CN114317317A - Salt-tolerant Bacillus belgii with high lipopeptide yield and application thereof - Google Patents

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides a salt-tolerant Bacillus velezensis with high lipopeptide yield and application thereof. The Bacillus beleisi sieve is selected from northeast traditional naturally fermented soybean paste, and the preservation number is CGMCC No. 23402. The strain can produce lipopeptide with high yield, can effectively inhibit common pathogenic bacteria in food 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

Salt-tolerant Bacillus belgii with high lipopeptide yield and application thereof

Technical Field

The invention relates to the technical field of functional microorganism screening and application, in particular to a novel salt-tolerant Bacillus belgii with high lipopeptide yield and application thereof.

Background

Lipopeptide is a secondary metabolite of bacillus and is produced and synthesized by a non-ribosomal polypeptide synthetase system formed by combining certain functional modules. Lipopeptide has the characteristics of low toxicity, high isoelectric point, stable physicochemical property, good heat resistance and acid-base property, safety, no toxicity, high stability and the like. Lipopeptides have strong biological effects, such as plant disease control, anti-tumor, anti-viral activity and broad antimicrobial spectrum, and thus lipopeptides are widely used in the fields of food, biomedicine, agriculture, and technology. Lipopeptides can be divided into different classes according to their amino acid composition and fatty acid chain length, the main classes of which are Surfactin (Surfactin), Iturin (Iturin) and Fengycin (Fengycin). The Surfactin lipopeptide is a substance with strong biological surface activity, can reduce the surface tension of plant roots, can be used as a detergent of a biological membrane, and has the effects of resisting ulcer, reducing cholesterol and resisting inflammation. The Iturin lipopeptide has good inhibitory action on plant pathogenic bacteria, has bacteriostatic action on most fungi and a small part of bacteria, is safe and nontoxic, and cannot damage the ecological environment, so that the Iturin 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 lipopeptide has strong hemolytic activity and fungus inhibiting effect, so that the Fengycin lipopeptide has good biocontrol value.

The most remarkable characteristic and advantage of the lipopeptide is that the lipopeptide has good antibacterial property and has 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 eat food by mistake to cause food poisoning and harm human health. Of all food safety incidents, staphylococcus aureus and listeria monocytogenes are the two major food-borne pathogenic microorganisms responsible for food-borne diseases. At present, most of methods for killing two food-borne pathogenic microorganisms adopt antibiotics for treatment, but with the abuse of the antibiotics, various drug-resistant bacteria appear, and the health of human beings is continuously harmed. Research shows that the antibacterial mechanism of lipopeptide is completely different from that of antibiotics, and lipopeptide destroys cell wall structure or changes the permeability of cell membrane to generate antibacterial effect through antagonistic action. In addition, fengycins type lipopeptides similar to the structure of AIP signal transduction molecules of staphylococcus aureus Agr quorum sensing channels are published in the journal of nature in 2018, and can be competitively combined with staphylococcus aureus Agr C receptor proteins to further inhibit staphylococcus aureus toxin synthesis and intestinal tract colonization. Therefore, the application of the lipopeptide can hopefully replace the traditional antibiotic treatment, has the antibacterial effect on staphylococcus aureus with drug resistance, effectively inhibits the problem of food-borne disease pollution, and ensures the food safety.

Currently, lipopeptide-producing microorganisms are mainly 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 producing lipopeptides, in a greater amount and variety than other microbial genera. And the bacillus is safe and low in toxicity, has no pathogenicity, is nontoxic and harmless to human beings and livestock, does not cause pollution to the environment and can produce lipopeptide with strong bacteriostasis, so that the bacillus is considered to be a safe probiotic microorganism and has been widely applied to food. Among the Bacillus species that produce lipopeptides in the food processing field are Bacillus subtilis, Bacillus licheniformis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens. The lipopeptide produced by the bacillus has low yield, narrow bacteriostatic spectrum and less obvious bacteriostatic effect, so that the application range and the application effect of the lipopeptide in food production are greatly limited. Therefore, screening out the bacillus with high yield and good antibacterial lipopeptide is a research hotspot in the field of current food processing.

Disclosure of Invention

The invention provides a salt-tolerant Bacillus velezensis (Bacillus velezensis) with high lipopeptide yield and application thereof for solving the problems of the prior art. The Bacillus beleisi sieve is selected from traditional naturally fermented soybean paste in northeast China, can produce lipopeptide at high yield, can effectively inhibit common pathogenic bacteria in food such as staphylococcus aureus, Listeria monocytogenes, penicillium, escherichia coli, salmonella and the like, and can be widely applied to the field of food production.

