Natural antiviral liquid as well as preparation method and application thereof

Abstract

The invention discloses a natural antiviral liquid and a preparation method and application thereof, and relates to the technical field of agricultural biotechnology and plant protection.The natural antiviral liquid is prepared through sequential mixed fermentation of bacillus subtilis and bacillus atrophaeus, corncob hydrolysate and corn steep liquor are used as a composite culture medium, and the natural antiviral liquid is prepared through fermentation. A pH response type attapulgite carrier which is accurately modified by chitosan and specific double precursors are added; the method comprises the following steps: inoculating bacillus subtilis, and inoculating bacillus atrophaeus when the pH value of fermentation liquor is naturally reduced to 6.0-6.2, thereby constructing a'metabolism interlocking 'and'signal space-time regulation' double closed-loop system. The method is simple in process and low in cost, the product synergistic interaction effect is remarkable, the prepared antiviral liquid can be self-assembled to form intelligent micelles, the inhibition effect on tobacco mosaic virus (TMV) is excellent, the lasting period is long, and the application prospect in green plant protection is wide.

Description

Technical Field

[0001] This invention relates to the fields of agricultural biotechnology and plant protection technology, specifically a natural antiviral liquid, its preparation method, and its application. Background Technology

[0002] Plant viral diseases are among the major threats to global agricultural production, often leading to severe yield reductions and quality declines. Currently, control still relies heavily on chemical pesticides, which presents problems such as pesticide residues, environmental pollution, and the development of pesticide resistance by viruses. Developing biopesticides using microorganisms and their metabolites is an important green alternative.

[0003] While existing technologies have reported the use of Bacillus subtilis or Bacillus atrophaeus alone for fermentation to produce antimicrobial substances, the resulting antiviral spectrum is relatively narrow, and the control effect is generally limited. Furthermore, some studies have involved mixed fermentation, but most are still limited to simple co-culture models, lacking efficient and controllable synergistic mechanisms between strains. This leads to poor process stability, high production costs, and difficulty in meeting the needs of industrial production. On the other hand, microbial metabolites prepared by existing technologies are easily degraded rapidly by environmental factors in field applications, resulting in generally short-lived efficacy, which greatly limits their commercial application.

[0004] Therefore, the development of a natural antiviral liquid based on a novel mechanism of action, with synergistic effects, and easy to industrialize has become an urgent need in this field. Summary of the Invention

[0005] This invention provides a natural antiviral liquid, its preparation method, and its application to solve the above-mentioned problems.

[0006] The core of the technical solution of this invention lies in the construction of a multi-layered collaborative system:

[0007] Strain combination and metabolic interlocking basis: Bacillus subtilis and Bacillus atrophus were selected to utilize their inherent metabolic complementarity.

[0008] Intelligent Carrier and Sensing Control System: Attapulgite modified with chitosan hydrochloride is used as a pH-responsive intelligent carrier, with its isoelectric point precisely designed within the pH range of 6.0-6.3. Combined with specifically added dual precursors (mevaleric acid and triglyceride derivatives), a dual closed-loop system is spontaneously constructed during fermentation, thereby achieving spatiotemporal control of quorum sensing signal molecules (such as ComX) and synergists (such as Surfactin).

[0009] Sequential fermentation control: The natural pH value of the fermentation broth is used as an indicator signal of metabolic state to precisely control the inoculation timing of the second strain.

[0010] Intelligent delivery system: Through simple pH adjustment, fermentation products self-assemble into stable micelles, improving adhesion to plant surfaces and environmental stability.

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] A method for preparing a natural antiviral liquid includes the following steps:

[0013] (1) Bacillus subtilis (strain A) was inoculated into the fermentation medium for the first stage of fermentation;

[0014] The fermentation medium contains corn cob hydrolysate, corn steep liquor, mevalonic acid, and chitosan hydrochloride-modified attapulgite carrier.

[0015] (2) Monitor the pH value of the first stage fermentation broth. When the pH value drops to 6.0 to 6.2, inoculate the fermentation broth with Bacillus atrophus (strain B) and triglyceride derivatives to carry out the second stage of mixed fermentation.

[0016] (3) After the second stage of fermentation is completed, the pH of the fermentation broth is adjusted to 4.0 to precipitate some lipopeptides and polyketides. Then, the pH is adjusted to 7.0 to allow another part of the active substances to form a "self-assembled" nanoscale micelle system with the extracellular polysaccharides in the fermentation broth. After being sprayed on the plant surface, this micelle system can penetrate the waxy layer of the plant surface more efficiently and exert antiviral and disease resistance-inducing effects for a long time. The solution is filtered to obtain the natural antiviral liquid.

