A kind of Bacillus Velezii with high production of lipopeptide substances and its application

Abstract

The invention belongs to the field of biotechnology, and discloses a Bacillus Velezii with high yield of lipopeptide substances. After fermentation, the total yield of Surfactin obtained by simple purification is about 10 854 μg / mL, and the total yield of Iturin is about 537 μg / mL; the results of antibacterial experiments show that the fermentation broth has a certain inhibitory effect on Escherichia coli, Pseudomonas aeruginosa and Fusarium; liquid chromatography tandem mass spectrometry detects that the fermentation product contains two types of lipopeptide antibacterial active ingredients, Surfactin and Iturin, indicating that the isolated strain has the potential for broad-spectrum antibacterial. This study is expected to provide a new bacterial source library for the development of green microecological preparations and enrich the acquisition of Bacillus.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and more specifically, relates to a Bacillus Velezii capable of producing high amounts of lipopeptide substances and an application thereof. Background Art

[0002] The scientific name of red fish is Lutjanus erythrocephalus, which is an important marine economic fish in the South my country Sea. Its dried products are favored by the general public for their unique flavor, bringing huge business opportunities to the local fishery in Zhanjiang. However, most of the production processes of dried red fish retain traditional methods such as sun drying and pickling, which are inevitably affected by environmental factors and contaminated by bacteria, fungi and their toxins, resulting in a decline in the quality of dried red fish and even food safety incidents. The use of additives has a significant effect on ensuring product quality and extending the storage period, but there are frequent news reports about the excessive use of artificial additives and their hazards. Finding safe, reliable and green microecological preparations is more in line with consumers' needs for healthy diets.

[0003] Based on the research of many scholars, it is found that Bacillus has broad-spectrum antibacterial properties, and the existence of the spore structure makes the strain have strong stress resistance. Under appropriate culture conditions, many Bacillus can secrete lipopeptide metabolites with a broad antibacterial spectrum. These metabolites have no strict requirements on pH and are highly thermally stable, which undoubtedly makes this strain a leading strain of probiotics and biological control bacteria. The most widely studied lipopeptide substances are mainly surfactins and iturins, and their antibacterial mechanisms are also different. For example, surfactins can destroy the lipid membrane structure of bacteria, release intracellular substances and cause bacterial death; for example, iturins can create tiny pores in the fungal cell membrane, causing some important ions in the cell to leak out, thereby killing the microorganisms.

[0004] In recent years, marine Bacillus with broad-spectrum antibacterial activity has been continuously discovered, but there are few reports on the research of red fish-derived Bacillus. Red fish lives in an extreme marine environment characterized by low water temperature, low oxygen content, high salt content, and fewer nutrients than on land. This makes the Bacillus living in it have stronger environmental adaptability and contains the potential for rich antimicrobial lipopeptide components, which provides a good strain for the development of green and safe microecological preparations and expands the bacterial source library for marine Bacillus. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the above problems existing in the prior art. Firstly, a Bacillus Velezii with high yield of lipopeptide substances is provided.

[0006] The second object of the present invention is to provide the use of the above-mentioned Bacillus Velez.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A Bacillus Velezii strain, which is deposited in the Guangdong Provincial Microbiological Collection Center, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, the preservation number is GDMCC No: 62769, and the preservation date is September 6, 2022.

[0009] The invention obtains a bacillus having certain inhibitory activity on some gram-negative bacteria and fungi by separating the intestine of red fish, and the bacillus is identified as Bacillus Velezii.

[0010] Therefore, the present invention also provides the use of the Bacillus Velezii in inhibiting bacteria and fungi, wherein the bacteria are Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus; and the fungi are Fusarium oxysporum.

[0011] In addition, the present invention also provides the use of the Bacillus Velezii in the preparation of a microecological preparation for inhibiting bacteria and fungi, wherein the bacteria are Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus; and the fungus is Fusarium oxysporum.

[0012] The antibacterial substances produced by the red fish-derived Bacillus isolated this time were detected to contain two lipopeptide components, Surfactin and Iturin, through LC-MS.

[0013] Therefore, the present invention also provides the use of the Bacillus Velezii in producing lipopeptide substances, and the lipopeptide substances are Surfactin and Iturin.

[0014] Bacillus Velezii produces high amounts of lipopeptides. The total yield of Surfactin obtained by the fermentation method of the present invention after simple purification is about 10 854 μg / mL, and the total yield of Iturin is about 537 μg / mL.

[0015] As a specific implementation, the above application is: after the strain is activated, it is inoculated into the culture medium according to a certain inoculum amount, liquid culture is carried out, and the bacteria are removed to obtain a crude extract containing lipopeptide substances.

