Streptomyces baubi GZ-10 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to streptomyces baubi GZ-10 and application thereof. The preservation number of the streptomyces baubi GZ-10 is GDMCC NO.65684, the strain name is Streptomyces boili, and the streptomyces baubi GZ-10 is separated from a soil sample in the dolomite area of Guangzhou City, Guangdong Province, is obtained through separation and purification by adopting a Gause No.1 agar culture medium, has red-based internal hyphae and white aerial hyphae, and can produce red soluble pigments. The streptomyces baubi disclosed by the invention has broad-spectrum bacteriostatic activity on various pathogenic bacteria and putrefying bacteria; according to the invention, antibacterial metabolites generated by the streptomyces baubi are brevibacterium peptide and cinereorubicin B; the streptomyces is simple in culture condition and easy for industrial production, and has potential application value in the field of microbial source preservatives.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a Streptomyces bobili GZ-10 and its application. Background Art

[0002] In food safety, food poisoning caused by foodborne pathogenic microorganisms and spoilage microorganisms has the largest number of cases. The main foodborne pathogenic microorganisms and spoilage microorganisms include Escherichia coli, Salmonella spp., Vibrio parahaemolyticus, Bacillus cereus, Staphylococcus aureus, as well as Aspergillus, Penicillium, etc.

[0003] Microbial-derived preservatives have a series of outstanding advantages such as high efficiency and safety, easy extraction of fermented active metabolites, more suitable for large-scale industrial production, easy control of economic costs, and meeting the requirements of high efficiency, safety, and health as natural preservatives. They have become the most promising development direction of natural preservatives in the future food industry. Among microbial resources, there are abundant active metabolite-producing strains, especially actinomycetes as representatives. Actinomycetes are a type of microbial resource that produces the most antibacterial compounds and other important secondary metabolites, and have a close relationship with human production and life. They have been widely used in medical, sanitary, agricultural and other fields. Therefore, among microbial resources, taking actinomycetes as the research object, screening for active actinomycetes with potential value for the development of microbial-derived preservatives against foodborne pathogenic bacteria and spoilage microorganisms, and excavating the antibacterial active metabolites of these strains have important research and application values for the development of new, highly efficient, safe, and healthy microbial-derived preservatives, and have broad development prospects. Summary of the Invention

[0004] To overcome the above defects, the purpose of the present invention is to provide a Streptomyces bobili GZ-10 and its application.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A Streptomyces bobili GZ-10, with a preservation number of GDMCC NO.65684.

[0007] Preferably, the Streptomyces bobili GZ-10 is isolated from a soil sample in the Baiyun Mountain area of Guangzhou City, Guangdong Province. The isolation and purification media are both Gao's No.1 agar media. It has red substrate mycelia and white aerial mycelia, and can produce red soluble pigments.

[0008] Application of Streptomyces bobili GZ-10 in preparing microbial source preservative.

[0009] Preferably, the microbial source preservative is a microbial preservative against foodborne pathogenic bacteria and foodborne spoilage fungi.

[0010] Preferably, the foodborne pathogenic bacteria are Escherichia coli, Salmonella, Staphylococcus aureus or Listeria monocytogenes.

[0011] Preferably, the foodborne spoilage fungi are Aspergillus niger or Aspergillus flavus.

[0012] Preferably, the genome size of Streptomyces bobili GZ-10 is 8993450 bp, which is a microbial group with a high GC content of 71.04%. The antibacterial metabolites produced by Streptomyces bobili GZ-10 are gramicidin and cinerubin B.

[0013] Positive and beneficial effects of the present invention:

[0014] 1. Compared with the prior art, Streptomyces bobili GZ-10 of the present invention has broad-spectrum antibacterial activity, especially can effectively inhibit foodborne pathogenic bacteria, including: Escherichia coli CMCC 44102, Salmonella typhimurium CMCC 50115, Staphylococcus aureus GDMCC 1.644, Listeria monocytogenes GDMCC1.347, and foodborne spoilage fungi Aspergillus niger CMCC98003, Aspergillus flavus GDMCC 3.626.

[0015] Based on the whole-genome sequencing analysis of the Illumina NEXTSEQ sequencing platform, the genome size of the strain is 8993450 bp. It is a microbial group with a high GC content of 71.04%. At the same time, based on the antiSMASH 4.0 database, the integrated genomic data is compared and analyzed with several databases such as GO, COG, and KEGG to predict the main functions and metabolic pathways of the strain. It is speculated that the antibacterial metabolites produced by the strain are Germicidin and Cinerubin B. Moreover, the culture conditions of this Streptomyces are simple and easy for industrial production, showing potential application value in the field of developing microbial-derived preservatives, filling the gap in the research of microbial-derived food preservatives of this type of strain, and providing a new approach and potential strains for developing new microbial-derived food preservatives.