[0017] Bacillus subtilis can efficiently synthesize lipopeptide surfactants (surfactin), which are amphiphilic and can effectively disrupt the lipid layer of the Bacillus subtilis cell membrane, significantly increasing its membrane permeability. Surfactin itself is also an elicitor that can induce systemic resistance in plants. Bacillus subtilis produces abundant polyketide antibiotics (such as difficidin) and various antimicrobial proteins that interfere with nucleic acids. This application, through in-depth research, found that the surfactin produced by Bacillus subtilis can significantly enhance the permeability of the Bacillus subtilis cell membrane, thereby greatly promoting the synthesis and secretion of polyketide antiviral substances, which are originally produced in lower quantities. This synergistic effect results in a significant synergistic effect in the antiviral activity of the metabolites from both strains.

[0018] Preferably, the corn cob hydrolysate is prepared as follows:

[0019] Take dried corn cobs (moisture content ≤10%) and crush them into 40-60 mesh particles (to ensure contact area for enzymatic hydrolysis);

[0020] Rinse the granules twice with deionized water to remove surface dust and soluble impurities, and dry them at 60°C to constant weight for later use.

[0021] Pretreated corn cobs and deionized water were added to the reactor at a solid-liquid ratio of 1:10 (g:mL).

[0022] Adjust the pH of the system to 4.8 (the optimal pH for cellulase and xylanase);

[0023] Add compound enzyme preparations: the amount of cellulase (enzyme activity 5000U / g) added is 0.5% of the dry weight of corn cob, and the amount of xylanase (enzyme activity 10000U / g) added is 0.3% of the dry weight of corn cob (the two enzymes work synergistically to efficiently degrade hemicellulose into xylose).

[0024] Heat to 50℃ and maintain constant temperature stirring at 150 rpm for 48 hours for enzymatic hydrolysis. During the enzymatic hydrolysis process, take samples every 12 hours to monitor the reducing sugar content and ensure that the xylose concentration is gradually increased to 35-45 g / L.

[0025] After enzymatic hydrolysis, heat to 100℃ and boil for 10 minutes to inactivate the enzymes (to terminate the reaction and avoid enzyme residue affecting subsequent fermentation).

[0026] Centrifuge at 4000 rpm for 20 minutes to remove undegraded solid residue;

[0027] Take the supernatant and filter it through a 0.22μm filter membrane to remove bacteria, and obtain corn cob hydrolysate (xylose content 35-45g / L, glucose content 2-5g / L, no phenol inhibitors);

[0028] Store at 4℃ for later use, or use directly in the preparation of compound culture media.

[0029] The addition of corn cob hydrolysate primarily provides xylose. Xylose is metabolized by Bacillus subtilis to produce D-xylose-5-phosphate. This intermediate provides the carbon skeleton for the synthesis of Surfactin and can also pass through a permeable membrane into Bacillus subtilis, serving as a precursor for the synthesis of polyketides.

[0030] The corn steep liquor used in the fermentation medium was industrial grade (purchased from publicly available channels and ready to use without additional purification), with a total nitrogen content of 8%-10%, providing sufficient nitrogen source and growth factors for *Bacillus subtilis* and *Bacillus atrophicus*, promoting rapid cell growth. It also contained ≥0.3% (w / v) L-proline and ≥0.005% (w / v) manganese ions, purchased from publicly available channels. L-proline is a direct precursor to surfactin synthesis, significantly increasing its yield. Manganese ions (Mn)...2+ As a key cofactor of Surfactin synthase, it contains glutamate, acetic acid, etc., and can serve as a precursor of acetyl-CoA, indirectly promoting the synthesis of polyketides. It solves the supply problems of carbon source, nitrogen source, key precursor, and cofactor simultaneously at extremely low cost (as an industrial byproduct), providing the material basis for achieving efficient metabolic interlocking.

[0031] Preferably, the chitosan hydrochloride-modified attapulgite carrier is prepared by the following method:

[0032] Attapulgite clay was mixed with a 1.0% (w / v) chitosan hydrochloride solution (dissolved in 1% acetic acid solution) at a mass ratio of 1:1.8-2.1.

[0033] Stir at 40-45℃ for 30-40 minutes, filter, dry at 55-65℃, and pulverize through a 200-mesh sieve.

[0034] Attapulgite is a natural porous silicate clay that provides a huge specific surface area, serving as a physical support for biofilms and guiding Bacillus subtilis to form dense colonies on its surface.

[0035] The chitosan hydrochloride-modified attapulgite carrier has an isoelectric point precisely designed within the pH range of 6.0-6.3. The principle behind this precise isoelectric point design is as follows:

[0036] This invention achieves precise control of the carrier's isoelectric point within the pH range of 6.0-6.3 by adjusting the mass ratio of attapulgite clay to chitosan hydrochloride solution. The core mechanism is based on the charge balance principle of colloidal surfaces.

[0037] Charge source and interaction: Attapulgite is a natural porous silicate clay, and its surface is negatively charged due to the dissociation of silanol groups (-SiOH); when chitosan hydrochloride is dissolved in 1% acetic acid solution, the amino groups (-NH2) on the molecular chain are protonated to form positively charged -NH3. + The two are attracted by electrostatics to form a composite structure of "negatively charged substrate - positively charged coating".