[0016] More specifically, in the above application, the inoculation amount of the Bacillus seed solution is 5%, and the fermentation medium is Landy medium (g / L): glucose 20.0g, L-glutamic acid 5.0g, MgSO4 0.5g, KCl 0.5g, KH2PO4 1.0g, FeSO4 0.15mg, MnSO4 5.0mg, CuSO4 0.16mg, distilled water 1000mL, pH 7.0. The liquid culture is carried out at 30°C for 12h at a rotation speed of 180rpm.

[0017] The invention also provides a microecological preparation containing the Velez subspore, and the microecological preparation has an inhibitory effect on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Fusarium oxysporum.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a Bacillus Velezii with high yield of lipopeptide substances. After fermentation, the total yield of surfactin obtained by simple purification is about 10 854 μg / mL, and the total yield of iturin is about 537 μg / mL; the results of antibacterial experiments show that the fermentation broth has a certain inhibitory effect on Escherichia coli, Pseudomonas aeruginosa and Fusarium; liquid chromatography tandem mass spectrometry detects that the fermentation product contains two types of lipopeptide antibacterial active ingredients, surfactin and iturin, indicating that the isolated strain has the potential for broad-spectrum antibacterial. This study is expected to provide a new bacterial source library for the development of green microecological preparations and enrich the acquisition of Bacillus. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the plate culture picture of the isolated strain;

[0021] Figure 2 The figure is a microscopic image of the isolated strain (×40);

[0022] Figure 3 The PCR amplification results of the 16sRNA gene of the isolated strain;

[0023] Figure 4 is the phylogenetic tree of the isolated strains;

[0024] Figure 5 The antibacterial activity of the isolated strains (Oxford cup method);

[0025] Figure 6 The antibacterial effect of the isolated strain on Fusarium oxysporum;

[0026] Figure 7 This is the ion chromatogram of Surfactin standard;

[0027] Figure 8 This is the ion chromatogram of Iturin standard;

[0028] Fig. 9 is the standard curve of 6 Surfactin components;

[0029] Fig.10 is the standard curve of the five Iturin components;

[0030] Fig.11It is the content of lipopeptide substances in each component of the fermentation product of the isolated strain. DETAILED DESCRIPTION

[0031] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0032] Unless otherwise specified, the test methods used in the following embodiments and experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels; the equipment used, unless otherwise specified, are all conventional experimental equipment.

[0033] Experimental samples: The redfin snapper samples were purchased from Xiashan Aquatic Products Wholesale Market near Zhanjiang waters. The red fish was bled, skinned, intestines removed, the fish meat was cut into pieces, packaged, and stored in a -20℃ refrigerator for later use.

[0034] Indicator bacteria: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Fusarium oxysporum. The indicator bacteria used in this experiment were all collected from the Aquatic Products Green Control Laboratory of the School of Food Science and Technology of Guangdong Ocean University.

[0035] Example 1

[0036] 1. Identification of strains

[0037] (1) Weigh fish intestines and fish meat respectively, cut them into pieces, put them into LB broth liquid culture medium, and culture them in a shaking incubator at 37°C for 24 hours to obtain enriched bacterial liquid; place the enriched bacterial liquid in an 80°C water bath for 20 minutes to kill heat-sensitive bacteria, take 1 mL of the bacterial liquid and add it to a test tube containing 9 mL of physiological saline, and dilute it in sequence to obtain 10 -3 -10 -8 For each dilution of the suspension, 100 μL was pipetted and poured onto a nutrient agar plate for culture.

[0038] (2) Morphological observation: Pick a single colony and purify it, and observe the colony morphology under natural light; according to the method in the Gram staining kit, pick a single colony for smearing, fixation, staining, and microscopic examination to observe the staining characteristics and cell morphology of the bacteria.

[0039] (3) Molecular biological identification: 16S rRNA identification was performed using universal primers, strain PCR reaction system (30 μL): upstream primer 5'-GAGAGTTTGATCCTGGCTCAG-3' (27F) and downstream primer 5'-CGGCTACCTTGTTACGAC-3' (1492R) (10 μmol / μL, 0.75 μL each), ddH2O (12.75 μL), 2×MightAmp Buffer (15 μL), MightyAmp DNA polymerase (0.75 μL); pick the purified single colony, add it to the system and mix well, as a DNA template. PCR amplification program: 98℃ pre-denaturation for 2 min; 98℃ 10 s, 58℃ 15 s, 68℃ 1 min, a total of 40 cycles; 72℃ extension for 10 min; 4℃ storage.

