Lipopeptide extract with bacteriostasis and toxicity control activity and application of lipopeptide extract

Abstract

The invention aims to provide a lipopeptide extract with antibacterial and toxin-controlling activity and application of the lipopeptide extract, the lipopeptide extract provided by the invention is produced by bacillus amyloliquefaciens XJ-BV2007, the lipopeptide extract prepared by adopting the strain XJ-BV2007 has a remarkable inhibition effect on growth of alternaria alternata, and the lipopeptide extract can be used for preparing the lipopeptide extract with the antibacterial and toxin-controlling activity. The growth of alternaria alternata on the tomatoes can be effectively inhibited, the inhibition rate reaches 89.97%, and the inhibition rates on alternaria alternata toxins TeA, AOH, AME and TEN in the tomatoes are 100%, 98%, 99% and 53% (Plt; 0.01) of the substrate; meanwhile, the compound has a relatively strong control effect on synthesis of alternaria toxin, can be applied to biological prevention and control of alternaria toxin pollution in fruit and vegetable planting and post-harvesting stages, and has a relatively good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to the technical field of a lipopeptide extract with antibacterial and toxicity-controlling activities and its application. Background Art

[0002] Tomato (Lycopersicum esculentum Mill.) is an annual herbaceous plant of the Solanaceae family. It has strong antioxidant capacity and can lower blood pressure, clear away heat and detoxify. Tomatoes are usually divided into fresh tomatoes and processing tomatoes. my country is a major producer of processed tomatoes. According to data from the Food and Agriculture Organization of the United Nations, my country provided about 67 million tons of tomatoes in 2021, accounting for about 35% of the global production. With the improvement of processing and manufacturing levels, the market demand for tomatoes has gradually increased. However, processing tomatoes are susceptible to contamination by pathogenic microorganisms during growth, harvesting, storage, transportation, and processing. Especially during storage, tomatoes caused by Alternaria sp. suffer from severe post-harvest rot, which not only causes great yield losses, but also accumulates Alternaria toxins in tomatoes and their products that pose a potential threat to human health. Among them, tenuazonic acid (TeA) is the most toxic and detected among all Alternaria toxins, and is listed in the toxic chemical register by the US Food and Drug Administration. Alternariol (AOH) and alternariol monomethylether (AME) are acutely toxic and can show synergistic effects (Pinto et al., 2017). In 2011, according to the European Food Safety Authority, using the threshold of toxicological concern (TTC), the tolerable upper limit of intake for AOH and AME is 2.5 ng / kg body weight (bw), and the tolerable upper limit of intake for TeA and TEN is 1500 ng / kg body weight (bw). In 2022, the European Union (EU) issued Proposal 202 / 553 to amend Regulation (EC) No 401 / 2006 on the monitoring of Alternaria toxins in food and set guideline limit values ​​for the toxin content in processed tomato products, with AOH, AME and TeA not exceeding 10, 5 and 500 μg / kg respectively. Currently, there are few reports on the prevention and control of Alternaria fungus and its toxins in processing tomatoes.

[0003] Lipopeptide antibiotics are important active substances produced by Bacillus that have antibacterial and toxic effects. They have good antibacterial properties and are widely used in agriculture, food, pharmaceuticals, biotechnology and other fields. They have multiple biological activities such as anti-inflammatory, anti-tumor, antifungal, antiviral and antiplatelet. They have the advantages of high yield, simple production process, good stability, not easy to produce drug resistance, low toxicity to humans and animals, and environmental friendliness. In addition, lipopeptide antibiotics can promote bacterial colonization in plant roots and induce plant resistance. Some lipopeptides produced by Bacillus can inhibit the growth of plant pathogenic microorganisms in food as biological preservatives. Among them, the heterogeneous lipopeptides produced by Bacillus 6spp.LM7 have extremely high stability and a broad antibacterial spectrum, which may be closely related to the antibacterial activity of Chinese herbal medicines; Bacillus subtilis strain NCD-2 and its lipopeptide extracts have a strong inhibitory effect on the pathogens of cotton damping-off in vitro. These studies have remained on the inhibition of pathogenic fungi, and there have been no reports on biological toxins.

[0004] At present, spraying chemical fungicides is the main measure to prevent and control tomato fungal diseases and fungal toxin pollution. However, due to unfavorable factors such as high carcinogenicity, long degradation cycle, and severe environmental pollution, the use of chemical synthetic fungicides is becoming more and more standardized and even restricted. Relatively speaking, the use of safe, environmentally friendly, and efficient biological fungicides is more scientific, reasonable, and urgent. Summary of the invention

[0005] In order to solve the problem of controlling Alternaria and its toxins, and there is no technical problem reported for biological toxins, the present invention aims to provide a lipopeptide extract with antibacterial and toxin-controlling activity and its application. The lipopeptide extract provided by the present invention is produced by Bacillus amyloliquefaciens XJ-BV2007. The lipopeptide extract prepared by the strain XJ-BV2007 has a significant inhibitory effect on the growth of Alternaria, and can effectively inhibit the growth of Alternaria on tomatoes, with an inhibition rate of 89.97%. The inhibition rates of Alternaria toxins TeA, AOH, AME, and TEN in tomatoes are 100%, 98%, 99%, and 53% (P<0.01), respectively; at the same time, it has a strong control effect on the synthesis of Alternaria toxins, and can be used in the biological control of Alternaria toxin pollution in fruit and vegetable planting and post-harvest stages, and has good application prospects.