[0038] Quantitative correlation between solid-liquid ratio and isoelectric point:

[0039] When the mass ratio of attapulgite clay to chitosan hydrochloride solution is 1:1.8: the chitosan loading is low, the positive charge density on the carrier surface is low, and only a low concentration of H+ is required. + (i.e., a higher pH) can neutralize the negative charge on the surface, raising the isoelectric point to pH 6.3;

[0040] When the mass ratio increases to 1:2.1: the chitosan loading increases, the surface positive charge density increases, and a higher concentration of H+ is required. + Neutralization requires a lower pH (i.e., the isoelectric point drops to pH 6.0);

[0041] When the mass ratio is in the range of 1:1.8-2.1, the isoelectric point naturally falls in the pH range of 6.0-6.3, which completely overlaps with the fermentation trigger pH (6.0-6.2).

[0042] This carrier exhibits distinctly different electrical properties at different stages of fermentation, thereby enabling precise spatiotemporal control of signal molecules:

[0043] Early stage of fermentation (ambient pH > 6.5, higher than the carrier's isoelectric point):

[0044] The deprotonation of the chitosan amino groups on the carrier surface makes it negatively charged. At this time, the quorum sensing signal peptide ComX secreted by Bacillus subtilis (whose isoelectric point pI is typically higher than 8.0) is positively charged at the ambient pH. Therefore, it is electrostatically attracted to the negatively charged carrier and temporarily immobilized on the carrier surface, preventing premature signal diffusion and storing potential energy for the subsequent concentrated burst of response. Meanwhile, Surfactin (whose isoelectric point pI is approximately 2.5-3.0) produced at the same time is negatively charged at the ambient pH and electrostatically repelled by the carrier, thus diffusing freely without interfering with the initial metabolic initiation of Bacillus subtilis itself.

[0045] During the mid-to-late stages of fermentation (when the ambient pH naturally drops to 6.0-6.2, close to or slightly below the carrier's isoelectric point):

[0046] The surface charge of the carrier reverses, changing from negative to positive. At this point, ComX, still positively charged, electrostatically repels the carrier, causing it to be rapidly released into the fermentation broth and strongly activating the global quorum sensing system. Simultaneously, the negative charge of Surfactin electrostatically attracts the positive charge of the carrier, and, in conjunction with the hydrophobic interactions of the biofilm matrix on the carrier surface and the hydrogen bonding of chitosan molecules, is efficiently captured and enriched around the carrier. This creates a high-concentration synergist microenvironment near the subsequently introduced Bacillus atrophus (strain B), significantly enhancing its cell membrane permeability and initiating a highly efficient metabolic interlock.

[0047] Preferably, the xylose content of the corn cob hydrolysate in the fermentation medium is 35-45 g / L;

[0048] The amount of corn steep liquor added is 8%-10% (v / v);

[0049] The addition amount of mevalonic acid is 0.003%-0.008% (w / v);

[0050] The amount of chitosan hydrochloride-modified attapulgite carrier added is 0.1%-0.2% (w / v).

[0051] Mevaleric acid is an upstream precursor of ComX, the quorum sensing signaling molecule synthesized by Bacillus subtilis. Exogenous addition can bypass its own metabolic regulation, significantly improving the synthesis efficiency of ComX, thereby advancing and strengthening the quorum sensing signal, promoting biofilm formation and the synthesis of surfactin.

[0052] Preferably, the amount of the triglyceride derivative added is 0.05%-0.1% (w / v);

[0053] The triglyceride derivative is a product obtained by alkaline hydrolysis of kitchen waste oil. The preferred preparation method for the triglyceride derivative is as follows: kitchen waste oil is filtered to remove impurities, and mixed with sodium hydroxide solution (5% w / v) at a ratio of 1:3 (v / v). The mixture is stirred at 70°C for 2 hours, cooled, centrifuged to separate the lower layer product, washed until neutral, and dried at 60°C. The main component is sodium salt of short-chain fatty acids.

[0054] The addition of triglyceride derivatives during Bacillus atrophicus inoculation allows the short-chain fatty acids produced from its decomposition to act as signaling molecules, activating the quorum sensing master regulator Spo0A in Bacillus atrophicus. This makes Bacillus atrophicus more sensitive to signals from Bacillus subtilis (ComX, Surfactin), enabling it to rapidly initiate the synthesis of antiviral substances. This forms a "cross-strain signal synergy." Mevalonate enhances the "calling out" (signal generation) of Bacillus subtilis from upstream, while the triglyceride derivatives improve the "hearing" (receiving and responding to signals) of Bacillus atrophicus, maximizing the communication efficiency and coordination between the two bacteria.

[0055] Preferably, in step (1), the inoculation amount of Bacillus subtilis is 3%-5% (v / v);

[0056] In step (2), the inoculation amount of Bacillus atrophus is 3%-5% (v / v).

[0057] Preferably, in step (2), the conditions for the second stage of mixed fermentation are: temperature 28-30℃, dissolved oxygen maintained at 10%-20% (dissolved oxygen is maintained by adjusting the aeration rate (0.1-0.3vvm) and stirring speed (120-180rpm)), and fermentation time 20-28 hours.

