Method for extracting antibacterial components from entomopathogenic nematode fermentation liquor

Abstract

The invention belongs to the technical field of biopesticides, and particularly relates to a method for extracting antibacterial components in entomopathogenic nematode fermentation broth, and the method comprises the following steps: (1) propagation culture of entomopathogenic nematodes; (2) inoculating and fermenting; (3) preparing a fermentation liquor crude extract; and (4) extracting the fermentation liquor crude extract by using a mixed solvent of chloroform and diethyl ether to obtain extract liquor, removing the solvent from the extract liquor, and drying to obtain the antibacterial component. The extraction method disclosed by the invention has remarkable beneficial effects in the aspects of improving the extraction efficiency of the antibacterial components, environmental protection sustainability, application prospect, resource recycling and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological pesticides, and particularly relates to a method for extracting antibacterial components from the fermentation broth of entomopathogenic nematodes. Background Art

[0002] With the increasing global attention to food safety, environmental protection, and sustainable development issues, the use of traditional chemically synthesized pesticides and antibacterial agents is facing unprecedented challenges. Although these chemical substances effectively control pests and diseases in agricultural production, their long-term use has brought many problems, such as pesticide residues, ecological imbalance, water pollution, and human health risks. Therefore, finding efficient, environmentally friendly, and sustainable antibacterial and pest control technologies has become an important direction in current scientific research.

[0003] 1. Existing Antibacterial Technologies and Their Limitations

[0004] Currently, the mainstream antibacterial products on the market mainly rely on chemically synthesized substances, such as antibiotics, preservatives, and chemical pesticides. Although these products have significant antibacterial effects, their long-term use can lead to the emergence of drug resistance in pathogenic bacteria, and their non-specific bactericidal effects also disrupt the balance of the ecosystem. In addition, chemical antibacterial agents pose high environmental risks during production and use, which is not conducive to the sustainable development of the agricultural and food industries. Take taro as an example. To prevent and control diseases such as Phytophthora colocasiae, Erwinia carotovora subsp. carotovora, and Fusarium oxysporum f. sp. colocasiae, as well as pests such as underground pests and Spodoptera litura, farmers often use various chemical pesticides and bactericides, such as 600-fold liquid of 30% copper oxychloride suspension or 3000-fold liquid of 72% streptomycin sulfate soluble powder for agriculture, phoxim, trichlorfon, etc. These chemical antibacterial products achieve the purpose of quickly controlling pests and diseases by destroying the physiological functions of pathogenic bacteria and pests. However, behind this rapid effect, there are hidden concerns about long-term use. For example, the use of these chemical pesticides may lead to pesticide residues on the surface and inside of taro; chemical pesticides may spread into the environment through wind, water, etc. during application, causing pollution to soil, water sources, etc.; while killing pests and diseases, chemical pesticides may also harm beneficial organisms (such as natural enemy insects, microorganisms, etc.) in the farmland ecosystem, affecting the balance and stability of the farmland ecosystem.

[0005] 2. Development and Utilization of Biological Resources

[0006] To overcome the above problems, in recent years, scientists have begun to explore the use of biological resources, especially natural organisms such as entomopathogenic nematodes, to develop new antibacterial technologies. Entomopathogenic nematodes have strong antibacterial abilities, and their bodies or fermentation broths contain a variety of natural components with broad-spectrum antibacterial activities. These components not only have high antibacterial effects but also have less impact on the environment and human health, showing great application potential.

[0007] 3. Bottlenecks in Extraction Technology

[0008] However, although biological resources such as entomopathogenic nematodes have significant advantages, the extraction of their antibacterial components faces many technical bottlenecks. Traditional extraction methods often have problems such as low efficiency, poor stability, and easy loss of components, making it difficult to achieve industrial production. In addition, some extraction processes require the use of a large amount of harmful chemical reagents, increasing the risk of environmental pollution.

