJ 0013) and the specific procedure was as described in the kit.

(3) Re-screening results

According to the amplification result, only one bacillus strain of 11 bacillus strains obtained through preliminary screening of the invention simultaneously produces the surfactant, the fenethamine and the iturin type lipopeptides, and is named SNBV-20.

EXAMPLE 2 identification of SNBV-20 Strain

1. Colony morphology identification

The colony morphology of the SNBV-20 strain is shown in FIG. 3, and the colony is microcolonies, white, round and convex, smooth, bright, opaque, moist and neat in edge.

2. Physiological and biochemical identification

After the SNBV-20 strain is activated for three generations, physiological and biochemical characteristics of the strain are identified by referring to the Bonji bacteria identification Manual (ninth edition) and the common bacteria System identification Manual which is compiled by Dongxiu beads and Cai Miaoying (2001). The specific results are shown in Table 4.

TABLE 4 physiological and biochemical identification results

Note that: "+" indicates positive reaction or growth, and "-" indicates negative reaction or no growth.

3. Molecular biological identification

(1) Extraction of genomic DNA:

genomic DNA of SNBV-20 strain was extracted using bacterial genomic kit (Solarbio D1600).

(2) And (3) PCR amplification:

PCR upstream primer 23F and downstream primer 1492R were designed and synthesized by Shanghai Bioengineering Co.

23F：AGAGTTTGATCCTGGCTCAG(5'---3')；

1492R：GGTTACCTTGTTACGACTT(5'---3')。

The PCR reaction conditions were: preheating for 5min at 95 ℃, denaturing for 1min at 94 ℃, annealing for 1min at 50 ℃, extending for 1min at 32 ℃ for 20s, and circulating for 36 times; keeping at 32 ℃ for 8min and preserving heat at 4 ℃.

(3) Recovery of PCR products:

the PCR product was recovered using AxyPrep DNA gel recovery kit (Boyao, ASJ 0013) and the specific procedure was as described in the kit.

(4) 16S rDNA sequencing and sequence alignment

The positive PCR product was sampled to the Shanghai Pair Nuo Biotech Co., ltd and sequenced to obtain the 16s rDNA sequence of SNBV-20 strain as SEQ ID NO:1, as shown below.

cgtggcggggtgcctaatacatgcaagtcgagcggacagatgggagcttgctccctgatgttagcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatggttgtttgaaccgcatggttcagacataaaaggtggcttcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctcaccaaggcgacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaagaacaagtgccgttcaaatagggcggcaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattgggcgtaaagggctcgcaggcggtttcttaagtctgatgtgaaagcccccggctcaaccggggagggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtctgtaactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgcattaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtgaggtaaccttttaggagccagccgccgaagggggatcagag。

Setting SEQ ID NO: 1A BLAST tool was used in NCBI database to align with existing sequences in GenBank database. The results show that the 16s rDNA sequence of SNBV-20 strain SEQ ID NO:1 has the highest similarity with bacillus belgium (Bacillus velezensis). Further, a phylogenetic tree was constructed using MEGA3.0, and the result is shown in FIG. 4, in which the SNBV-20 strain of the present invention has the highest homology with Bacillus bailii (Bacillus velezensis).

In summary, by combining the colony morphology, the physiological and biochemical characteristics and the molecular biological identification results of the strain, it can be concluded that the SNBV-20 strain screened by the invention is a novel bacillus bailii, named bacillus bailii SNBV-20 (Bacillus velezensis SNBV-20).

The applicant reserves Bacillus berryis SNBV-20 (Bacillus velezensis SNBV-20) in the China general microbiological culture Collection center (CGMCC, address: north Xielu No. 1, 3, institute of microbiology, china academy of sciences) at 9 and 13 days 2021, and the preservation number is CGMCC No.23402.

EXAMPLE 3 salt tolerance analysis of Bacillus bailii

NaCl is added into LB liquid culture medium according to a proportion to prepare culture media with salt concentration (w/v) of 1%, 10%, 15% and 20% respectively.

Bacillus belicus SNBV-20 was inoculated into the LB liquid medium with different salt concentrations according to the inoculation amount of 2% (v/v), and cultured for 24 hours at the rotation speed of 160r/min of the shaking table at 33 ℃, and the culture medium without the inoculated thalli was used as a blank control group. After the completion of the incubation, absorbance values of each group were measured at a wavelength of 240nm using an enzyme-labeled instrument.