[0058] System startup and operation process:

[0059] Sequential fermentation and metabolic interlocking set the basic process. Bacillus subtilis initiates fermentation in a complex medium, metabolizing xylose and L-proline from corn steep liquor, starting the synthesis of surfactin, and causing a decrease in pH.

[0060] When the pH naturally drops to 6.0-6.2, this signal triggers two actions simultaneously:

[0061] Inoculate with Bacillus atrophus to initiate physical mixed fermentation.

[0062] When the pH of the fermentation broth drops to near the isoelectric point of the carrier, the surface charge of the carrier changes from negative to positive. At this time, ComX is still positively charged and electrostatically repels the carrier, and is quickly released into the fermentation broth. Surfactin is enriched through the hydrophobic interaction of the biofilm and the hydrogen bonds of chitosan, forming a high-concentration microenvironment around Bacillus atrophus.

[0063] Simultaneously with vaccination, two precursors (mevaleric acid and triglyceride derivatives) are introduced.

[0064] With the support of mevalonic acid, Bacillus subtilis continuously produces ComX with a strong signal.

[0065] Newly inoculated Bacillus atrophus exhibits a highly sensitive Spo0A system under the pre-activation of triglyceride derivatives.

[0066] Thus, the high concentrations of ComX and Surfactin locally enriched by the intelligent carrier can form a highly efficient microenvironment around Bacillus atrophus, strongly activating its quorum sensing system, thereby rapidly initiating and enhancing the synthesis pathway of polyketide antiviral substances.

[0067] Forming a self-reinforcing closed loop and ultimate effect:

[0068] Polyketides synthesized by Bacillus atrophus and Surfactin synthesized by Bacillus subtilis together form a synergistic antiviral combination.

[0069] Intermediate products such as acetyl-CoA produced during polyketide synthesis can enhance the quorum sensing signal of Bacillus subtilis, thereby promoting the synthesis of Surfactin, forming a complete closed loop of metabolic interlocking and signal feedback, and further stabilizing and optimizing the biofilm structure.

[0070] Thus, a self-driven and self-optimizing metabolic interlocking closed loop (Bacillus subtilis → xylose intermediate → Bacillus atrophus → polyketide → Bacillus subtilis) and a signal regulation closed loop (mevaleric acid → ComX → biofilm → signal enrichment → metabolic activation) are formed and continuously operated, ultimately producing a composition with excellent antiviral effects.

[0071] The present invention also provides a natural antiviral liquid, which is obtained by the above preparation method and contains surfactantin and polyketide antiviral substances, wherein the surfactantin content is not less than 1000 mg / L.

[0072] The present invention also provides the application of the above-mentioned natural antiviral liquid in the prevention and control of plant viral diseases, including but not limited to viral diseases caused by tobacco mosaic virus or cucumber mosaic virus.

[0073] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0074] Outstanding synergistic antiviral effect: By constructing a dual closed-loop system of "metabolic interlock" and "signal temporal regulation", it synergistically and efficiently activates and enhances the synthesis of two key antiviral substances, Surfactin and Difficidin, greatly improving the inhibition rate of TMV.

[0075] The raw materials are green and economical, enabling high-value utilization of waste: the core culture medium components use corn cob hydrolysate, corn steep liquor, and derivatives of waste cooking oil, eliminating the need for expensive chemical inducers or organic solvents throughout the process. This not only significantly reduces production costs but also achieves high-value and resource-based utilization of agricultural waste, meeting the requirements of a green circular economy.

[0076] Excellent stability and applicability: Through a unique "pH-adjusted post-treatment," the active substances in the fermentation products can self-assemble into a stable nanoscale micelle system. This system can effectively improve the adhesion, spreading, and penetration of the drug solution on the plant surface, and achieve sustained release of active ingredients, thereby extending the duration of efficacy. At the same time, the biofilm structure formed in the system also enhances the colonization ability of the microbial community in complex environments, ensuring the stability of the control effect. Attached Figure Description

[0077] Figure 1 A flowchart illustrating a method for preparing a natural antiviral liquid provided by this invention.

[0078] Figure 2 The flowchart illustrates the preparation method of the chitosan hydrochloride-modified attapulgite carrier provided by this invention. Detailed Implementation

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0080] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0081] The Bacillus subtilis and Bacillus atrophaeus used in this invention are conventional industrial strains. Bacillus subtilis CGMCC 1.1086 and Bacillus atrophaeus CGMCC 1.1849 are selected, both of which are publicly available wild-type strains.

[0082] Example 1

[0083] In this embodiment of the invention, a natural antiviral liquid, such as Figures 1-2 As shown, its preparation method includes the following steps:

[0084] Preparation of S1 corn cob hydrolysate:

[0085] S1.1 Take dried corn cobs (moisture content ≤10%) and crush them into 40-60 mesh particles;

[0086] S1.2 Rinse the particles twice with deionized water to remove surface dust and soluble impurities, and dry at 60°C to constant weight for later use.