[0009] 4. The Need for Technological Innovation

[0010] Based on the above background, there is an urgent need to develop an efficient, stable, and environmentally friendly extraction technology for antibacterial components to maximize the utilization of biological resources such as entomopathogenic nematodes. This can not only improve the purity and effectiveness of antibacterial products but also reduce environmental pollution and promote the sustainable development of agriculture and the food industry.

[0011] In view of this, the present invention is committed to developing a fine fermentation and extraction method based on macroporous adsorption resin and gradient ethanol elution technology, aiming to efficiently enrich and separate the antibacterial components in the fermentation broth of entomopathogenic nematodes. This method realizes the stable extraction of antibacterial components and significantly reduces the negative impact on the environment by optimizing steps such as fermentation conditions, solid-liquid separation, pH adjustment, adsorption and elution, and solvent extraction. At the same time, this method will provide new ideas and technical support for the biological control of agricultural pests and food preservation and other fields, promoting the green upgrading and sustainable development of related industries. Summary of the Invention

[0012] The present invention aims to solve the above technical problems and provides an extraction method for antibacterial components in the fermentation broth of entomopathogenic nematodes, which shows significant effects in terms of extraction efficiency of antibacterial components, environmental protection and sustainability, application prospects, and resource recycling.

[0013] The technical solution of the present invention is as follows:

[0014] An extraction method for antibacterial components in the fermentation broth of entomopathogenic nematodes, comprising the following steps:

[0015] (1) Propagation and culture of entomopathogenic nematodes

[0016] Inoculate infective-stage entomopathogenic nematodes onto host insects for propagation. After the host insects die, collect the nematodes in their bodies and wash the collected nematodes to remove impurities;

[0017] (2) Inoculation and fermentation

[0018] Inoculate the washed nematodes into the prepared liquid medium and carry out fermentation under suitable temperature, pH value, and stirring conditions. After the fermentation is completed, collect the fermentation broth containing entomopathogenic nematodes and their metabolites;

[0019] (3) Preparation of the crude extract of the fermentation broth

[0020] Perform solid-liquid separation on the fermentation broth to obtain the supernatant. Adjust the pH value to 7 - 10, then adsorb it through macroporous adsorption resin, and then elute it successively with ethanol aqueous solutions with a volume fraction of 90%, 80%, 70%, and 60%. Collect the elution fraction with 60% ethanol, remove the ethanol, and obtain the crude extract of the fermentation broth;

[0021] (4) Extract the crude extract of the fermentation broth with a mixed solvent of chloroform and ether to obtain an extract. Remove the solvent from the extract and dry it to obtain the antibacterial component.

[0022] Furthermore, in step (1), the entomopathogenic nematode is a nematode of the family Steinernematidae, specifically Steinernema carpocapsae.

[0023] Furthermore, in step (1), the host insect is Galleria mellonella.

[0024] Furthermore, in step (2), the liquid medium is TSB medium.

[0025] Furthermore, in step (2), the fermentation conditions are 25°C - 30°C, the pH value is 7.0 - 7.4, and the fermentation time is 3 - 21 days.

[0026] Furthermore, in step (3), the macroporous adsorption resin is D101 macroporous adsorption resin.

[0027] Furthermore, in step (4), in the mixed solvent of chloroform and ether, the volume ratio of chloroform to ether is 7:3.

[0028] The present invention provides the application of the antibacterial component in the entomopathogenic nematode fermentation broth described above in the preparation of pesticides for controlling diseases of sweet potatoes and taros.

[0029] Furthermore, the disease of taro is soft rot of taro, and the disease of sweet potato is black rot of sweet potato.

[0030] Due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:

[0031] (1) Through delicate fermentation and extraction steps, especially by using macroporous adsorption resin and gradient ethanol elution technology, the present invention can efficiently enrich and separate the antibacterial component in the fermentation broth. This method not only improves the purity of the antibacterial component but also ensures the stability and repeatability of the extraction process.