[0006] In order to achieve the above technical effects, the present invention is implemented through the following technical solutions.

[0007] In a first aspect, the present invention provides a lipopeptide extract with antibacterial and toxic control activity, wherein the lipopeptide extract is produced by Bacillus amyloliquefaciens XJ-BV2007; the lipopeptide extract consists of surfactin A, iturin A2, iturin A4, iturin A6 and fengycin.

[0008] In a second aspect, the present invention provides a method for preparing a lipopeptide extract having antibacterial and antitoxic activity, the method for preparing the lipopeptide extract comprising the following steps:

[0009] (1) Bacillus amyloliquefaciens XJ-BV2007 was inoculated onto NA solid medium with an inoculation needle for activation, and cultured in an incubator at 25-30° C. for 24-48 h;

[0010] (2) picking up a single colony of Bacillus amyloliquefaciens XJ-BV2007 activated in step (1) with an inoculating needle, transferring it to a flask containing NB liquid culture medium, placing the flask in a constant temperature shaker for shaking culture, and culturing it in a shaking incubator at 25-30° C. at a rate of 125-175 r / min for 24 h;

[0011] (3) The seed liquid of Bacillus amyloliquefaciens XJ-BV2007 obtained in step (2) was inoculated into SPY medium at an inoculum amount of 3.5-4.5%, and cultured at a rate of 125-175 r / min in a shaking incubator at 25-36° C. for 36-60 h. The supernatant was collected after centrifugation at 8000 g for 15 min, and filtered through a 0.22 μm sterile filter membrane to obtain a cell-free fermentation liquid of Bacillus amyloliquefaciens XJ-BV2007. The bacterial cell concentration was measured to be 1-9×10 8 CFU / mL, the desired fermentation broth is obtained;

[0012] (4) Take 300 mL of the cell-free fermentation broth of Bacillus amyloliquefaciens XJ-BV2007 obtained in step (3), adjust the pH to ≤ 2.0 with 6.5-7.5 mol / L hydrochloric acid, and store it in a refrigerator at 4°C overnight; centrifuge at 10000 g for 10 min, collect the precipitate; extract at least twice with methanol of 3 times the volume of the precipitate, and adjust the pH to neutral with 1 mol / L sodium hydroxide; dry the methanol extract by rotary evaporator at 40°C, and filter it through a hydrophilic PTFE double-layer membrane with a pore size of 0.22 μm to finally obtain 2 mL of lipopeptide extract.

[0013] The SPY medium used in the preparation of the fermentation broth in the present invention is: 6 g / L maltose, 5 g / L yeast extract, and 9 g / L peptone.

[0014] In the step (3) of the present invention, the seed liquid inoculation amount of Bacillus amyloliquefaciens XJ-BV2007 is 4%, the fermentation temperature is 34° C., and the culture is carried out in a shaking incubator at a rate of 125 to 175 r / min for 48 hours.

[0015] The lipopeptide extract with antibacterial and antitoxic activity described in the present invention is in the form of liquid.

[0016] In a third aspect, the present invention provides the use of the above-mentioned lipopeptide extract with antibacterial and toxic control activity in preventing and controlling Alternaria toxin contamination in fruits and vegetables.

[0017] Furthermore, the application of the lipopeptide extract described in the present invention in preventing and treating Alternaria toxin contamination in fruits and vegetables comprises the following steps:

[0018] The concentration is 1~9×10 8 The lipopeptide extract with antibacterial and toxic control activity produced by Bacillus amyloliquefaciens XJ-BV2007 at CFU / mL was directly sprayed on the surface of biological samples during the growth stage of fruits and vegetables.

[0019] In a fourth aspect, the present invention provides the use of the above-mentioned lipopeptide extract with antibacterial and toxic control activity in preventing and controlling Alternaria toxin contamination in processing tomatoes.

[0020] Furthermore, the prevention and control of Alternaria toxin contamination in processing tomatoes in the present invention includes inhibiting at least one of black spot disease or Alternaria toxin synthesis.

[0021] By implementing the above technical solution provided by the present invention, the present invention obtains the following technical effects:

[0022] (1) The lipopeptide extract obtained by the present invention has antibacterial and antitoxic activity, and has a strong inhibitory effect on the main fungal toxins in Altemariaaltemata XJ-FQ72, such as tenuazonic acid (TeA), alternariol (AOH) and alternariol monomethyl ether (AME). The lipopeptide extract produced by Bacillus amyloliquefaciens XJ-BV2007 showed a good inhibitory effect on the growth of Alternaria in a plate confrontation test, with an inhibition rate of 56%, and the inhibition rates of TeA, AOH, AME and TEN were 99%, 46%, 95% and 63% respectively (P<0.01).