[0087] S1.3 Add pretreated corn cobs and deionized water to the reactor at a solid-liquid ratio of 1:10 (g:mL);

[0088] S1.4 Adjust the pH of the system to 4.8 (the optimal pH for cellulase and xylanase);

[0089] S1.5 Add compound enzyme preparation: The amount of cellulase (enzyme activity 5000U / g) added is 0.5% of the dry weight of corn cob, and the amount of xylanase (enzyme activity 10000U / g) added is 0.3% of the dry weight of corn cob (the two enzymes work together to efficiently degrade hemicellulose into xylose).

[0090] S1.6 was heated to 50℃ and kept at a constant temperature of 150 rpm for 48 hours for enzymatic hydrolysis. The reducing sugar content was sampled and monitored every 12 hours during the enzymatic hydrolysis process.

[0091] After the enzymatic hydrolysis of S1.7 is completed, heat to 100℃ and boil for 10 minutes to inactivate the enzyme (to terminate the reaction and avoid enzyme residue affecting subsequent fermentation);

[0092] S1.8 was centrifuged at 4000 rpm for 20 minutes to remove undegraded solid residue;

[0093] S1.9 Take the supernatant and filter it through a 0.22μm filter membrane to remove bacteria, and obtain corn cob hydrolysate (xylose content was measured to be 40g / L, glucose content to be 3g / L, and no phenol inhibitors were found).

[0094] Preparation of S2 chitosan-modified attapulgite smart carrier

[0095] S2.1 Mix attapulgite soil with a chitosan hydrochloride solution with a mass-volume concentration of 1.0% at a mass ratio of 1:1.8;

[0096] S2.2 was stirred at 40℃ for 30 minutes, filtered, dried at 55℃, pulverized, and passed through a 200-mesh sieve to obtain chitosan-modified attapulgite smart carrier. Zeta potential analysis (measured by Malvern Zetasizer Nano ZS, test conditions: 0.01mol / L NaCl buffer, carrier concentration 0.1% (w / v), temperature 25℃, each sample was tested in parallel 3 times, and the average value was taken as the isoelectric point) showed that its isoelectric point was pH=6.3.

[0097] Preparation of S3 Natural Antiviral Liquid

[0098] S3.1 Fermentation medium: Corn cob hydrolysate as the base, with the addition of 8% (v / v) corn steep liquor, 0.003% (w / v) mevalonic acid, and 0.1% (w / v) chitosan-modified attapulgite smart carrier. Natural pH (approximately 6.8), sterilized at 121℃ for 20 minutes.

[0099] S3.2 Fermentation Process: Activated Bacillus subtilis was inoculated at a rate of 3% (v / v) and cultured at 30℃ and 200 rpm. When the pH of the fermentation broth naturally decreased to 6.0-6.2, activated Bacillus atrophus was inoculated at a rate of 3% (v / v), and 0.05% (w / v) of a triglyceride derivative (alkaline hydrolysate of kitchen waste oil) was added simultaneously. The temperature was maintained at 28℃, dissolved oxygen at 10%, and the fermentation time was 20 hours.

[0100] S3.3 Post-treatment: After fermentation, adjust the pH of the fermentation broth to 4.0 with 6M HCl and let it stand for 2 hours; then adjust the pH back to 7.0 with 4M NaOH, stir evenly and filter (using a 0.45μm filter membrane) to obtain the natural antiviral liquid stock solution.

[0101] In this embodiment, the following method is preferably used for strain activation:

[0102] Bacillus subtilis activation: CGMCC 1.1086 strain was inoculated into LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0) and cultured at 30℃ with constant temperature shaking at 200 rpm for 12 hours to obtain activated bacterial solution;

[0103] Activation of Bacillus atrophus: CGMCC 1.1849 strain was inoculated into LB medium and cultured at 28°C and 180 rpm for 12 hours to obtain activated bacterial solution.

[0104] Example 2

[0105] In this embodiment of the invention, a natural antiviral liquid, such as Figures 1-2 As shown, its preparation method includes the following steps:

[0106] The preparation of S1 corn cob hydrolysate is the same as in Example 1.

[0107] Preparation of S2 chitosan-modified attapulgite smart carrier

[0108] S2.1 Mix attapulgite soil with a chitosan hydrochloride solution with a mass-volume concentration of 1.0% at a mass ratio of 1:1.9;

[0109] S2.2 was stirred at 41℃ for 32 minutes, filtered, dried at 58℃, pulverized and passed through a 200-mesh sieve to obtain chitosan-modified attapulgite smart carrier. Zeta potential analysis showed that its isoelectric point was pH=6.2.

[0110] Preparation of S3 Natural Antiviral Liquid

[0111] S3.1 Fermentation medium: Corn cob hydrolysate as the base, with the addition of 8.5% (v / v) corn steep liquor, 0.004% (w / v) mevalonic acid, and 0.12% (w / v) chitosan-modified attapulgite smart carrier. pH natural (approximately 6.8), sterilized at 121℃ for 20 minutes.