The results show that: the culture medium with the salt concentration of 15% and 20% is clear, and no bacterial cells are found when the absorbance value is smaller. The culture medium with the salt concentration of 10% is turbid, the absorbance value is larger than that of the culture medium with the salt concentration of 15% and 20%, and the bacterial cells are found to grow well during microscopic examination. Therefore, the bacillus belicus SNBV-20 strain provided by the invention has stronger salt tolerance and can grow in a high-salt environment.

Example 4 determination of bacteriostatic Functions of Bacillus bailii SNBV-20

Culturing Bacillus bailii SNBV-20LB liquid medium at 33deg.C with shaking table rotation speed of 160r/min for 24 hr. The activated bacillus is centrifuged for 10min at 15300 Xg and filtered by a 0.22 μm needle filter membrane filter to obtain supernatant.

Five pathogenic bacteria of staphylococcus aureus, listeria monocytogenes, escherichia coli, salmonella and penicillium are respectively inoculated into an LB liquid culture medium according to the inoculation amount of 3 percent, and the three generations of the bacteria are used after activation.

100 mu L of pathogenic bacteria culture solution is respectively sucked by a pipette and is pumped into a plate filled with LB solid culture medium for coating, and each pathogenic bacteria is repeated three times in parallel, and 15 plates are total. The sterilized filter paper sheets are cut into circles with the diameter of 0.3cm, and five filter paper sheets, each of which is 4 layers, are uniformly placed in a plate coated with pathogenic bacteria. The filter paper pieces were then added with 20. Mu.L of the supernatant of Bacillus belicus and incubated at 33℃for 24 hours. After culturing, measuring and calculating the diameter of the bacteriostasis ring of each oxford cup by using a vernier caliper.

The results show that: the bacillus belicus SNBV-20 has different inhibition effects on five pathogenic bacteria, wherein the bacillus belicus SNBV-20 has the strongest inhibition effect on staphylococcus aureus, listeria monocytogenes and penicillium, and the average inhibition zone diameter is more than 20mm; has certain inhibiting effect on colibacillus and salmonella, but the diameter of the inhibition zone reaches more than 12 mm.

Example 5 fermentation production method of Bacillus bailii SNBV-20

And (3) performing activation culture on bacillus bailii SNBV-20, taking a test tube inclined plane liquid LB culture medium as a starting point, expanding the culture by seed expansion, expanding a constant temperature culture link to a secondary seed tank culture, taking a fermentation tank as an end point, and providing sufficient liquid strain for production so as to meet the production requirement.

Inoculating the activated bacillus belicus SNBV-20 strain into 1kg of LB liquid medium, and culturing at a constant temperature of 33 ℃ for 24 hours to obtain first-stage seeds; the primary seeds were inoculated into a secondary seed tank containing 10kg of LB liquid medium, the pH of the liquid medium was adjusted to 3, and the culture was continued at 33℃for 24 hours. After the fermentation, the fermentation broth was centrifuged at 5000rpm at 4℃for 5 minutes, and the weight of the cells in the fermentation broth was measured.

The results show that: after the seed expansion culture and the constant temperature culture link are expanded to the secondary seed tank culture, the weight of the strain contained in the secondary seed tank is 356g.

EXAMPLE 6 extraction of lipopeptid produced by Bacillus bailii SNBV-20

The lipopeptid in bacillus beijerinus SNBV-20 fermentation broth is extracted by adopting an acid precipitation separation method and an organic solvent extraction method. The specific method comprises the following steps:

the Bacillus bailii SNBV-20 fermentation broth obtained in example 5 was centrifuged at 10000rpm at 4℃for 10min, and the supernatant was retained. The pH of the fermentation supernatant was adjusted to 2.0,4 ℃with hydrochloric acid and placed in a refrigerator for 12 hours. And further centrifuging the fermentation broth after acid precipitation for 10min at 4 ℃ and 10000rpm, removing supernatant, and collecting precipitate. Extracting with methanol for 3 times repeatedly, mixing the extractive solutions, adjusting pH to neutrality with sodium hydroxide, and filtering with 0.22 μm needle type filter membrane to obtain lipopeptide crude extract. And (3) carrying out reduced pressure suspension evaporation on the methanol solution by a reduced pressure rotary evaporator at the temperature of 45-55 ℃ to obtain a brown yellow solid substance which is the extracted lipopeptide, weighing, and calculating the extraction quantity of the lipopeptide. The lipopeptides were redissolved in methanol after weighing and stored in a refrigerator at 4 ℃.

Lipopeptide extraction amount = weight of post extraction flask-weight of empty flask prior to extraction.

The results show that: through calculation, the extraction amount of lipopeptid in bacillus bailii SNBV-20 fermentation broth reaches 0.135g/L, namely the estimated extraction amount of lipopeptid is 0.11g per g of thallus, and unexpected technical effects are obtained.