[0032] (2) By multiplying and culturing entomopathogenic nematodes and extracting the antibacterial components from their fermentation broth, the present invention realizes the effective utilization and recycling of biological resources. This not only helps to reduce resource waste but also promotes the maintenance of ecological balance. The entire extraction process mainly uses physical and chemical methods such as solid-liquid separation, pH adjustment, adsorption and elution, solvent extraction, etc., avoiding the extensive use of harmful chemical reagents and reducing the risk of environmental pollution. At the same time, using entomopathogenic nematodes as a biological resource provides a new approach for the biological control of agricultural pests and promotes the sustainable development of agriculture.

[0033] (3) The antibacterial components extracted by the present invention have extensive potential application values. They can be used as natural antibacterial agents and play important roles in fields such as food preservation, medicine, and agriculture. Especially in the agricultural field, these antibacterial components can be used as substitutes for biological pesticides, reducing the use of chemical pesticides, lowering pesticide residues, and improving the safety and quality of agricultural products.

[0034] In summary, the extraction method of the present invention shows significant beneficial effects in terms of improving the extraction efficiency of antibacterial components, environmental protection and sustainability, application prospects, and resource recycling. Detailed implementation manners

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0036] Example 1

[0037] A method for extracting antibacterial components from the fermentation broth of entomopathogenic nematodes, comprising the following steps:

[0038] (1) Multiplication and culture of entomopathogenic nematodes

[0039] Inoculate infective-stage entomopathogenic nematodes onto host insects for multiplication culture. After the host insects die, collect the nematodes in their bodies and wash the collected nematodes to remove impurities; the entomopathogenic nematodes are nematodes of the family Steinernematidae, specifically Steinernema carpocapsae; the host insects are Galleria mellonella;

[0040] (2) Inoculation and fermentation

[0041] Inoculate the washed nematodes into the prepared TSB medium and ferment for 5 days at 28 °C and a pH value of 7.3. After the fermentation ends, collect the fermentation broth containing entomopathogenic nematodes and their metabolites;

[0042] (3) Preparation of the crude extract of the fermentation broth

[0043] The fermentation broth was subjected to solid-liquid separation to obtain the supernatant. The pH value was adjusted to 8, and then it was adsorbed through D101 macroporous adsorption resin. Subsequently, it was eluted successively with ethanol aqueous solutions with a volume fraction of 90%, 80%, 70%, and 60%. The 60% ethanol elution fraction was collected, and the ethanol was removed to obtain the crude extract of the fermentation broth;

[0044] (4) The crude extract of the fermentation broth was extracted three times with an equal volume of a mixed solvent of chloroform and ether. The extraction solutions were combined, the solvent was removed from the extraction solutions, and they were dried to obtain the antibacterial component. Among them, in the mixed solvent of chloroform and ether, the volume ratio of chloroform to ether was 7:3.

[0045] Example 2

[0046] A method for extracting the antibacterial component from the fermentation broth of entomopathogenic nematodes, comprising the following steps:

[0047] (1) Mass propagation and cultivation of entomopathogenic nematodes

[0048] The infective-stage entomopathogenic nematodes were inoculated onto host insects for mass cultivation. After the host insects died, the nematodes in their bodies were collected, and the collected nematodes were washed to remove impurities; the entomopathogenic nematodes were Steinernema nematodes, specifically Steinernema carpocapsae; the host insects were Galleria mellonella;

[0049] (2) Inoculation and fermentation

[0050] The washed nematodes were inoculated into the prepared TSB medium and fermented for 3 - 21 days at 25°C and a pH value of 7.0. After fermentation, the fermentation broth containing entomopathogenic nematodes and their metabolites was collected;

[0051] (3) Preparation of the crude extract of the fermentation broth

[0052] The fermentation broth was subjected to solid-liquid separation to obtain the supernatant. The pH value was adjusted to 7, and then it was adsorbed through D101 macroporous adsorption resin. Subsequently, it was eluted successively with ethanol aqueous solutions with a volume fraction of 90%, 80%, 70%, and 60%. The 60% ethanol elution fraction was collected, and the ethanol was removed to obtain the crude extract of the fermentation broth;

[0053] (4) The crude extract of the fermentation broth was extracted three times with an equal volume of a mixed solvent of chloroform and ether. The extraction solutions were combined, the solvent was removed from the extraction solutions, and they were dried to obtain the antibacterial component. Among them, in the mixed solvent of chloroform and ether, the volume ratio of chloroform to ether was 7:3.