[0112] S3.2 Fermentation Process: Activated Bacillus subtilis was inoculated at a rate of 3.5% (v / v) and cultured at 30℃ and 200 rpm. When the pH of the fermentation broth naturally decreased to 6.0-6.2, activated Bacillus atrophus was inoculated at a rate of 3.5% (v / v), and 0.06% (w / v) of a triglyceride derivative (alkaline hydrolysate of kitchen waste oil) was added simultaneously. The temperature was maintained at 28.5℃, dissolved oxygen at 12%, and the fermentation time was 22 hours.

[0113] S3.3 Post-treatment: After fermentation, adjust the pH of the fermentation broth to 4.0 with 6M HCl and let it stand for 2 hours; then adjust the pH back to 7.0 with 4M NaOH, stir evenly and filter to obtain the natural antiviral liquid stock solution.

[0114] Example 3

[0115] In this embodiment of the invention, a natural antiviral liquid, such as Figures 1-2 As shown, its preparation method includes the following steps:

[0116] The preparation of S1 corn cob hydrolysate is the same as in Example 1.

[0117] Preparation of S2 chitosan-modified attapulgite smart carrier

[0118] S2.1 Mix attapulgite soil with a chitosan hydrochloride solution with a mass-volume concentration of 1.0% at a mass ratio of 1:1.95;

[0119] S2.2 was stirred at 42.5℃ for 35 minutes, filtered, dried at 60℃, pulverized and passed through a 200-mesh sieve to obtain chitosan-modified attapulgite smart carrier. Zeta potential analysis showed that its isoelectric point was pH=6.15.

[0120] Preparation of S3 Natural Antiviral Liquid

[0121] S3.1 Fermentation medium: Corn cob hydrolysate as the base, with the addition of 9% (v / v) corn steep liquor, 0.005% (w / v) mevalonic acid, and 0.15% (w / v) chitosan-modified attapulgite smart carrier. pH natural (approximately 6.8), sterilized at 121℃ for 20 minutes.

[0122] S3.2 Fermentation Process: Activated Bacillus subtilis was inoculated at a rate of 4% (v / v) and cultured at 30℃ and 200 rpm. When the pH of the fermentation broth naturally decreased to 6.0-6.2, activated Bacillus atrophus was inoculated at a rate of 4% (v / v), and 0.08% (w / v) of a triglyceride derivative (alkaline hydrolysate of kitchen waste oil) was added simultaneously. The temperature was maintained at 29℃, dissolved oxygen at 15%, and the fermentation time was 24 hours.

[0123] S3.3 Post-treatment: After fermentation, adjust the pH of the fermentation broth to 4.0 with 6M HCl and let it stand for 2 hours; then adjust the pH back to 7.0 with 4M NaOH, stir evenly and filter to obtain the natural antiviral liquid stock solution.

[0124] Example 4

[0125] In this embodiment of the invention, a natural antiviral liquid, such as Figures 1-2 As shown, its preparation method includes the following steps:

[0126] The preparation of S1 corn cob hydrolysate is the same as in Example 1.

[0127] Preparation of S2 chitosan-modified attapulgite smart carrier

[0128] S2.1 Mix attapulgite soil with a chitosan hydrochloride solution with a mass-volume concentration of 1.0% at a mass ratio of 1:2;

[0129] S2.2 was stirred at 44℃ for 38 minutes, filtered, dried at 62℃, pulverized and passed through a 200-mesh sieve to obtain chitosan-modified attapulgite smart carrier. Zeta potential analysis showed that its isoelectric point was pH=6.1.

[0130] Preparation of S3 Natural Antiviral Liquid

[0131] S3.1 Fermentation medium: Corn cob hydrolysate as the base, with the addition of 9.5% (v / v) corn steep liquor, 0.007% (w / v) mevalonic acid, and 0.18% (w / v) chitosan-modified attapulgite smart carrier. pH natural (approximately 6.8), sterilized at 121℃ for 20 minutes.

[0132] S3.2 Fermentation Process: Activated Bacillus subtilis was inoculated at a rate of 4.5% (v / v) and cultured at 30℃ and 200 rpm. When the pH of the fermentation broth naturally decreased to 6.0-6.2, activated Bacillus atrophus was inoculated at a rate of 4.5% (v / v), and 0.09% (w / v) of a triglyceride derivative (alkaline hydrolysate of kitchen waste oil) was added simultaneously. The temperature was maintained at 29.5℃, dissolved oxygen at 18%, and the fermentation time was 26 hours.

[0133] S3.3 Post-treatment: After fermentation, adjust the pH of the fermentation broth to 4.0 with 6M HCl and let it stand for 2 hours; then adjust the pH back to 7.0 with 4M NaOH, stir evenly and filter to obtain the natural antiviral liquid stock solution.