[0040] The amplified products were detected by agarose gel electrophoresis, and the expected products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were analyzed for homology in the NCBI official website database, and the related strain sequences with the highest similarity were downloaded, and the phylogenetic tree was constructed using MEGA5.1 software.

[0041] from Figure 1 and Figure 2 It can be seen that the isolated strain initially isolated from the red fish sample in the marine environment has round colonies, rough and opaque surface, slightly yellow, and wrinkled edges; after Gram staining, it is purple rod-shaped under a microscope, and is a Gram-positive bacterium. It was named HY02 and originated from the intestine of red fish.

[0042] After the isolated strains were amplified by PCR, the products were observed in a gel imaging system after electrophoresis. Figure 3 As shown, the length of the target band gene fragment is about 1500bp. The amplified product was sent to the detection sequence to obtain the 16S rRNA sequence suspected to be Bacillus.

[0043] According to the feedback of the 16S rRNA nucleotide sequence, a comparison was performed on the NCBI official website database. The results showed that the marine red fish-derived Bacillus HY02 had the highest similarity with Bacillus velezensis (MG752876.1), with a comparative similarity of 99.51%. Therefore, the strain was identified as Bacillus velezensis. The strain is deposited in the Guangdong Provincial Microbiological Collection Center, with the preservation address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The preservation number is GDMCC No: 62769, and the preservation date is September 6, 2022.

[0044] The sequences of the isolated strains were compared and analyzed using MEGA5.1 software, and then a phylogenetic tree was constructed. Figure 4 .

[0045] 2. Determination of antibacterial activity of isolated strains

[0046] (1) Preparation of fermentation broth: Activate the isolated strain stored on the slant, pick 1-2 rings of activated bacteria and inoculate them into 100 mL LB liquid medium, and culture them at 37°C and 150 r / min for 24 h. Then inoculate them into Landy medium at a 5% (v / v) inoculum, and culture them at 30°C and 180 r / min for 36 h. Centrifuge at 10,000 r / min for 20 min to remove the bacteria and set aside.

[0047] (2) Preparation of bacterial suspension: Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli were inoculated on agar slants, cultured for 12-24 hours, and then an appropriate amount of physiological saline was added and diluted and mixed. The OD was measured using a UV-visible spectrophotometer. 600 The concentration was about 1×10 7 CFU / mL bacterial suspension.

[0048] (3) Determination of bacterial antibacterial activity: The double-layer agar Oxford cup antibacterial test method was used. 1.5 mL of the indicator bacterial suspension was taken and added to the nutrient agar cooled to 55-60°C. The mixture was mixed to prepare the indicator bacterial culture medium. A layer of plain agar was poured on the culture dish. After solidification, an Oxford cup was placed on the agar. The indicator bacterial culture medium was spread on the plain agar as the second layer of agar. After cooling, the Oxford cup was taken out. 150 μL of fermentation liquid was added to the cup hole. The culture was cultured at 37°C for 12-24 h. The transparent antibacterial zone around the cup hole was observed and its diameter was measured and recorded.

[0049] (4) Determination of fungal antibacterial activity: 2 mL of Bacillus Velez fermentation broth was added to 50 mL of PDA medium cooled to 55-60°C, and mixed to prepare fermentation broth-PDA medium. A Fusarium cake was placed in the center of the medium plate using a hole puncher (inner diameter 4 mm), and cultured in a biochemical incubator at 28°C. The diameter of the growing hyphae was measured and compared with the blank group. The hyphae inhibition rate after 7 days of culture was calculated. The formula for determining the hyphae inhibition rate is: inhibition rate = (control colony diameter - treated colony diameter) / (control colony diameter - cake diameter) × 100%.

[0050] like Figure 5 As shown, the fermentation broth of the isolated strain has different degrees of antagonism against three common pathogens, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. It is speculated that the fermentation product of the strain contains antibacterial active substances, demonstrating the broad-spectrum antibacterial potential of marine red fish-derived Bacillus.

[0051] As shown in Table 1, strain HY02 had antibacterial activity against the above three indicator bacteria.

[0052] Table 1 Comparison of the antibacterial diameter of the fermentation broth of the strains in different indicator bacteria

[0053] Indicator bacteria Antibacterial diameter (mm) Staphylococcus aureus <![CDATA[11.29±0.28 a ]]> Escherichia coli <![CDATA[12.52±0.34 b ]]> Pseudomonas aeruginosa <![CDATA[12.61±0.92 b ]]>

[0054] Note: Different letters indicate significant differences among the data in the same row (p<0.05).