[0023] (2) The lipopeptide extract produced by Bacillus amyloliquefaciens XJ-BV2007 of the present invention can effectively inhibit the growth of Alternaria alternata on tomatoes under room temperature storage conditions, with an inhibition rate of 89.97%. The inhibition rates of Alternaria alternata toxins TeA, AOH, AME, and TEN in tomatoes are 100%, 98%, 99%, and 53% (P<0.01), respectively, with good prevention and control effects. Therefore, the lipopeptide extract produced by Bacillus amyloliquefaciens XJ-BV2007 can be used to prevent and control the contamination of tomato Alternaria alternata toxins.

[0024] (3) The lipopeptide extract obtained by the present invention was applied to the field spraying of processing tomatoes. With clean water as the control, it could effectively inhibit the growth of Alternaria toxin TeA on tomatoes, with an inhibition rate of 95.20%. At the same time, it could effectively reduce the incidence of black spot disease in processing tomatoes, from 17.94% in the control group to 6.25% in the lipopeptide treatment group.

[0025] (4) The lipopeptide extract produced by Bacillus amyloliquefaciens XJ-BV2007 of the present invention is applied to the prevention and control of Alternaria contamination in processing tomatoes, which can overcome a series of problems caused by the use of chemical pesticides, and is thus conducive to the pollution-free production of agricultural products to improve product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing the inhibitory effect of the lipopeptide extract produced by Bacillus amyloliquefaciens XJ-BV2007 on XJ-FQ72 hyphae in solid culture medium;

[0027] Figure 1 -a is the control group: Alternaria alternata cultured only on solid surface; Figure 1 -b is the experimental group: lipopeptide extract produced by solid culture of Bacillus amyloliquefaciens XJ-BV2007 and Alternaria alternata.

[0028] Figure 2 The effect of lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 on the biomass of XJ-FQ72;

[0029] Figure 2 -a is the control group: solid culture medium without addition of lipopeptide extract; Figure 2 -b is the experimental group: solid culture medium with lipopeptide extract added.

[0030] Figure 3 The inhibitory effect of the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 on XJ-FQ72;

[0031] Figure 3 The control group was: only Alternaria alternata was cultured in liquid; the experimental group was: the lipopeptide extract produced by Bacillus amyloliquefaciens XJ-BV2007 and Alternaria alternata were cultured in liquid at the same time.

[0032] Figure 4 After being treated with lipopeptide extracts, the mycelial structure of XJ-FQ72 collapsed and broke, and the spore morphology was twisted and broken, as observed by scanning electron microscopy;

[0033] Figure 4 -a and Figure 4 -b is mycelium; Figure 4 -c and Figure 4 -d is the characteristic diagram of spore morphology.

[0034] Figure 5 This is the effect of lipopeptide extract treatment on the expression of genes related to XJ-FQ72 synthesis.

[0035] Figure 6 The control effect of lipopeptide extract on Alternaria toxin in processing tomatoes by field spraying;

[0036] Figure 6 -a is the effect of field lipopeptide extract spraying experiment on the incidence of black spot disease in processing tomatoes; Figure 6 -b is the effect of lipopeptide extract on the content of Alternaria toxin TeA in processing tomatoes.

[0037] Figure 7 To process the tomato black spot disease incidence in the experimental group and the control group on the 7th day after the lipopeptide extract was treated in the tomato biocontrol experiment;

[0038] Figure 7 -a is the control group: inoculated with only the spore suspension of Alternaria alternata; Figure 7 -b is the experimental group: the lipopeptide extract produced by Bacillus amyloliquefaciens XJ-BV2007 and the spore suspension of Alternaria alternata were inoculated at the same time.

[0039] Figure 8 The diameter of tomato lesions in the experimental and control groups treated with lipopeptide extracts during storage for 7 days in a tomato biocontrol experiment.

[0040] Figure 8 The control group was inoculated with only the spore suspension of Alternaria alternata; the experimental group was inoculated with the lipopeptide extract produced by Bacillus amyloliquefaciens XJ-BV2007 and the spore suspension of Alternaria alternata at the same time.

[0041] Fig. 9 The inhibition rate of lipopeptide extract on Alternaria toxin in processing tomatoes after 7 days of storage in the biocontrol experiment of processing tomatoes; Fig. 9 -a is the inhibition rate of lipopeptide extract treatment on Alternaria toxin TeA in processing tomatoes after storage for 7 days; Fig. 9 -b is the inhibition rate of lipopeptide extract treatment on Alternaria toxin AOH in processing tomatoes after storage for 7 days; Fig. 9 -c is the inhibition rate of lipopeptide extract treatment on Alternaria toxin AME in processing tomatoes after 7 days of storage; Fig. 9 -d is the inhibition rate of lipopeptide extract treatment on Alternaria toxin TEN in processing tomatoes after 7 days of storage; Fig. 9 The control group was inoculated with only the spore suspension of Alternaria alternata; the experimental group was inoculated with the lipopeptide extract produced by Bacillus amyloliquefaciens XJ-BV2007 and the spore suspension of Alternaria alternata at the same time. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. In the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. If not specifically stated, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0043] The Bacillus amyloliquefaciens XJ-BV2007 strain involved in the present invention has a deposit number of CGMCC NO.23669, a deposit date of October 26, 2021, and a deposit unit of the General Microbiology Center of the China Microorganism Culture Collection Administration. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The general public can contact the General Microbiology Center of the China Microorganism Culture Collection Administration to purchase it.