[0134] Example 5

[0135] In this embodiment of the invention, a natural antiviral liquid, such as Figures 1-2 As shown, its preparation method includes the following steps:

[0136] The preparation of S1 corn cob hydrolysate is the same as in Example 1.

[0137] Preparation of S2 chitosan-modified attapulgite smart carrier

[0138] S2.1 Mix attapulgite soil with a chitosan hydrochloride solution with a mass concentration of 1.0% at a mass ratio of 1:2.1;

[0139] S2.2 was stirred at 45℃ for 40 minutes, filtered, dried at 65℃, pulverized and passed through a 200-mesh sieve to obtain chitosan-modified attapulgite smart carrier. Zeta potential analysis showed that its isoelectric point was pH=6.0.

[0140] Preparation of S3 Natural Antiviral Liquid

[0141] S3.1 Fermentation medium: Corn cob hydrolysate as the base, with the addition of 10% (v / v) corn steep liquor, 0.008% (w / v) mevalonic acid, and 0.2% (w / v) chitosan-modified attapulgite smart carrier. pH natural (approximately 6.8), sterilized at 121℃ for 20 minutes.

[0142] S3.2 Fermentation Process: Activated Bacillus subtilis was inoculated at a rate of 5% (v / v) and cultured at 30℃ and 200 rpm. When the pH of the fermentation broth naturally decreased to 6.0-6.2, activated Bacillus atrophus was inoculated at a rate of 5% (v / v), and 0.1% (w / v) of a triglyceride derivative (alkaline hydrolysate of kitchen waste oil) was added simultaneously. The temperature was maintained at 30℃, dissolved oxygen at 20%, and the fermentation time was 28 hours.

[0143] S3.3 Post-treatment: After fermentation, adjust the pH of the fermentation broth to 4.0 with 6M HCl and let it stand for 2 hours; then adjust the pH back to 7.0 with 4M NaOH, stir evenly and filter to obtain the natural antiviral liquid stock solution.

[0144] Example 6

[0145] The difference from Example 3 is that, in preparing the chitosan-modified attapulgite smart carrier, the attapulgite and chitosan hydrochloride solution are mixed at a mass ratio of 1:1.8.

[0146] Example 7

[0147] The difference from Example 3 is that, in preparing the chitosan-modified attapulgite smart carrier, the attapulgite and chitosan hydrochloride solution are mixed at a mass ratio of 1:1.9.

[0148] Example 8

[0149] The difference from Example 3 is that, in preparing the chitosan-modified attapulgite smart carrier, the attapulgite and chitosan hydrochloride solution are mixed at a mass ratio of 1:2.

[0150] Example 9

[0151] The difference from Example 3 is that, in preparing the chitosan-modified attapulgite smart carrier, the attapulgite and chitosan hydrochloride solution are mixed at a mass ratio of 1:2.1.

[0152] Comparative Example 1: Fermentation by a single strain

[0153] Only Bacillus subtilis was inoculated, and other conditions were the same as in Example 3.

[0154] Comparative Example 2: Simple Mixed Fermentation

[0155] Bacillus subtilis and Bacillus atrophus were inoculated simultaneously, and other conditions were the same as in Example 3.

[0156] Comparative Example 3: Unmodified Carrier

[0157] The original attapulgite clay that has not been modified with chitosan was used, and other conditions were the same as in Example 3.

[0158] Comparative Example 4: No mevalonic acid

[0159] Mevaleric acid was not added to the fermentation medium, and other conditions were the same as in Example 3.

[0160] Comparative Example 5: No triglyceride derivatives

[0161] No triglyceride derivatives were added when inoculating strain B, and other conditions were the same as in Example 3.

[0162] Comparative Example 6: Incorrect pH Inoculation

[0163] Strain B was inoculated when the pH of the fermentation broth was 7.0, and other conditions were the same as in Example 3.

[0164] Comparative Example 7: No intelligent carrier

[0165] No carrier was added to the fermentation medium, and other conditions were the same as in Example 3.

[0166] Comparative Example 8

[0167] When preparing the chitosan-modified attapulgite smart carrier, the attapulgite and chitosan hydrochloride solution were mixed at a mass ratio of 1:1.6, and other conditions were the same as in Example 3.

[0168] Comparative Example 9

[0169] When preparing the chitosan-modified attapulgite smart carrier, the attapulgite and chitosan hydrochloride solution were mixed at a mass ratio of 1:1.7, and other conditions were the same as in Example 3.

[0170] Comparative Example 10

[0171] When preparing the chitosan-modified attapulgite smart carrier, the attapulgite and chitosan hydrochloride solution were mixed at a mass ratio of 1:2.2, and other conditions were the same as in Example 3.

[0172] Comparative Example 11

[0173] When preparing the chitosan-modified attapulgite smart carrier, the attapulgite and chitosan hydrochloride solution were mixed at a mass ratio of 1:2.3, and other conditions were the same as in Example 3.

[0174] Comparative Example 12: The difference from Example 3 is that there is no post-treatment of pH adjustment, and the mixture is directly filtered after fermentation without the "pH 4.0 settling → pH 7.0 correction" step.