On one hand, the invention provides a strain of Bacillus belgii, which is named as Bacillus velezensis SNBV-20(Bacillus velezensis SNBV-20), and the strain is preserved in 13 days 2021 to China general microbiological culture Collection center at 09.13 months, the preservation address is the microbial research institute of China academy of sciences No. 3 North road 1 institute of Shangyng, Beijing, and the preservation number is CGMCC No. 23402.

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

the invention provides an application of Bacillus belgii SNBV-20 in preparing a food leavening agent.

The invention provides an application of Bacillus belgii SNBV-20 in preparing an antibacterial composition.

The invention also provides the application of the Bacillus belgii SNBV-20 in the production of lipopeptide.

The invention also provides a method for producing lipopeptide, which comprises the following steps:

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

(2) centrifuging the Bacillus belgii SNBV-20 fermentation liquid at 4 ℃ and 10000rpm for 10min, and reserving supernatant;

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

(4) centrifuging the acid-precipitated fermentation broth at 4 deg.C and 10000rpm for 10min, removing supernatant, and collecting precipitate;

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

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

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

The lipopeptide contains Surfactin (Surfactin), fengycins (fengycins) and Iturin (Iturin), and has a remarkable inhibitory effect on staphylococcus aureus.

The invention also provides an antibacterial composition comprising the lipopeptide.

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

The invention has the beneficial effects that:

the Bacillus beleisi SNBV-20 provided by the invention is screened from northeast naturally fermented soybean paste, has strong salt resistance and can grow well in a high-salt environment with the concentration of 10%. The strain has obvious inhibition effect on five common pathogenic bacteria in food, namely staphylococcus aureus, listeria monocytogenes, penicillium, escherichia coli and salmonella, wherein the inhibition effect on the staphylococcus aureus, the listeria monocytogenes and the penicillium is strongest, and the average diameter of an inhibition zone exceeds 15 mm.

The Bacillus belgii SNBV-20 can ferment high-yield lipopeptide, the extraction amount of the lipopeptide in the fermentation liquid is up to 0.135g/L, namely the extraction amount of the lipopeptide of each g of thallus is about 0.11g, and unexpected technical effects are achieved. The components with antibacterial effect in lipopeptide produced by Bacillus belgii SNBV-20 are Surfactin (Surfactin), fengycin (fengycins) and Iturin (Iturin). The three types of lipopeptides have significant inhibitory effects on staphylococcus aureus. The observation under a scanning electron microscope shows that lipopeptide can damage partial cell walls of staphylococcus aureus, form holes and even break cells, and the antibacterial effect is very obvious. The Bacillus belgii SNBV-20 can be widely applied to the production of natural lipopeptide, has short production period, high yield, safety and no toxic or side effect, and meets the requirements of the food industry.

The Bacillus belgii SNBV-20, the fermentation product thereof or the extracted lipopeptide can be used for producing the antibacterial composition singly or in combination and can be widely used for preparing food or medicines.

Drawings

FIG. 1 is a gram stain microscopy picture;

FIG. 2 is an electrophoretic detection map of PCR products;

FIG. 3 is a colony morphology map;

FIG. 4 is a phylogenetic tree;

FIG. 5 shows the results of thin layer chromatography for producing lipopeptide from Bacillus belgii SNBV-20;

FIG. 6 is a MALDI-TOF-MS and analysis table of the production of lipopeptides by Bacillus belgii SNBV-20;

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

Detailed Description

The screening method of the present invention is not limited to the examples, and any known method capable of achieving the screening purpose is possible, and the screening description of the examples is only illustrative of the present invention and is not limiting the scope of the present invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

The culture medium used in the embodiment of the invention and the formula thereof are as follows:

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

EXAMPLE 1 isolation and screening of lipopeptide-producing strains

1. Sample source

Traditional fermented soybean paste collected in Shenyang City of Liaoning province.

2. Primary screen for bacillus

A soybean paste sample (1 g) was taken, and placed in a test tube containing 9ml of 0.9% physiological saline, followed by thorough mixing. And (3) in order to only retain the bacillus, putting the uniformly mixed test tube into a water bath kettle at 85 ℃, heating in a water bath for 20-25min, and taking out. The test tube is taken out and then sequentially subjected to gradient dilution of 10 times, and the diluent is sterilized 0.9% physiological saline and is continuously diluted for 6 times. From a dilution gradient of 10^-5And 10^-6The two tubes in (2) each aspirate 100. mu.L of the diluted sample, which is plated onto LB solid plates using sterilized applicator rods. Observing the plate after culturing for 18-36h, picking part of single colony, transferring into liquid LB culture medium for culturing, simultaneously performing gram staining microscopy on the other same part of single colony, and observing morphological characteristics.