[0016] Drawings of the specification

[0017] Figure 1 This is a scanning electron micrograph of the mycelium of Streptomyces bobili of the present invention. A - The resolution is 5 nm, and B - The resolution is 3 nm;

[0018] Figure 2 This is a growth diagram of Streptomyces bobili of the present invention on Gao's No. 1 agar medium;

[0019] Figure 3 This is an adjacent-joining phylogenetic tree based on the 16S rDNA gene sequence.

[0020] Figure 4 Using the Oxford cup method to test that the strain GZ-10 has a good antibacterial effect on Staphylococcus aureus (GDMCC 1.644). Detailed implementation manners

[0021] The present invention will be further described below in conjunction with some specific implementation manners.

[0022] A Streptomyces bobili GZ-10 was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on December 26, 2024, with the deposit number: GDMCC NO.65684; the address of the deposit unit is: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0023] Example 1

[0024] Identification of Streptomyces bobili GZ-10 strain

[0025] 1) Morphological characteristics of the strain

[0026] S1. Observation of the mycelium of strain GZ-10 by scanning electron microscope. The pretreatment operation method of the mycelium is as follows: Streptomyces bobiliae GZ-10 was isolated from soil samples in the Baiyun Mountain area of Guangzhou City, Guangdong Province. Strain GZ-10 was inoculated into Czapek-Dox liquid medium and cultured for 48 hours. The medium formula: soluble starch 20 g / L, KNO3 1 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.5 g / L, FeSO4·7H2O 0.01 g / L, pH = 7.4 - 7.6. 10 mL of the culture broth of strain GZ-10 with vigorous growth was centrifuged at 6000 r / min for 10 min in a centrifuge, and the supernatant was discarded to obtain the cultured mycelium. 2 mL of 2.5% glutaraldehyde fixative was aspirated with a 1000 mL pipette and added to the mycelium obtained after centrifugation, and after standing for 5 hours for fixation, the upper glutaraldehyde solution was aspirated away with a pipette;

[0027] S2. Dehydration was carried out according to the following steps: The mycelium obtained from the treatment in step S1 was eluted 6 times with PBS buffer, 20 min each time; ethanol gradient elution, eluted 2 times with 30% and 50% ethanol, 10 min each time; eluted 1 time with 70% and 90% ethanol, 15 min each time; eluted 3 times with absolute ethanol, 15 min each time; finally, continuously eluted 2 times with tert-butanol, 20 min each time;

[0028] S3. Critical point drying: Qualitative filter paper was cut into strips with a size of 35.0 mm × 18.0 mm with scissors. The part with a long side of 35.0 mm was evenly divided into 3 parts and folded into a pocket shape. The bacterial solution after the treatment in step S2 was dropped into the above filter paper and placed in a critical point dryer for CO2 critical point drying;

[0029] S4. Ion sputtering gold: The dried powdery pure mycelium obtained in step S3 was poured into a clean petri dish and gently shaken to disperse the mycelium. One side of the carbon conductive tape was stuck on a 1 / 4 cover glass, and the other side was gently pressed upside down on the mycelium powder. After turning it over, the mycelium on the cover glass was gently scraped thin and flattened. After ion sputtering gold, it can be observed on a HITACHI S-3000N type scanning electron microscope. Figure 1 It is the scanning electron micrograph of this strain.

[0030] By observing the strain under a scanning electron microscope, see Figure 1 , it was found that the spores formed in chains; the spore chains were long and flexuous; the spores were columnar, of uniform size, and smooth on the surface, indicating that this strain had the mycelial morphology of Streptomyces.

[0031] 2) Culture characteristics

[0032] The spores of strain GZ-10 were diluted and then spread on Gao's No.1 agar medium (soluble starch 20 g / L, KNO3 1 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.5 g / L, FeSO4·7H2O 0.01 g / L, agar 20 g / L, pH = 7.4 - 7.6), soy peptone medium (tryptone 15.0 g / L, soy papain hydrolyzate 5.0 g / L, sodium chloride 5.0 g / L, agar 15.0 g / L, pH 7.3 ± 0.2) and ISP1-3 series agar media (ISP1: tryptone 10 g / L, yeast extract powder 5 g / L, agar 15.0 g / L; ISP2: yeast extract powder 4.0 g / L, glucose 4.0 g / L, malt extract powder 10 g / L, agar 15.0 g / L; ISP3: oatmeal 20 g / L, NaCl 1 g / L, FeSO4·7H2O 0.001 g / L, MnCl2·4H2O 0.001 g / L, ZnSO4·7H2O 0.001 g / L, agar 15.0 g / L), and placed in an incubator at 28 °C for 5 - 7 days. Observe the color, shape and growth degree of aerial mycelium and substrate mycelium, whether there is pigment production, and colony characteristics. Among them, Figure 2 Figure 0000075 is the growth situation diagram of the strain on Gao's No.1 agar medium, and Table 1 shows the growth situation of strain GZ-10 on different media.