[0044] The pathogen involved in the present invention is Altemaria altemata XJ-FQ72, the deposit number of the strain is CGMCC NO.40624, the deposit date is May 12, 2023, the deposit unit is the General Microbiology Center of the China Microorganism Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The general public can contact the General Microbiology Center of the China Microorganism Culture Collection Administration to purchase it. Hereinafter referred to as XJ-FQ72.

[0045] The culture medium used in the present invention is a NA culture medium, the components of which are: 0.2-0.5% beef extract, 0.2-0.8% peptone, 0.5-1.5% glucose, 1.5-2.0% agar, prepared with water, pH 7.0-7.2, and the percentages in the culture medium components are weight ratios;

[0046] The NB culture medium comprises: 0.2-0.5% beef extract, 0.2-0.8% peptone, 0.5-1.5% glucose, prepared with water, pH 7.0-7.2, and the percentages in the culture medium components are all weight ratios.

[0047] The components of SPY culture medium are: 0.2%-0.3% maltose, 0.15%-0.3% yeast extract powder, 0.5%-0.9% peptone, and 0.01%-0.05% sodium chloride, prepared with water, pH 7.0-7.2, and the percentages in the culture medium components are all weight ratios.

[0048] The PDA culture medium components are: 0.3%-0.5% potato powder, 1.5%-2% glucose, 1.5%-2% agar, 0.01%-0.03% chloramphenicol, prepared with water, pH 7.0-7.2, and the percentages in the culture medium components are all weight ratios.

[0049] The components of the PDB culture medium are: 0.3%-0.5% potato extract powder, 1%-2% peptone, 1.5%-3% glucose, 0.3%-0.5% sodium chloride, prepared with water, pH 7.0-7.2, and the percentages in the culture medium components are all weight ratios.

[0050] Example 1: Preparation and identification of lipopeptide extracts

[0051] (I) Preparation of lipopeptide extract

[0052] (1) Activation of strains: Bacillus amyloliquefaciens XJ-BV2007 was inoculated onto NA solid medium using an inoculation needle for activation and cultured in an incubator at 27°C for 24 h;

[0053] (2) Preparation of seed solution: Use an inoculation needle to pick up a single colony of Bacillus amyloliquefaciens and transfer it to a flask containing NB liquid culture medium. Place the flask in a constant temperature shaker for shaking culture and culture at a shaking incubator at 28°C and a rate of 150 r / min for 24 h.

[0054] (3) Preparation of fermentation broth: The carbon source and its addition amount, nitrogen source and its addition amount, fermentation time, temperature, inoculation amount and liquid volume in SPY fermentation broth were optimized by single factor experiment and response surface experiment, and the optimal fermentation process was obtained: maltose 6 g / L, yeast extract 5 g / L, peptone 9 g / L, fermentation time 48 h, inoculation amount 4%, liquid volume 300 mL and fermentation temperature 34 ° C; Bacillus amyloliquefaciens obtained in step (2) was added to the broth. The seed liquid of Bacillus amyloliquefaciens XJ-BV2007 was inoculated into SPY medium with an inoculation amount of 4%. After culturing at 130 r / min in a shaking incubator at 34°C for 48 h, the supernatant was collected after centrifugation at 8000 g for 15 min and filtered through a 0.22 μm sterile filter membrane to obtain the cell-free fermentation broth of Bacillus amyloliquefaciens XJ-BV2007 with a cell concentration of 1.98×10 8 CFU / mL;

[0055] (4) Preparation of lipopeptide extract: Take 300 mL of the cell-free fermentation broth prepared in step (3), adjust the pH to 2.0 with 7 mol / L hydrochloric acid, and store in a 4°C refrigerator overnight; centrifuge at 10,000 g for 10 min, collect the precipitate; extract at least twice with methanol 3 times the volume of the precipitate, and adjust the pH to neutral with 1 mol / L sodium hydroxide; dry the methanol extract with a rotary evaporator at 40°C, and filter through a hydrophilic PTFE double-layer membrane with a pore size of 0.22 μm to finally obtain 2 mL of lipopeptide extract;

[0056] (5) Purification of lipopeptide extracts: The lipopeptide extracts were purified by semi-preparative HPLC using a PREP HS C18 (5 μm, 10 × 250 mm) column. The mobile phase was methanol and water (80:20, v / v), the flow rate was 3 mL / min, and the total running time was 30 min. The eluent was collected every 1 min, and the purified fractions were identified by ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry.

[0057] (II) Identification of lipopeptide extracts

[0058] (1) Experimental Materials

[0059] The 2 g / mL lipopeptide extract was purified using step (5) of preparing the lipopeptide extract in (I).

[0060] (2) Experimental methods

[0061] The lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 was identified by UHPLC-MS. The test conditions were as follows: an HSS T3 (1.7 μm, 2.1 × 100 mm) column was used, and the mobile phase was acetonitrile and ultrapure water. The flow rate was set to 0.3 mL / min. QTOF MS analysis was performed in positive ion mode (ESI+) at a capillary voltage of 3.5 kV, a desolvation temperature of 370 °C, a source temperature of 115 °C, a desolvation gas flow of 650 L / h, and a conical nitrogen flow of 45 L / h, and the injection volume was 10 μL.