In the microscopic examination, 24 strains of blue, slender and rod-shaped single or chain-shaped bacteria which can partially have spore-like substances and have spores positioned in the middle of the bacteria and are circular are selected, and the microscopic examination result is shown in FIG. 1. Continuously activating and culturing the bacteria for three generations until the bacteria are pure bacteria.

Extracting DNA of each strain by using a bacterial DNA extraction kit, measuring and calculating the DNA concentration of each strain by using a ultramicro nucleic acid protein quantifier after extraction, and configuring a PCR system according to the concentration calculation.

TABLE 1 PCR System

The volume of DNA added and extracted is measured by a ultramicro nucleic acid protein quantifier, and the total amount of DNA added is required to be 100ng, but the maximum volume of DNA added is 10 ul. After the addition, the rest extracted DNA is put into a refrigerator at the temperature of 20 ℃ below zero for preservation, and is reserved for the subsequent re-screening of the bacillus producing lipopeptide.

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

23F：AGAGTTTGATCCTGGCTCAG；

1492R：TACGGCTACCTTGTTACGACTT。

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

The corresponding electrophoretic bands of about 1500bp were sent to Biotechnology engineering (Shanghai) GmbH for sequencing. The sequencing results were logged into the NCBI website for BLAST analysis. The sequence alignment results showed a total of 11 strains of Bacillus (Bacillus sp.).

3. Rescreening of lipopeptide-producing bacillus

And (3) detecting related genes for synthesizing the bacillus cyclic lipopeptide by adopting a PCR (polymerase chain reaction) technology, and re-screening the 11 strains of bacillus obtained by primary screening so as to screen out the bacillus mainly producing surfactin, camellin and iturin type lipopeptide.

(1) Configuring a 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 respectively taking the genome DNA of 11 strains of bacillus obtained by preliminary screening as a template. The primers were synthesized by Shanghai Senno biomedical science and technology, Inc.

The primer sequences are 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) And (3) recovering a PCR product:

the PCR product was recovered using AxyPrep DNA gel recovery kit (Boyao, ASJ0013), and the detailed procedures were performed according to the kit instructions.

(3) Re-screening the results

According to the amplification result, only one of 11 bacillus strains obtained by primary screening of the invention produces surfactin, camellin and iturin type lipopeptide simultaneously, and is named as SNBV-20.

Example 2 identification of SNBV-20 Strain

1. Colony morphology identification

The colony morphology of the SNBV-20 strain is shown in figure 3, and the colony is a microcolony which is white, circular and convex, and has a smooth, bright, opaque and wet surface and regular edges.

2. Physiological and biochemical identification

After activation of the SNBV-20 strain for three generations, the physiological and biochemical characteristics of the strain were identified with reference to "handbook of bacterial identification of Bergey (ninth edition)" and "Manual of identification of common bacterial systems" published by east elegans pearl and Zea Miaoying (2001) ". The specific results are shown in Table 4.

TABLE 4 physiological and biochemical identification results

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

3. Molecular biological identification

(1) Extraction of genomic DNA:

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

(2) And (3) PCR amplification:

PCR forward primer 23F and reverse primer 1492R were designed and synthesized by Shanghai bioengineering, Inc.

23F：AGAGTTTGATCCTGGCTCAG(5'---3')；

1492R：GGTTACCTTGTTACGACTT(5'---3')。

The PCR reaction conditions are as follows: preheating at 95 deg.C for 5min, denaturing at 94 deg.C for 1min, annealing at 50 deg.C for 1min, extending at 32 deg.C for 1min for 20s, and circulating for 36 times; keeping at 32 deg.C for 8min, and keeping at 4 deg.C.

(3) And (3) recovering a PCR product:

the PCR product was recovered using AxyPrep DNA gel recovery kit (Boyao, ASJ0013), and the detailed procedures were performed according to the kit instructions.