[0175] Comparative Example 13

[0176] A commercially available single Bacillus subtilis preparation.

[0177] Performance tests were performed on the products obtained in each embodiment and comparative example:

[0178] Surfactin content was determined by high-performance liquid chromatography (HPLC). Chromatographic conditions were as follows: C18 column; mobile phase: acetonitrile / water (containing 0.1% trifluoroacetic acid) gradient elution; flow rate: 1.0 mL / min; column temperature: 30℃; detection wavelength: 205 nm. Quantification was performed using the external standard method (Surfactin standard, Sigma, purity ≥98%).

[0179] The content of polyketides (calculated as Difficidin) was determined by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: C18 column; mobile phase: gradient elution of acetonitrile / 10 mM ammonium acetate aqueous solution; flow rate: 0.3 mL / min; column temperature: 40℃; detection wavelength: 210 nm. Quantification was performed using external standard method (Difficidin standard with a purity ≥95%).

[0180] The TMV inhibition rate was determined using the half-leaf necrotic spot method. The simplified procedure was as follows: the antiviral solution obtained in each example and comparative example was evenly sprayed onto one side of the heart leaf of the tobacco plant, while the other side was sprayed with water as a control. After drying, the TMV virus solution was inoculated by friction. After culturing for 5-7 days, the number of necrotic spots was counted, and the inhibition rate was calculated using the following formula: Inhibition rate (%) = (1 - number of necrotic spots on the treated side / number of necrotic spots on the control side) × 100%.

[0181] Determination of duration of efficacy: Potted tobacco plants in a greenhouse were sprayed with antiviral solution once, and TMV was inoculated on the 7th, 10th, 13th and 16th days after treatment. The duration of efficacy was recorded as the maximum number of days with a necrotic spot inhibition rate of more than 50%.

[0182] The results are shown in Table 1 below:

[0183] Table 1 Performance Test Results

[0184]

[0185]

[0186] The results above show that the synergistic system constructed in this invention significantly improves the antiviral effect of wild-type dual strains compared to single strains and commercially available products.

[0187] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0188] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

Claims

1. A method for preparing a natural antiviral liquid, characterized in that, Includes the following steps: (1) Bacillus subtilis was inoculated into a fermentation medium for the first stage of fermentation; the fermentation medium contained corn cob hydrolysate, corn steep liquor, mevalonic acid and attapulgite carrier modified with chitosan hydrochloride. (2) Monitor the pH value of the first stage fermentation broth. When the pH value drops to 6.0 to 6.2, inoculate the fermentation broth with Bacillus atrophus and triglyceride derivatives to carry out the second stage of mixed fermentation. (3) After the second stage of fermentation is completed, the pH of the fermentation liquid is adjusted to 4.0, left to stand, then adjusted to pH 7.0, filtered, and the natural antiviral liquid is obtained.

2. The method for preparing the natural antiviral liquid according to claim 1, characterized in that, The chitosan hydrochloride-modified attapulgite carrier was prepared by the following method: Attapulgite soil was mixed with a 1.0% (w / v) chitosan hydrochloride solution at a mass ratio of 1:1.8-2.

1. Stir at 40-45℃ for 30-40 minutes, filter, dry at 55-65℃, and pulverize through a 200-mesh sieve.

3. The method for preparing the natural antiviral liquid according to claim 1, characterized in that, The xylose content of the corn cob hydrolysate in the fermentation medium is 35-45 g / L; The amount of corn steep liquor added is 8%-10% (v / v); The addition amount of mevalonic acid is 0.003%-0.008% (w / v); The amount of chitosan hydrochloride-modified attapulgite carrier added is 0.1%-0.2% (w / v).

4. The method for preparing the natural antiviral liquid according to claim 1, characterized in that, The amount of the triglyceride derivative added is 0.05%-0.1% (w / v); The triglyceride derivative is a product obtained by alkaline hydrolysis of kitchen waste oil.

5. The method for preparing the natural antiviral liquid according to claim 1, characterized in that, In step (1), the inoculation amount of Bacillus subtilis is 3%-5% (v / v); In step (2), the inoculation amount of Bacillus atrophus is 3%-5% (v / v).

6. The method for preparing the natural antiviral liquid according to claim 1, characterized in that, In step (2), the conditions for the second stage of mixed fermentation are: temperature 28-30℃, dissolved oxygen maintained at 10%-20%, and fermentation time 20-28 hours.

7. A natural antiviral liquid prepared by the method according to any one of claims 1-6.

8. The natural antiviral liquid according to claim 7, characterized in that, It contains surfactantin and polyketide antiviral substances, with surfactantin content not less than 1000 mg / L.

9. The application of the natural antiviral liquid according to claim 7 or 8 in the prevention and control of plant viral diseases.

10. The application according to claim 9, characterized in that, The plant viral diseases mentioned include viral diseases caused by tobacco mosaic virus or cucumber mosaic virus.

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