[0062] The acquisition mode was high definition mass spectrometry (HDMSE), and the low and high collision energies were set to 6 eV and 60 eV, respectively. Data were collected between 50 and 1500 m / z, and a leucine enkephalin solution (0.2 ng / L) was injected into the ion source every 5 minutes for lock mass calibration (m / z 556.2766 was used as a reference ion in positive ion mode). Ion mobility and mass calibration were performed using the Major Mix IMS / TOF calibration kit (Waters Corporation).

[0063] (3) Experimental results

[0064] The lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 was identified by UHPLC-MS. As shown in Table 1, the components of different lipopeptide extracts were qualitatively and quantitatively identified, including surfactin A, iturin A2, iturin A4, iturin A6 and fengycin, indicating that the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 produced surfactin, iturin and fengycin, among which fengycin was the main antagonist.

[0065] Table 1: Composition of lipopeptide extract of strain XJ-BV2007

[0066]

[0067] Example 2: Antagonistic experiment of lipopeptide extract on XJ-FQ72

[0068] (1) Experimental Materials

[0069] The preparation method of the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 is as described in Example 1. The pathogen XJ-FQ72 was cultured on a plate for 5-7 days in advance.

[0070] Preparation of spore suspension: Take the XJ-FQ72 PDA medium that has been cultured for 7 days, gently scrape the XJ-FQ72 spores with a disposable sterile applicator, add an appropriate amount of sterile water to rinse the surface of the medium, filter it into a 50mL glass triangular flask using four layers of gauze, vortex quickly for 30 seconds to mix the spores evenly, count the number of spores using a hemocytometer, and add sterile deionized water to adjust the XJ-FQ72 spore concentration to 10 5 Pieces / mL.

[0071] (2) Experimental methods

[0072] Solid culture medium inhibition experiment: The colony diameter was measured by the cross method. The inhibition rate was calculated by measuring the diameter of mycelial growth. 5 mL of lipopeptide extract and PDA medium were mixed evenly in a ratio of 1:3. After cooling and solidification, sterile glass paper was spread on the surface of the PDA medium. XJ-FQ72 cakes with a diameter of 5 mm were inoculated in the center of the medium and cultured at 28°C for 7 days. The colony diameter was measured every day, and the culture medium without the addition of Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract was used as a control. The effect of Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract on the growth of XJ-FQ72 was detected. Three replicates were made in each group, and the culture was cultured at 28°C for 7 days, and the diameter of the inhibition zone was measured.

[0073] Liquid medium inhibition experiment: 6 mL of Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract (4 μg / mL) and 6 mL of XJ-FQ72 spore suspension (10 5 spores / mL) were mixed into 6 mL of PDB liquid medium, and only 6 mL of XJ-FQ72 spore suspension (10 5 100 mL / mL) and 12 mL PDB were used as controls. The cells were cultured at 28°C and 150 rpm / min in a shaking incubator for 7 days, and the growth of XJ-FQ72 was observed. Three replicates were performed for each group.

[0074] (3) Experimental results

[0075] The inhibition rate was calculated by measuring the mycelium growth diameter according to the following formula. The test results are shown in Figure 1 As shown in Table 2, the results show that the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 has an inhibitory effect on XJ-FQ72, with an inhibition rate of 56%.

[0076] Bacterial inhibition rate (%) = [(mycelium diameter of control group - mycelium diameter of experimental group) / mycelium diameter of control group] × 100%

[0077] Table 2: Inhibition rate of XJ-FQ72 growth diameter by lipopeptide extract on day 7 of inoculation

[0078]

[0079]

[0080] In order to further explore the inhibitory effect of Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract on XJ-FQ72, a liquid shaking culture experiment was conducted. The results are shown in the attached Figure 2 As shown, in the control group without the addition of lipopeptide extract, XJ-FQ72 grew significantly in large quantities, showing gray-brown spherical shapes, and the color of the PDB liquid culture medium became darker; as shown in Table 2, after treatment with the lipopeptide extract, there was a significant inhibitory effect on XJ-FQ72, with an inhibition rate of 56%. Only a small amount of XJ-FQ72 grew in the PDB, and the color of the culture medium was yellow and transparent, which was lighter than that of the control group.

[0081] Example 3: Effect of lipopeptide extract on XJ-FQ72 biomass

[0082] (1) Experimental Materials

[0083] The lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 was prepared by the method described in Example 1. The pathogen XJ-FQ72 was cultured on a plate for 5-7 days in advance.

[0084] (2) Experimental methods

[0085] 5 mL of Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract was mixed with PDA medium at a ratio of 1:3. After cooling and solidification, sterile cellophane was spread on the surface of PDA medium, and XJ-FQ72 bacterial cake with a diameter of 5 mm, which was cultured at 28°C for 7 days, was inoculated in the center of the medium. After culturing at 28°C for 7 days, the cellophane was removed, weighed, and XJ-FQ72 mycelium was collected for subsequent experiments. The weight difference of the bacterial cake and the cellophane before and after cultivation was used to characterize the biomass. The culture medium without lipopeptide extract was used as the control, and 3 replicates were made in each group.