(4)16S rDNA sequencing and sequence alignment

The positive PCR product is sent to Shanghai Pesenuo Biotech Co., Ltd for sequencing, and the 16s rDNA sequence of the obtained SNBV-20 strain is SEQ ID NO: 1, as follows.

cgtggcggggtgcctaatacatgcaagtcgagcggacagatgggagcttgctccctgatgttagcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatggttgtttgaaccgcatggttcagacataaaaggtggcttcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctcaccaaggcgacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaagaacaagtgccgttcaaatagggcggcaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattgggcgtaaagggctcgcaggcggtttcttaagtctgatgtgaaagcccccggctcaaccggggagggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtctgtaactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgcattaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtgaggtaaccttttaggagccagccgccgaagggggatcagag。

Converting SEQ ID NO: 1 in NCBI database using BLAST tools with GenBank database existing sequences for alignment. The results show that the 16s rDNA sequence of the SNBV-20 strain SEQ ID NO: 1 has the highest similarity with Bacillus velezensis. Further, a phylogenetic tree was constructed using MEGA3.0, and as a result, as shown in FIG. 4, the SNBV-20 strain of the present invention has the highest homology with Bacillus subtilis (Bacillus velezensis).

In conclusion, according to the colony morphology, physiological and biochemical characteristics and molecular biological identification results of the strains, the SNBV-20 strain screened by the invention is a novel Bacillus belgii strain named as Bacillus velezensis SNBV-20.

The applicant reserves Bacillus subtilis SNBV-20(Bacillus velezensis SNBV-20) in China general microbiological culture Collection center (CGMCC for short, address: Beijing area Korean area Beichen Xilu No. 1 institute of microbiology, China academy of sciences, zip code 100101) at 13.9.2021 with the preservation number of CGMCC No. 23402.

Example 3 salt tolerance analysis of Bacillus belgii

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

Inoculating Bacillus belgii SNBV-20 into LB liquid culture medium with different salt concentrations according to the inoculation amount of 2% (v/v), respectively, culturing at the rotating speed of a shaking table at 33 ℃ of 160r/min for 24h, and taking the culture medium without inoculated bacteria as a blank control group. After the completion of the culture, the absorbance of each group was measured at a wavelength of 240nm using a microplate reader.

The results show that: the culture medium with salt concentration of 15% and 20% is relatively clear, and no thallus is found when the light absorption value is slightly microscopic. The medium with 10% salt concentration is turbid, the light absorption value is larger than that of the medium with 15% and 20% salt concentration, and the thalli are found to grow well in microscopic examination. Therefore, the Bacillus belgii SNBV-20 strain provided by the invention has stronger salt tolerance and can grow in a high-salt environment.

Example 4 measurement of bacteriostatic function of Bacillus belgii SNBV-20

Culturing Bacillus belgii in SNBV-20LB liquid culture medium at 33 deg.C and table rotation speed of 160r/min for 24 h. The activated bacillus was centrifuged at 15300 Xg for 10min and filtered through a 0.22 μm needle filter to obtain the supernatant.

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

Respectively sucking 100 mu L of pathogenic bacteria culture solution by using a liquid transfer gun, and pumping the pathogenic bacteria culture solution into a flat plate filled with LB solid culture medium for coating, wherein each pathogenic bacteria is repeatedly and parallelly repeated for three times, and 15 flat plates are counted. The sterilized filter paper sheets are cut into a circle with the diameter of 0.3cm, and five filter paper sheets are uniformly placed in a plate coated with pathogenic bacteria, wherein each filter paper sheet is provided with 4 layers. mu.L of the supernatant of Bacillus beiLeisi was added to the four filter paper sheets and cultured at 33 ℃ for 24 hours. And after the culture, the diameter of the inhibition zone of each oxford cup is measured and calculated by using a vernier caliper.

The results show that: the Bacillus belgii SNBV-20 has different inhibiting effects on five pathogenic bacteria, wherein the inhibiting effect on staphylococcus aureus, Listeria monocytogenes and penicillium is strongest, and the average inhibiting ring diameter exceeds 20 mm; the antibacterial agent also has certain inhibition effect on escherichia coli and salmonella, but the diameter of an inhibition zone reaches more than 12 mm.

Example 5 fermentation production method of Bacillus beilesiensis SNBV-20

The Bacillus beilis SNBV-20 is subjected to activated culture, a test tube slant liquid LB culture medium is taken as a starting point, the seed amplification culture is carried out, a constant temperature culture link is expanded to a secondary seed tank for culture, a fermentation tank is taken as an end point, sufficient liquid strains are provided for production, and the production requirement is met.