EXAMPLE 7 isolation and purification and component identification of lipopeptid produced by Bacillus bailii SNBV-20

1. Thin layer chromatography analysis

Taking a silica gel GF254 thin layer chromatography plate of the group A and the group B, heating at 105 ℃ for 30min for activation, spotting 10 mu L of bacillus bailii lipopeptid extracted in the example 5 on the thin plate, and mixing chloroform in a volume ratio: methanol: water (65:25:4) is used as a layer spreading agent for chromatography, and after drying, the layer spreading agent is sprayed with a solution containing 0.5 percent ninhydrin for color development observation. And B, treating the sheet by adopting an in-situ acid hydrolysis-ninhydrin color development method, putting the sheet after the layer development is finished and drying into a high-temperature resistant sealed container filled with 2mL of concentrated hydrochloric acid, fumigating for 1h at 110 ℃ in an oven, cooling in a fume hood, developing by using a ninhydrin reagent after the hydrochloric acid volatilizes, and calculating the mobility (Rf) value.

Mobility (Rf) =d1/d 2.

d1: the distance from the origin to the center of the spot; d2: the distance from the origin to the solvent front.

As shown in FIG. 6, the result of thin layer chromatography shows that the bacillus belicus SNBV-20 lipopeptide of the invention has a non-single component and contains cyclic lipopeptides and ring-opened or linear small molecular peptides through mobility judgment.

2. HPLC analysis

Using Shimadzu high performance liquid chromatograph, using C ₁₈ And (3) separating and purifying components in the lipopeptid produced by the bacillus bailii SNBV-20 by using a reverse phase column.

The water/TFA is used as a mobile phase A, the methanol/TFA is used as a mobile phase B, and the concentration of the B is increased from 30% to 30% in 10min, and then the concentration is finally increased to 100% in 25min for separation and purification. The HPLC components are collected manually, concentrated by a centrifugal rotary evaporator, and then the components are subjected to antibacterial activity measurement of staphylococcus aureus by a filter paper sheet method, so that only one component has an inhibitory effect on staphylococcus aureus. The components are separated and purified for the second time, and four components are collected again after purification, and the activity of inhibiting staphylococcus aureus is measured again respectively. From the antibacterial effect, it is seen that there is an inhibitory effect of one component on staphylococcus aureus, wherein the diameter of the inhibition zone of one component reaches 11mm, and the component may contain lipopeptide substances due to the good antibacterial effect.

2. MALDI-TOF-MS authentication

And identifying the two components with good antibacterial effect after the second purification by adopting a Matrix-assisted laser desorption ionization time-of-flight mass spectrum (Matrix-Assisted Laser Desorption Ionization Time of Flight Mass, MALDI-TOF-MS).

The CHCA (alpha-cyano-4-hydroxy cinnamic acid) matrix was dissolved in a 30% acetonitrile aqueous solution containing 0.1% TFA (trifluoroacetic acid) at a matrix concentration of 16mg/mL and a sample concentration of 10mg/mL, the sample was treated, 1. Mu.L of the matrix solution was mixed with 1. Mu.L of the sample solution, and 0.5. Mu.L of the mixed solution was spotted on the sample target and naturally dried. The spectrogram is obtained by adopting a positive ion reflection mode, the detection range is 800-3000Da, the voltage is 20kV, and the reflection voltage is 18KV.8kV pulsed ions. The identity of the bacteriostatic component in the lipopeptides was determined analytically in combination with the mass spectrum (fig. 6) and mass spectrometry tables (table 5) and molecular weight information for bacillus lipopeptides in the relevant references (table 5).

Table 5 mass spectrometry table

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The results show that the lipopeptide components with antibacterial effect in the bacillus belicus SNBV-20 lipopeptide provided by the invention are surface active agents (Surfactin), fencine (fengycins) and Iturin (Iturin). The three types of lipopeptides have remarkable inhibitory effects on staphylococcus aureus.

Example 8 determination of the bacteriostatic function of lipopeptid produced by Bacillus bailii SNBV-20

1. Bacteriostasis observation of bacillus bailii SNBV-20 lipopeptid on staphylococcus aureus under scanning electron microscope

Inoculating activated staphylococcus aureus strain into 100mL of LB liquid medium according to 2% inoculum size, adding lipopeptid produced by bacillus beijerinckii SNBV-20 into the LB liquid medium to make the final concentration of the lipopeptid be 0.8 xMIC (the minimum inhibitory concentration of the lipopeptid on staphylococcus aureus is 1.25 mg/mL), culturing the lipopeptid at 33 ℃ at a shaking table rotating speed of 160r/min for 12h, and setting a control group.