[0054] Example 3

[0055] A method for extracting antibacterial components from the fermentation broth of entomopathogenic nematodes, comprising the following steps:

[0056] (1) Propagation and cultivation of entomopathogenic nematodes

[0057] Inoculate the infective-stage entomopathogenic nematodes onto the host insects for propagation. After the host insects die, collect the nematodes in their bodies and wash the collected nematodes to remove impurities; the entomopathogenic nematodes are Steinernema nematodes, specifically Steinernema carpocapsae; the host insects are Galleria mellonella;

[0058] (2) Inoculation and fermentation

[0059] Inoculate the washed nematodes into the prepared TSB medium and ferment for 21 days at 30 °C and a pH value of 7.4. After the fermentation is completed, collect the fermentation broth containing entomopathogenic nematodes and their metabolites;

[0060] (3) Preparation of the crude extract of the fermentation broth

[0061] Perform solid-liquid separation on the fermentation broth to obtain the supernatant. Adjust the pH value to 10, then adsorb it through D101 macroporous adsorption resin, and then elute it successively with ethanol aqueous solutions with a volume fraction of 90%, 80%, 70%, and 60%. Collect the elution part with 60% ethanol, remove the ethanol, and obtain the crude extract of the fermentation broth;

[0062] (4) Extract the crude extract of the fermentation broth three times with an equal volume of a mixed solvent of chloroform and ether, combine the extraction solutions, remove the solvent from the extraction solutions, and dry to obtain the antibacterial components. Among them, in the mixed solvent of chloroform and ether, the volume ratio of chloroform to ether is 7:3.

[0063] To illustrate the technical effects of the present invention, the inventors set up the following comparative examples and comparative tests:

[0064] Comparative Example 1

[0065] The difference from Example 1 is that in step (4), the fermentation broth is extracted with n-butanol, specifically as follows:

[0066] A method for extracting antibacterial components from the fermentation broth of entomopathogenic nematodes, comprising the following steps:

[0067] (1) Propagation and cultivation of entomopathogenic nematodes

[0068] During the infection stage, entomopathogenic nematodes are inoculated onto host insects for mass cultivation. After the host insects die, the nematodes in their bodies are collected and the collected nematodes are washed to remove impurities. The entomopathogenic nematodes are nematodes of the family Steinernematidae, specifically Steinernema carpocapsae; the host insects are Galleria mellonella;

[0069] (2) Inoculation and fermentation

[0070] The washed nematodes are inoculated into the prepared TSB medium and fermented for 5 days at 28 °C with a pH value of 7.3. After fermentation, the fermentation broth containing entomopathogenic nematodes and their metabolites is collected;

[0071] (3) Preparation of the crude extract of the fermentation broth

[0072] The fermentation broth is subjected to solid-liquid separation to obtain the supernatant. The pH value is adjusted to 8, then it is adsorbed through D101 macroporous adsorption resin, and then eluted successively with ethanol aqueous solutions with a volume fraction of 90%, 80%, 70%, and 60%. The 60% ethanol elution fraction is collected, the ethanol is removed, and the crude extract of the fermentation broth is obtained;

[0073] (4) The crude extract of the fermentation broth is extracted three times with an equal volume of water-saturated n-butanol. The extraction solutions are combined, the solvent is removed from the extraction solutions, and they are dried to obtain the antibacterial component.