Inoculating the activated Bacillus belgii SNBV-20 strain into 1kg of LB liquid culture medium, and culturing at the constant temperature of 33 ℃ for 24h to form first-stage seeds; inoculating the primary seeds into a secondary seed tank filled with 10kg of LB liquid culture medium, adjusting the pH of the liquid culture medium to 3, and culturing at the constant temperature of 33 ℃ for 24 h. After the fermentation is finished, the fermentation liquor is centrifuged for 5min at the temperature of 4 ℃ and the rpm of 5000, and the weight of the thalli in the fermentation liquor is measured.

The results show that: after the seed amplification culture and the constant temperature culture link are expanded to the second-stage seeding tank culture, the weight of strains contained in the second-stage seeding tank is 356 g.

Example 6 extraction of lipopeptides produced by Bacillus belgii SNBV-20

The invention adopts a method of acid precipitation separation and organic solvent extraction to extract lipopeptide in the Bacillus belius SNBV-20 fermentation liquor. The specific method comprises the following steps:

the Bacillus belgii SNBV-20 fermentation broth obtained in example 5 was centrifuged at 10000rpm at 4 ℃ for 10min, and the supernatant was retained. Adjusting pH of the fermentation supernatant to 2.0 with hydrochloric acid, and standing in a refrigerator at 4 deg.C for 12 h. And then continuously centrifuging the fermentation liquor after acid precipitation under the conditions of 10min, 4 ℃ and 10000rpm, removing supernatant, and collecting the precipitate. Repeatedly extracting with methanol for 3 times, mixing extractive solutions, adjusting pH to neutral with sodium hydroxide, and filtering with 0.22 μm needle filter to obtain crude lipopeptide extractive solution. And (3) carrying out reduced pressure suspension evaporation on the crude extract to a methanol solution at the temperature of 45-55 ℃ by using a reduced pressure type rotary evaporator to obtain a brown yellow solid substance, namely the extracted lipopeptide, weighing, and calculating the extraction amount of the lipopeptide. After weighing, the lipopeptides were redissolved in methanol and stored in a refrigerator at 4 ℃.

The amount of lipopeptide extracted-the weight of the bottle after extraction-the weight of the empty bottle before extraction.

The results show that: through calculation, the extraction amount of lipopeptide in the SNBV-20 fermentation liquor of the Bacillus belgii reaches 0.135g/L, namely the expected extraction amount of lipopeptide per g of thallus is 0.11g, and unexpected technical effects are achieved.

Example 7 isolation and purification and characterization of the fractions of the lipopeptide produced by Bacillus beilesiensis SNBV-20

1. Thin layer chromatography

Two groups of silica gel GF254 thin layer chromatography plates A and B were taken, heated at 105 ℃ for 30min for activation, 10. mu.L of Bacillus beiensis lipopeptide extracted in example 5 was spotted on the plates, and the volume ratio of chloroform: methanol: water (65: 25: 4) as a spreading agent, drying, spraying 0.5% ninhydrin solution, and observing. And B, treating the thin plate by adopting an in-situ acid hydrolysis-ninhydrin color development method, putting the thin plate after the layer spreading 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 the color of a ninhydrin reagent after the hydrochloric acid is volatilized, and calculating the mobility (Rf) value.

Mobility (Rf) d1/d 2.

d 1: distance from origin to center of spot; d 2: distance from origin to solvent front.

The thin layer chromatography color development result is shown in figure 6, and the judgment of the mobility proves that the Bacillus belgii SNBV-20 produces lipopeptide which has not only cyclic lipopeptide but also open-loop or linear micromolecular peptide.

2. HPLC analysis

Using Shimadzu high performance liquid chromatograph, using C₁₈Separating and purifying components in the lipopeptide produced by the Bacillus beiLeisi SNBV-20 by a reverse phase column.

The separation and purification were carried out with an elution gradient of water/TFA as mobile phase A and methanol/TFA as mobile phase B, with the concentration of B increasing from 30% to 30% in 10min, followed by a final increase to 100% in 25 min. Manually collecting HPLC components, concentrating by using a centrifugal rotary evaporator, and respectively measuring the antibacterial activity of staphylococcus aureus by using a filter paper method, wherein only one component has an inhibiting effect on staphylococcus aureus. And (3) carrying out secondary separation and purification on the component, collecting four components again after purification, and respectively carrying out activity determination on the four components again. As seen from the bacteriostatic effect, the component has the inhibitory effect on staphylococcus aureus, wherein the diameter of the bacteriostatic circle of one component reaches 11mm, and the component possibly contains lipopeptide substances due to the good bacteriostatic effect.