The bacterial cells were collected by centrifugation to ensure the bacterial cell amount, washed with 0.9% physiological saline for 2 times, fixed with 2.5% glutaraldehyde at 4℃for 12 hours, the smear was air-dried, and the inhibitory effect of lipopeptid on Staphylococcus aureus was observed under a scanning electron microscope after plating, and the results are shown in FIG. 3.

As is evident from FIG. 3, the lipopeptid has a good inhibitory effect on staphylococcus aureus, and the lipopeptid can damage part of cell walls of staphylococcus aureus to form holes and even break cells, so that the lipopeptid produced by bacillus belicus SNBV-20 provided by the invention has a good antibacterial effect.

Besides staphylococcus aureus, the lipopeptid disclosed by the invention can effectively inhibit listeria monocytogenes, penicillium, escherichia coli and salmonella, and has a remarkable effect.

Sequence listing

<110> Shenyang agricultural university

<120> Bacillus bailii strain resistant to salt and high in lipopeptide yield and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1455

<212> DNA

<213> Bacillus bailii (Bacillus velezensis)

<400> 1

cgtggcgggg tgcctaatac atgcaagtcg agcggacaga tgggagcttg ctccctgatg 60

ttagcggcgg acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg 120

ggaaaccggg gctaataccg gatggttgtt tgaaccgcat ggttcagaca taaaaggtgg 180

cttcggctac cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct 240

caccaaggcg acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac 300

acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg 360

acggagcaac gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga 420

agaacaagtg ccgttcaaat agggcggcac cttgacggta cctaaccaga aagccacggc 480

taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg 540

gcgtaaaggg ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg 600

gagggtcatt ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta 660

gcggtgaaat gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt 720

aactgacgct gaggagcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca 780

cgccgtaaac gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaac 840

gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg 900

gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960

ggtcttgaca tcctctgaca atcctagaga taggacgtcc ccttcggggg cagagtgaca 1020

ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080

cgcaaccctt gatcttagtt gccagcattc agttgggcac tctaaggtga ctgccggtga 1140

caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200

cacgtgctac aatggacaga acaaagggca gcgaaaccgc gaggttaagc caatcccaca 1260

aatctgttct cagttcggat cgcagtctgc aactcgactg cgtgaagctg gaatcgctag 1320

taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380

acaccacgag agtttgtaac acccgaagtc ggtgaggtaa ccttttagga gccagccgcc 1440

gaagggggat cagag 1455

Claims (7)

1. The bacillus beleiensis (Bacillus velezensis) is characterized in that the bacillus beleiensis is preserved in the China general microbiological culture Collection center (China general microbiological culture Collection center) on the 09 th month 13 of 2021, and the preservation address is CGMCC No.23402 of the national institute of microbiology, national institute of sciences No. 3, north Chen West Lu 1, the Korean region of Beijing.

2. The bacillus belgium of claim 1, wherein the bacillus belgium has a 16s rDNA sequence of SEQ ID NO:1.

3. use of bacillus belgium according to claim 1 for the preparation of a food starter.

4. Use of bacillus belgium according to claim 1 for the preparation of an antibacterial composition.

5. Use of bacillus belgium according to claim 1 for lipopeptide production.

6. A method for producing a lipopeptide, comprising the steps of:

(1) Inoculating the activated bacillus beljalis of claim 1 into an LB liquid culture medium, and culturing for 24 hours at a constant temperature of 33 ℃ to obtain a fermentation broth;

(2) Centrifuging Bacillus bailii fermentation broth at 4deg.C and 10000rpm for 10min, and keeping supernatant;

(3) Adjusting pH value of the fermentation supernatant with hydrochloric acid to 2.0,4 ℃ and placing in a refrigerator for 12 hours;

(4) Centrifuging the fermentation broth after acid precipitation at 4deg.C and 10000rpm for 10min, removing supernatant, and collecting precipitate;

(5) Extracting the precipitate with methanol for 3 times repeatedly, mixing the extractive solutions, and adjusting pH to neutrality with sodium hydroxide;

(6) Filtering the methanol extracting solution by using a 0.22 mu m needle type filter membrane filter to obtain a lipopeptide crude extracting solution;

(7) And (3) carrying out reduced pressure suspension evaporation on the methanol solution by a reduced pressure rotary evaporator at the temperature of 45-55 ℃ to obtain a brown yellow solid substance, namely the lipopeptide.

7. The method of claim 6, wherein the lipopeptides comprise a surfactant, a phetamine, and an iturin.

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