[0074] Comparative Example 2

[0075] The difference from Example 1 is that in step (3), the 80% ethanol elution fraction is collected, specifically as follows:

[0076] A method for extracting antibacterial components from an entomopathogenic nematode fermentation broth, comprising the following steps:

[0077] (1) Mass propagation and cultivation of entomopathogenic nematodes

[0078] During the infection stage, entomopathogenic nematodes are inoculated onto host insects for mass cultivation. After the host insects die, the nematodes in their bodies are collected and the collected nematodes are washed to remove impurities. The entomopathogenic nematodes are nematodes of the family Steinernematidae, specifically Steinernema carpocapsae; the host insects are Galleria mellonella;

[0079] (2) Inoculation and fermentation

[0080] The washed nematodes are inoculated into the prepared TSB medium and fermented for 5 days at 28 °C with a pH value of 7.3. After fermentation, the fermentation broth containing entomopathogenic nematodes and their metabolites is collected;

[0081] (3) Preparation of the crude extract of the fermentation broth

[0082] Perform solid-liquid separation on the fermentation broth to obtain the supernatant. Adjust the pH value to 8, then adsorb it through D101 macroporous adsorption resin, and then elute it successively with ethanol aqueous solutions with a volume fraction of 90%, 80%, 70%, and 60%. Collect the elution fraction with 80% ethanol, remove the ethanol, and obtain the crude extract of the fermentation broth;

[0083] (4) Extract the crude extract of the fermentation broth three times with an equal volume of a mixed solvent of chloroform and ether, combine the extraction solutions, remove the solvent from the extraction solutions, and dry them to obtain the antibacterial component. Among them, in the mixed solvent of chloroform and ether, the volume ratio of chloroform to ether is 7:3.

[0084] Comparative Example 3

[0085] The difference from Example 1 is that in step (4), the fermentation broth is extracted with pure chloroform, specifically as follows:

[0086] A method for extracting antibacterial components from an entomopathogenic nematode fermentation broth, comprising the following steps:

[0087] (1) Propagation and cultivation of entomopathogenic nematodes

[0088] Inoculate infective-stage entomopathogenic nematodes onto host insects for propagation. After the host insects die, collect the nematodes in their bodies and wash the collected nematodes to remove impurities; the entomopathogenic nematodes are Steinernematidae entomopathogenic nematodes, specifically Steinernema carpocapsae; the host insects are Galleria mellonella;

[0089] (2) Inoculation and fermentation

[0090] Inoculate the washed nematodes into the prepared TSB medium and ferment for 5 days at 28 °C and a pH value of 7.3. After fermentation, collect the fermentation broth containing entomopathogenic nematodes and their metabolites;

[0091] (3) Preparation of the crude extract of the fermentation broth

[0092] Perform solid-liquid separation on the fermentation broth to obtain the supernatant. Adjust the pH value to 8, then adsorb it through D101 macroporous adsorption resin, and then elute it successively with ethanol aqueous solutions with a volume fraction of 90%, 80%, 70%, and 60%. Collect the elution fraction with 60% ethanol, remove the ethanol, and obtain the crude extract of the fermentation broth;

[0093] (4) Extract the crude extract of the fermentation broth three times with an equal volume of pure chloroform, combine the extraction solutions, remove the solvent from the extraction solutions, and dry them to obtain the antibacterial component.

[0094] Comparative experiment

[0095] Pot experiment

[0096] The pot experiment was carried out with reference to the literature "Li Qian, Liu Xia, He Jun, et al. Study on the antibacterial activities of different crude extracts from the fermentation broth of entomopathogenic nematode symbiotic bacterium YL001 [J]. Acta Agriculturae Boreali-occidentalis Sinica, 2006. DOI: CNKI:SUN:XBNX.0.2006-06-059."

[0097] The test methods are as follows:

[0098] Determination of protective effect: First, dilute the antibacterial components obtained in the examples and comparative examples with water to 1 mg / ml respectively to obtain the aqueous solutions of the test samples, and spray them on the leaves of potted plants. After moisturizing for 24 h, inoculate the spore suspension of plant pathogens (spray method) on the leaves of potted plants. Use water treatment as the control, with 3 replicates for each treatment and 1 pot for each replicate.