[0055] Inoculate Fusarium on the fermentation liquid-PDA plate and observe the mycelial growth after 7 days of cultivation. Figure 6 As shown in Table 2, compared with the blank control group with vigorous mycelium growth, the fermentation broth of the isolated strain can inhibit the growth of mycelium of Fusarium, the dominant spoilage fungus in dried red fish, to a certain extent. This experiment confirmed that the antimicrobial active substances secreted by the isolated strain can hinder the growth of fungi, and also provided the possibility of its application in the prevention and control of disease microorganisms.

[0056] Table 2 Antifungal activity of fermentation broth of isolated strains

[0057]

[0058] 3. Identification of crude lipopeptide extracts from isolated strains

[0059] The fermentation supernatant of the isolated strain was taken, the pH was adjusted to 2.0 with formic acid, kept at low temperature overnight, centrifuged at 5 000 r / min for 15 min, the precipitate was dissolved in methanol, the pH was adjusted to 7.0 with ammonia water, and centrifuged at 5 000 r / min for 20 min at 4 ° C. The supernatant was blown with nitrogen until there was no flowing liquid to obtain a crude lipopeptide extract. The components of antibacterial lipopeptides in the product were detected by LS-MS / MS. The crude lipopeptide extract was added to an equal volume of 7:3 acetonitrile / water solution (containing 0.1% formic acid), ultrasonically dissolved for 30 min, filtered into a sampling vial with a 0.45 μm organic phase filter membrane, and sampled for determination. The parameters were set with reference to the method of DENG et al.

[0060] The Surfactin standard solution and Iturin standard solution with a concentration of 5 μg / mL were selected for detection, and the ion flow diagrams of Surfactin and Iturin standards were obtained, as shown in FIG. Figure 7 , Figure 8 As shown, the molecular ion peaks corresponding to the surfactin standard are 995, 1008, 1022, and 1058; the molecular ion peaks corresponding to the iturin standard are 1036, 1044, 1057, and 1071.

[0061] Surfactin working solutions with concentrations of 0, 0.5, 1.0, 2.0, and 5.0 μg / mL and Iturin working solutions with the same concentration gradient were prepared respectively, and the standard curves of the respective homologues were obtained with the abscissa as mass concentration (μg / mL) and the ordinate as peak area, as shown in the following figure: Fig. 9 , 10 As shown, the correlation coefficient r of the standard curves of the two lipopeptide homologues 2 All of them were greater than 0.994, indicating that they had a good linear relationship within this concentration range.

[0062] The peak area of ​​the components of the Bacillus fermentation product was calculated using the standard curve to obtain the lipopeptide yield of each component, such as Fig.11 As shown, surfactin and iturin lipopeptides were present in the fermentation products of the strains, with mass-to-charge ratios of 995, 1 008, 1 022, 1 058 (surfactin), 1 036, 1 044, 1 057, 1 071 (iturin), respectively.

[0063] The difference in the composition and content of lipopeptide homologues may be one of the reasons for the different antibacterial abilities of the strains. The total production of Bacillus Surfactin and Iturin was calculated based on the standard curve corresponding to the peak area, as shown in Table 3, indicating that it has good application potential in the research and development of microecological preparations.

[0064] Table 3 Total production of Surfactin and Iturin from Bacillus

[0065] strains Total Surfactin production (μg / mL) Iturin total yield (μg / mL) HY02 <![CDATA[10854.0740±52.5680 a ]]> <![CDATA[537.6520±5.2030 a ]]>

[0066] Note: Different letters indicate significant differences among the data in the same row (p<0.05).

[0067] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A Bacillus Velez, characterized in that The strain is preserved in the Guangdong Provincial Microbiological Collection Center, with the preservation address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The preservation number is GDMCC No: 62769, and the preservation date is September 6, 2022.

2. The use of the Bacillus Velez subtilis according to claim 1 in inhibiting bacteria and fungi, or the use of the Bacillus Velez subtilis according to claim 1 in preparing a microecological preparation for inhibiting bacteria and fungi, characterized in that: The bacteria are Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa; the fungus is Fusarium oxysporum Fusarium oxysporum .

3. The use of Bacillus Velezii according to claim 1 in high-yield lipopeptide substances, characterized in that: The lipopeptide substances are Surfactin and Iturin.

4. The microecological preparation containing the Bacillus Velezii according to claim 1, characterized in that: The microecological preparation is effective against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Fusarium oxysporum Fusarium oxysporum It has an inhibitory effect.

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