2. MALDI-TOF-MS identification

And identifying the two components with good bacteriostatic effect after the second purification by Matrix-Assisted Laser Desorption Ionization Time of Flight Mass spectrometry (MALDI-TOF-MS).

Dissolving CHCA (alpha-cyano-4-hydroxycinnamic acid) matrix in 30% acetonitrile aqueous solution containing 0.1% TFA (trifluoroacetic acid), the matrix concentration being 16mg/mL, the sample concentration being 10mg/mL, treating the sample, mixing 1. mu.L of the matrix solution with 1. mu.L of the sample solution, spotting 0.5. mu.L of the mixture on the sample target, and naturally drying. And obtaining a spectrogram in a positive ion reflection mode, wherein the detection range is 800-3000Da, the voltage is 20kV, and the reflection voltage is 18 KV. 8kV pulse ion. The types of bacteriostatic components in the lipopeptides were determined analytically in combination with mass spectra (FIG. 6) and mass spectrometry tables (Table 5) and molecular weight information of the Bacillus lipopeptides in the relevant references (Table 5).

TABLE 5 Mass Spectrometry

The result shows that lipopeptide components with antibacterial effect in the lipopeptide produced by the Bacillus beiLensis SNBV-20 are Surfactin (Surfactin), fengycin (fengycins) and Iturin (Iturin). The three types of lipopeptides have significant inhibitory effects on staphylococcus aureus.

Example 8 measurement of inhibitory function of SNBV-20 lipopeptide production by Bacillus belgii

1. Bacteriostatic observation of staphylococcus aureus by Bacillus beleisis SNBV-20 lipopeptide produced under scanning electron microscope

Inoculating activated staphylococcus aureus strains into 100mL LB liquid medium according to the inoculation amount of 2%, adding lipopeptide generated by Bacillus bleekii SNBV-20 into the LB liquid medium to ensure that the final concentration of the lipopeptide is 0.8 × MIC (the minimum inhibitory concentration of the lipopeptide to staphylococcus aureus is 1.25mg/mL), culturing at 33 ℃ and the rotating speed of a shaking table of 160r/min for 12h, and meanwhile, setting a control group.

The thalli is collected centrifugally to ensure the bacterial quantity, the thalli is washed for 2 times by 0.9 percent of normal saline, the thalli is fixed for 12 hours by 2.5 percent of glutaraldehyde at the temperature of 4 ℃, the smear is air-dried, the inhibition effect of the lipopeptide on the staphylococcus aureus is observed under a scanning electron microscope after film coating, and the result is shown in figure 3.

As is evident from FIG. 3, lipopeptide has a good inhibitory effect on Staphylococcus aureus, and can damage part of cell walls of Staphylococcus aureus, form pores, and even break cells, indicating that lipopeptide generated by Bacillus bleekii SNBV-20 provided by the invention has a good antibacterial effect.

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

Sequence listing

<110> Shenyang agriculture university

<120> salt-tolerant Bacillus belgii with high lipopeptide yield and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1455

<212> DNA

<213> Bacillus belgii (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 (9)

1. The bacillus belgii is characterized in that the bacillus belgii is preserved in 13 days 09 and 2021 to China general microbiological culture Collection center, the preservation address is microbial research institute of China academy of sciences No. 3 of West Lu No. 1 of Beijing Korean district, and the preservation number is CGMCC No. 23402.

2. The bacillus belgii of claim 1, wherein the 16s rDNA sequence of the bacillus belgii is SEQ ID NO: 1.

3. use of the bacillus belgii of claim 1 for the preparation of a food leavening agent.

4. Use of the bacillus belgii of claim 1 for the preparation of an antibacterial composition.

5. Use of Bacillus belgii according to claim 1 for the production of lipopeptides.

6. A lipopeptide, wherein the lipopeptide is produced by a process comprising the steps of:

(1) inoculating the activated Bacillus belgii of claim 1 into LB liquid culture medium, and culturing at 33 deg.C for 24h to obtain fermentation liquid;

(2) centrifuging Bacillus beiLeisi fermentation liquid at 4 deg.C and 10000rpm for 10min, and retaining supernatant;

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

(4) centrifuging the acid-precipitated fermentation broth at 4 deg.C and 10000rpm for 10min, removing supernatant, and collecting precipitate;

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

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

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

7. The lipopeptide according to claim 6, comprising surfactin, camellin and iturin.

8. An antimicrobial composition comprising the lipopeptide of claim 6 or 3.

9. Use of the antibacterial composition of claim 8 for the preparation of a food or pharmaceutical product.

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