[0033] Table 1 Growth situation of strain GZ-10 on different media

[0034]

[0035] Combining Table 1 and Figure 2 It shows that Streptomyces bobili GZ-10 of the present invention is a culturable strain with good growth characteristics. Its morphological characteristics are: rich aerial mycelium, presenting white, and substrate mycelium being red on Gao's No.1 agar medium, soy peptone medium and ISP1-3. Red soluble pigment will be secreted in the later growth stage, which conforms to the morphological characteristics of Streptomyces.

[0036] 3) 16S rDNA sequence analysis and construction of phylogenetic tree

[0037] Extraction of total DNA of the strain: Scrape an appropriate amount of the strain to be tested from the Gao's No. 1 medium stored at low temperature (4°C) in the laboratory, inoculate it into 100 ml of liquid seed expansion culture medium, and culture it at 28°C for 72 h. Then, use a bacterial genomic DNA extraction kit (BIOMIGA, USA) to extract the total DNA of the strain. The operation steps are carried out according to the kit instructions. The obtained genome is detected by 0.8% agarose electrophoresis on a gel imager for the extraction effect and used for genome sequencing.

[0038] When determining the 16S rDNA sequence of the strain, the cloning primers for the gene sequence are universal bacterial primers: forward is 27f, and reverse is 1492r. Set the reaction condition parameters in the PCR amplifier as follows: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 1 min, annealing at 55°C for 40 s, extension at 72°C for 2 min, 30 cycles, constant temperature at 72°C for 10 min, and termination at 22°C. Sequence the PCR reaction product to obtain the 16S rDNA sequence of the strain. The 16S rDNA sequence information of the strain is shown as SEQ ID 1.

[0039] The 16S rDNA sequence of strain GZ-10 is compared with the 16S rDNA sequences of known related closely related strains in the NCBI online database by BLAST. Download the 16S rDNA sequences of the strain's related closely related strains through the database, perform multiple alignments using Clustal X 1.8 software, and then perform clustering analysis using Mega 8.0. Use the neighbor-joining (N-J) method, set the parameter of 1000 times Bootstrap, construct a phylogenetic tree for strain GZ-10 and analyze its phylogenetic status. Figure 3 The phylogenetic tree constructed for the phylogenetic analysis of the strain of the present invention shows that strain GZ-10 is Streptomyces bobili according to the established phylogenetic tree.

[0040] Example 2

[0041] Antibacterial activity test of the fermentation broth of Streptomyces bobili strain GZ-10

[0042] 1) Activation and fermentation culture of the strain

[0043] In a test tube, Streptomyces bobili GZ-10 was inoculated into 10 mL of Gao's No. 1 liquid medium for activation. The culture conditions were: 28 °C, shaking culture on a shaker at 160 r / min for 3 days. The activated strain was inoculated into 100 mL of Gao's No. 1 liquid medium in a triangular flask at an inoculation amount of 10%, and cultured at 28 °C, shaking culture on a shaker at 160 r / min for 7 days; after centrifuging the fermented broth of the cultured strain at 8000 r / min and 4 °C for 10 min, the fermentation supernatant was obtained.

[0044] 2) Antibacterial activity test of the fermentation supernatant of the strain

[0045] Foodborne pathogenic bacteria and foodborne spoilage fungi were used as indicator bacteria. The foodborne pathogenic bacteria were Escherichia coli CMCC 44102, Salmonella typhimurium CMCC 50115, Staphylococcus aureus GDMCC 1.644, Listeria monocytogenes GDMCC 1.347, and the foodborne spoilage fungi were Aspergillus niger CMCC 98003 and Aspergillus flavus GDMCC 3.626.

[0046] The culture plates of the indicator bacteria were prepared. An appropriate amount of solid medium that had been sterilized and not cooled was poured into a sterile petri dish in a sterile operating table. After the medium cooled and solidified, 100 μL of the indicator bacteria liquid was spread. The initial concentration of the bacteria liquid was 10 7 cfu / mL. The antibacterial test was carried out using the Oxford cup method. The Oxford cup was placed on the petri dish coated with the indicator bacteria. 100 μL of the fermentation supernatant was taken with a pipette and added into the Oxford cup, and the number was recorded. Then it was placed in a constant temperature incubator for culture. The bacterial indicator bacteria were placed at 37 °C for 24 h, and the fungal indicator bacteria were placed at 28 °C for 48 h. The size of the antibacterial zone around the Oxford cup on the culture plate was observed, and the diameter of the antibacterial zone was measured with a digital caliper and recorded. Figure 4 Figure 1 is the antibacterial diagram using the Oxford cup method, and Table 2 is the result of statistically analyzing the antibacterial activity of the fermented broth of Streptomyces bobili GZ-10 strain.