[0086] (3) Experimental results

[0087] Biomass is one of the important indicators for detecting mycelial growth, reflecting the accumulation of substances during growth and metabolism. Figure 3As shown, the biomass of XJ-FQ72 was effectively reduced after treatment with the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007; during the 7-day culture period, the biomass of XJ-FQ72 treated with the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 was reduced by 18.5%, 28.2%, 42.6%, 49.3%, 53.6%, 57.5%, and 60.1% (P<0.05) compared with the control group, respectively. This indicates that the treatment with the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 significantly inhibited the growth of mycelium and spores of XJ-FQ72.

[0088] Example 4: Effect of lipopeptide extract on the biological structure of XJ-FQ72

[0089] (1) Experimental Materials

[0090] The lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 was prepared by the method described in Example 1. The spore suspension was prepared by the method in Experiment (1) of Example 2.

[0091] (2) Experimental methods

[0092] Add 1 mL of 10 5 The XJ-FQ72 spore suspension (without gauze filtration) with a concentration of 100 / mL was centrifuged at 8000g for 6 min at 20°C, the supernatant was discarded, 2 mL of Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract and 2 mL of PDB were added, and sterile deionized water was used as a control, and the mixture was cultured at 28°C for 24 h. Each group was repeated three times. Rinse three times with 0.1M PBS, place the treated samples in 2.5% glutaraldehyde, and fix them in a 4°C refrigerator for 24 h. After rinsing with PSB, fix them with 1% osmium tetroxide for 1 h, and dehydrate them with 50%, 70%, 80%, 90%, and 100% tert-butanol gradients in sequence, with each level being 15 min. Tert-butanol that covers the sample was placed in the sample box and placed at 4°C. After the tert-butanol was frozen, the sample was critically dried, bonded, and coated; finally, the obtained sample was used for scanning electron microscopy observation.

[0093] (3) Experimental results

[0094] Scanning electron microscopy was used to observe the structural changes of XJ-FQ72, and it was found that after being treated with the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007, the mycelium of XJ-FQ72 shrank and the morphology changed significantly. Figure 4 -a, under a 2000x microscope, it can be seen that the surface of XJ-FQ72 mycelium has slight wrinkles and the mycelium is slightly twisted, but the mycelium is round and plump, with uniform thickness. Figure 4 -b, the mycelium of XJ-FQ72 was shriveled and rough, with obvious ridges visible on the surface, the mycelium structure collapsed and broke, and lost its cylindrical structure.

[0095] After treatment with the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007, the morphology of XJ-FQ72 spores also changed significantly. Figure 4 -c, the spores in the control group were wrinkled and slightly concave; after being treated with the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007, the spores of the attached Figure 4 -d, the spores are distorted, with obvious depressions, and even large areas of cracks on the surface of the spores, losing their original structure. This is because the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 acts on the outer cell wall and cell membrane of XJ-FQ72, destroying its original integrity and normal metabolic function. The fungal nucleus may be wrapped inside, causing the cytoplasm to leak, hindering the normal growth and development of fungal hyphae and spores.

[0096] Example 5: Effect of lipopeptide extract on XJ-FQ72 toxin synthesis gene

[0097] (1) Experimental Materials

[0098] The lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 was prepared by the method described in Example 1. The pathogen XJ-FQ72 was cultured on a plate for 5-7 days in advance.

[0099] (2) Experimental methods

[0100] 5 mL of Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract and PDA medium were mixed evenly at a ratio of 1:3. After cooling and solidification, sterile cellophane was spread on the surface of the PDA medium, and XJ-FQ72 bacterial cake with a diameter of 5 mm, which was cultured at 28°C for 7 days, was inoculated in the center of the medium. After culturing at 28°C for 7 days, the cellophane was removed, and the mycelium of different treatments was collected in a 1.5 mL enzyme-free centrifuge tube, quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator for qRT-PCR verification.

[0101] (3) Experimental results

[0102] Real-time fluorescence quantitative PCR was used to analyze the expression levels of key genes for the synthesis of Alternaria toxins, such as omtI, aohR, AaMFS1, and AaTAS1. The relative gene expression was expressed using 2 -ΔΔCT Law, by the attached Figure 5 As shown, after adding the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007, the main synthesis genes of Alternaria toxin were all down-regulated, indicating that the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 inhibited the expression of the main synthesis genes of Alternaria toxin.

[0103] Example 6: Application of lipopeptide extracts in the tomato planting stage and their control of Alternaria toxins in processing tomatoes

[0104] (1) Experimental Materials

[0105] The lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 was prepared by the method described in Example 1.

[0106] (2) Experimental methods

[0107] 2mL, 0.2mL, 0.1mL and 0.02mL of Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract were taken and diluted with 200mL of clean water to four different concentrations. Therefore, the field test used low concentration, medium concentration, high concentration and ultra-high concentration of Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract for spraying, with the spraying of clean water as the control. Each treatment was repeated 3 times, and protective rows were set around the treatment group. Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract was sprayed once during the flowering period, fruiting period and maturity period of late-maturing processing tomatoes, with a dosage of 40L / mu, and applied 3 times in total. Other field management was the same as general production management.

[0108] There are 5 treatment groups in the field experiment, namely:

[0109] CK means clean water: spray clean water.