[0047] Table 2 Antibacterial situation of the active strains rescreened by the Oxford cup method

[0048]

[0049] From Table 2 and Figure 4It can be seen that the Streptomyces bobili strain of the present invention has broad-spectrum antibacterial activity against various pathogenic and spoilage bacteria such as Escherichia coli, Salmonella, Staphylococcus aureus, Listeria monocytogenes, Aspergillus niger, and Aspergillus flavus.

[0050] Example 3

[0051] Genome sequencing data processing and analysis of Streptomyces bobili GZ-10

[0052] 1) After extracting the total DNA of the strain according to Example 1, the whole genome sequencing was completed using the second-generation sequencing technology Illumina NEXTSEQ sequencing platform. The raw data obtained by sequencing was filtered, and all low-quality bases with a quality value ≤ 38, reads with N bases reaching 10 bp, and reads with an overlap with Adapter exceeding 15 bp were removed, and the sample host and duplication contamination were removed. Finally, the effective data (Clean Data) was obtained.

[0053] 2) The effective data (Clean Data) obtained after pretreatment was assembled using the SOAP denovo assembly software. According to the Paired-end information of the sequences, the Contig arrangement was determined. Sequences with a length greater than 500 bp were taken, and preliminary assembly results were obtained according to different K-mers. Then, the krskgf software was used to optimize and fill the holes in the preliminary assembly results, and the Contigs were connected into a genome sequence to obtain the final assembly result. The result was compared and analyzed with the COG, GO, and KEGG databases to obtain the genomic function annotation information of the strain. Based on the above data analysis and the comparison with the antiSMASH 4.0 database, the main functions and metabolic pathways of the strain were predicted, and the active metabolites of the strain were predicted.

[0054] 3) The SPADes assembly software was used to splice the Paired-end information data of the sequenced sequences. A total of 195 contigs with a total sequence information of 8,993,450 bp were obtained. Among them, the N50 size for judging the quality of genome splicing was 238,563 bp, the N80 size was 110,413 bp, and the N90 size was 64,620 bp; the GC content was 71.04%, the minimum length of the contigs was 128 bp, the maximum length was 952,940 bp, and the average length was 46,120 bp.

[0055] Secondary metabolites are a series of macromolecular substances that are non-essential for growth and are synthesized from primary metabolites by microorganisms during a certain growth period; see Table 3 for the annotation of secondary metabolite gene clusters and the prediction analysis of metabolites.

[0056] Table 3 Analysis of secondary metabolites of strain GZ-10 by antiSMASH 4.0

[0057]

[0058] As can be seen from Table 3, the Streptomyces bobiliae strain of the present invention has the ability to produce a variety of metabolites. Among them, the compounds Germicidin and Cinerubin B have obvious antibacterial abilities, verifying the broad-spectrum antibacterial ability of the strain. The compound Germicidin contains Germicidin A and Germicidin B and is a polyketide antibacterial compound, which has an obvious inhibitory effect on bacteria; Cinerubin B is an anthracycline antibacterial compound, which has an obvious inhibitory effect on fungi.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Any other modifications or equivalent replacements made by those of ordinary skill in the art to the technical solutions of the present invention shall be covered by the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solutions of the present invention.

Claims

1. A Streptomyces bobili GZ-10 with a preservation number of GDMCC NO. 65684.

2. The Streptomyces bobiliae GZ-10 according to claim 1, characterized in that, The Streptomyces bobili GZ-10 is isolated from a soil sample in the Baiyun Mountain area of Guangzhou City, Guangdong Province. The isolation and purification media are both Gao's No. 1 agar media. It has red substrate mycelium and white aerial mycelium and can produce red soluble pigments.

3. An application of Streptomyces bobili GZ-10 in preparing a microbial source preservative.

4. The application according to claim 3, characterized in that, The microbial source preservative is a microbial preservative against foodborne pathogenic bacteria and foodborne spoilage fungi.

5. The application according to claim 4, wherein The foodborne pathogenic bacteria are Escherichia coli, Salmonella, Staphylococcus aureus or Listeria monocytogenes.

6. The application according to claim 4, wherein The foodborne spoilage fungi are Aspergillus niger or Aspergillus flavus.

7. The application according to claim 3, wherein The genome size of the Streptomyces bobili GZ-10 is 8993450 bp. It is a microbial group with a high GC content of up to 71.04%. The antibacterial metabolites produced by the Streptomyces bobili GZ-10 are gramicidin and erythroglaucin B.

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