[0110] A is a very high concentration: 2 mL of Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract is dissolved in 200 mL of water (lipopeptide extract is diluted 10 2 times)

[0111] B is high concentration: 0.2 mL of Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract was dissolved in 200 mL of water (lipopeptide extract was diluted 10 3 times)

[0112] C is the medium concentration: 0.1 mL of Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract was dissolved in 200 mL of water (lipopeptide extract was diluted 5×10 3 times)

[0113] D is low concentration: 0.02 mL of Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract dissolved in 200 mL of water (lipopeptide extract diluted 104 times)

[0114] The "T / XAASS 002-2022 Detection of Alternaria toxins in tomatoes and their products by liquid chromatography-tandem mass spectrometry" was used to detect five common Alternaria toxins (AME, AOH, TEN, ALT and TeA) in healthy tomatoes and diseased tomatoes in different treatment groups.

[0115] (3) Experimental results

[0116] The results are shown in the attached Figure 6 -a, the black spot incidence rates of processing tomatoes treated with clear water, low concentration, medium concentration, high concentration and ultra-high concentration lipopeptide extract of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) XJ-BV2007 were 17.94%, 15.33%, 14.74%, 13.61% and 6.25%, respectively, and there were significant differences between the clear water group and the medium concentration, high concentration and ultra-high concentration treatment groups. The incidence rate of tomatoes sprayed with ultra-high concentration lipopeptide was the lowest.

[0117] Five toxins of Alternaria alternata (AME, AOH, TEN, ALT and TeA) have been detected. Since AME and AOH were not detected, they were not analyzed. The toxin content of TEN and ALT is very low, ranging from 0.78μg / kg to 5.95μg / kg, while the content of TeA toxin is very high, exceeding 2000μg / kg. Therefore, the analysis is mainly focused on TeA toxin. The TeA concentration of diseased tomatoes after spraying with different concentrations of lipopeptide extracts was significantly different from that of the CK group. Among them, the TeA concentration of diseased tomatoes after spraying with ultra-high concentration lipopeptide extracts was the lowest, and the control rate of TeA was as high as 95.02%. See attached Figure 6 -b, the TeA concentrations of healthy tomatoes after spraying at different concentrations were different from those of the CK group, but there were no significant differences among the four concentration groups, and the TeA content of healthy tomatoes was generally very low.

[0118] Example 7: Effect of lipopeptide extract on postharvest biocontrol of processing tomatoes

[0119] (1) Experimental Materials

[0120] The lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 was prepared by the method described in Example 1. The spore suspension was prepared by the method of Experiment (1) in Example 2.

[0121] (2) Experimental methods

[0122] Select healthy, surface-free, uniform-shaped and mature tomato fruits, immerse in 1% NaClO solution for disinfection for 2 minutes, rinse twice with sterile distilled water, wipe the surface of the tomato fruit with 75% ethanol solution, and air-dry. Use a sterile toothpick to create a wound with a diameter of 1 mm and a depth of 2.5 mm in the center of the tomato surface, and inoculate 10 μL of lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 at a concentration of 4 μg / mL. 10 μL of sterile deionized water was used as the control group. After 30 minutes, both the experimental group and the control group were inoculated with 10 μL of 10% ethanol solution at the wound. 5 / mL of XJ-FQ72 spore suspension. All processed tomatoes were placed in a fresh-keeping plastic box and stored at 28°C for 7 days. The diameter of the lesions was measured using the cross method and the appearance of the damage was observed. Five tomatoes were used in each group and repeated three times. The inhibition rate was calculated by measuring the diameter of the lesions. The common Alternaria toxins (AME, AOH, TEN, TeA) were detected in tomatoes in different treatment groups using "T / XAASS 002-2022 Detection of Alternaria toxins in tomatoes and their products by liquid chromatography-tandem mass spectrometry".

[0123] (3) Experimental results

[0124] The inhibition rate was calculated by measuring the mycelium growth diameter according to the following formula. The test results are shown in the attached Figure 7 and Table 3.

[0125] Bacterial inhibition rate (%) = [(mycelium diameter of control group - mycelium diameter of experimental group) / mycelium diameter of control group] × 100%

[0126] Table 3: Inhibition rate of XJ-FQ72 growth diameter by lipopeptide extract on day 7 of inoculation

[0127] Group Growth diameter of Alternaria alternata (mm) Inhibition rate Control group CK 29.9±0.2 / Lipopeptide extract 3±0.64 89.97％

[0128] The lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 and XJ-FQ72 were co-inoculated into processing tomatoes and stored for 7 days. The results are shown in the attached Figure 7 As shown in Figure 2, the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 can significantly inhibit the occurrence of black spot disease of processing tomatoes caused by XJ-FQ72. Figure 8As shown in the figure, with the increase of time, the lesions on the tomatoes inoculated with XJ-FQ72 alone increased significantly, while the lesions on the tomatoes inoculated with Bacillus amyloliquefaciens XJ-BV2007 lipopeptide extract and XJ-FQ72 grew more slowly. Fig. 9 As shown, the average lesion diameter of the processing tomatoes in the control group on the 7th day of storage was 30.8 mm, while the average lesion diameter of the processing tomatoes treated with the lipopeptide extract of Bacillus amyloliquefaciens XJ-BV2007 was 3.09 mm, a decrease of 89.97% (P<0.01). The inhibition rates of TeA, AOH, AME, and TEN in processing tomatoes were 100%, 98%, 99%, and 53%, respectively (P<0.01).

[0129] The above results indicate that Bacillus amyloliquefaciens XJ-BV2007 can not only inhibit the growth of XJ-FQ72 by producing lipopeptide extracts, showing strong antifungal activity, but also effectively control the accumulation of Alternaria toxins in processing tomatoes.

[0130] In the specification of the present invention, a lot of specific details are described. However, it is understood that embodiments of the present invention can be practiced without these specific details. In some embodiments, well-known methods, structures and techniques are not shown in detail so as not to obscure the understanding of this specification.

[0131] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0132] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

[0133] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. A lipopeptide extract with antibacterial and antitoxic activity, characterized in that: The lipopeptide extract is produced by Bacillus amyloliquefaciens XJ-BV2007; the lipopeptide extract consists of surfactinA, iturin A2, iturin A4, iturin A6 and fengycin.

2. A method for preparing a lipopeptide extract having antibacterial and antitoxic activity, characterized in that: The preparation method of the lipopeptide extract comprises the following steps: (1) Bacillus amyloliquefaciens XJ-BV2007 was inoculated onto NA solid medium with an inoculation needle for activation, and cultured in an incubator at 25 to 30° C. for 24 to 48 hours; (2) picking up a single colony of Bacillus amyloliquefaciens XJ-BV2007 activated in step (1) with an inoculating needle, transferring it to a flask containing NB liquid culture medium, placing the flask in a constant temperature shaker for shaking culture, and culturing it in a shaking incubator at 25 to 30° C. at a rate of 125 to 175 r / min for 24 h; (3) The seed liquid of Bacillus amyloliquefaciens XJ-BV2007 obtained in step (2) was inoculated into SPY medium at an inoculum amount of 3.5-4.5%, and cultured at a rate of 125-175 r / min in a shaking incubator at 25-36° C. for 36-60 h. The supernatant was collected after centrifugation at 8000 g for 15 min, and filtered through a 0.22 μm sterile filter membrane to obtain a cell-free fermentation liquid of Bacillus amyloliquefaciens XJ-BV2007. The bacterial cell concentration was measured to be 1-9×10 8 CFU / mL, the desired fermentation broth is obtained; (4) taking 300 mL of the cell-free fermentation broth of Bacillus amyloliquefaciens XJ-BV2007 obtained in step (3), adjusting the pH to ≤ 2.0 with 6.5-7.5 mol / L hydrochloric acid, and storing it in a refrigerator at 4°C overnight; centrifuging at 10000 g for 10 min, collecting the precipitate; extracting at least twice with methanol 3 times the volume of the precipitate, and adjusting the pH to neutral with 1 mol / L sodium hydroxide; The methanol extract was dried by a rotary evaporator at 40°C and filtered through a hydrophilic PTFE double-layer membrane with a pore size of 0.22 μm to finally obtain 2 mL of lipopeptide extract.

3. The method for preparing a lipopeptide extract having antibacterial and antitoxic activity according to claim 2, characterized in that: In the preparation of the fermentation liquid in step (3), the SPY culture medium comprises: 6 g / L maltose, 5 g / L yeast extract, and 9 g / L peptone.

4. The method for preparing a lipopeptide extract having antibacterial and toxicity-controlling activity according to claim 2, characterized in that: In the step (3), the seed liquid inoculation amount of Bacillus amyloliquefaciens XJ-BV2007 is 4%, the fermentation temperature is 34° C., and the culture is carried out in an oscillating incubator at a rate of 125 to 175 r / min for 48 hours.

5. The lipopeptide extract with antibacterial and antitoxic activity according to claim 1, characterized in that: The lipopeptide extract with antibacterial and toxicity-controlling activities is in the form of liquid.

6. Use of the lipopeptide extract with antibacterial and toxic-controlling activity as claimed in claim 1 in preventing and controlling Alternaria toxin contamination in fruits and vegetables.

7. Use of a lipopeptide extract with antibacterial and toxic-controlling activity in preventing and controlling Alternaria toxin contamination in fruits and vegetables as claimed in claim 6, characterized in that: The application of the lipopeptide extract in preventing and treating Alternaria toxin contamination in fruits and vegetables comprises the following steps: The bacterial concentration was 1~9×10 8 The lipopeptide extract with antibacterial and toxic control activity produced by Bacillus amyloliquefaciens XJ-BV2007 at CFU / mL was directly sprayed on the surface of biological samples during the growth stage of fruits and vegetables.

8. Use of the lipopeptide extract with antibacterial and toxic-controlling activity as claimed in claim 1 in preventing and controlling Alternaria toxin contamination in processing tomatoes.

9. Use of a lipopeptide extract with antibacterial and toxicity-controlling activity in preventing and controlling Alternaria toxin contamination in processing tomatoes according to claim 8, characterized in that: The method for preventing and controlling Alternaria toxin contamination in processing tomatoes comprises inhibiting at least one of black spot disease or Alternaria toxin synthesis.

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