[0099] Determination of therapeutic effect: First, inoculate the spore suspension of the test plant pathogens (spray method) under moisturizing conditions. After moisturizing for 24 h, spray the aqueous solution of the test sample on the plants. Each treatment has 3 replicates and 1 pot for each replicate. After 7 d, conduct disease investigation according to the corresponding grading standards, and calculate the disease index and control effect according to the following formulas.

[0100] Disease index = (∑ number of leaves at each level × disease level) / (highest representative level × total number of leaves investigated) × 100%.

[0101] Control effect = (control disease index - treatment disease index) / control disease index × 100%.

[0102] The results are shown in Table 1 and Table 2.

[0103] Table 1 Control effects of different antibacterial components on sweet potato black rot

[0104]

[0105] Table 2 Control effects of different antibacterial components on soft rot of taro

[0106]

[0107] The results of the pot experiment show that there are significant differences in the protective and therapeutic effects of the antibacterial components obtained by the preparation method of the present invention and those obtained by the comparative examples on sweet potato black rot and soft rot of taro at a concentration of 1 mg / mL. The therapeutic effects of the present invention on sweet potato black rot and soft rot of taro are better, reaching 91.35% and 89.67% respectively.

[0108] The above description is a detailed description of the preferred and feasible embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications made under the technical spirit disclosed by the present invention shall fall within the scope of the patent covered by the present invention.

Claims

1. A method for extracting antibacterial components from insect pathogenic nematode fermentation liquid, characterized in that: The following steps are involved: (1) Propagation and cultivation of insect pathogenic nematodes Inoculate the infective insect pathogenic nematodes onto the host insects for expansion, collect the nematodes in the host insects after they die, and wash the collected nematodes to remove impurities; (2) Inoculation and fermentation inoculating the cleaned nematodes into the prepared liquid culture medium, fermenting under suitable temperature, pH value and stirring conditions, and collecting the fermentation liquid containing the insect pathogenic nematodes and their metabolites after the fermentation is completed; (3) Preparation of crude fermentation broth extract The fermentation liquid is subjected to solid-liquid separation to obtain a supernatant, the pH value of which is adjusted to 7-10, and then adsorbed by a macroporous adsorption resin, followed by elution with ethanol aqueous solutions with volume fractions of 90%, 80%, 70%, and 60% in sequence, and the 60% ethanol elution portion is collected, and the ethanol is removed to obtain a crude extract of the fermentation liquid; (4) extracting the crude fermentation broth with a mixed solvent of chloroform and ether to obtain an extract, removing the solvent from the extract, and drying to obtain the antibacterial component.

2. The method for extracting antibacterial components from the entomopathogenic nematode fermentation liquid according to claim 1, characterized in that: In step (1), the insect pathogenic nematode is an insect pathogenic nematode of the family Steinernema, specifically Steinernema carinella.

3. The method for extracting antibacterial components from the entomopathogenic nematode fermentation liquid according to claim 1, characterized in that: In step (1), the host insect is greater wax moth.

4. The method for extracting antibacterial components from the entomopathogenic nematode fermentation liquid according to claim 2, characterized in that: In step (2), the liquid culture medium is TSB culture medium.

5. The method for extracting antibacterial components from the entomopathogenic nematode fermentation liquid according to claim 2, characterized in that: In step (2), the fermentation conditions are 25° C.-30° C., the pH value is 7.0-7.4, and the fermentation time is 3-21 days.

6. The method for extracting antibacterial components from the entomopathogenic nematode fermentation liquid according to claim 1, characterized in that: In step (3), the macroporous adsorption resin is D101 macroporous adsorption resin.

7. The method for extracting antibacterial components from insect pathogenic nematode fermentation liquid according to claim 1, characterized in that: In step (4), in the mixed solvent of chloroform and ether, the volume ratio of chloroform to ether is 7:

3.

8. Use of the antibacterial component in the entomopathogenic nematode fermentation liquid as claimed in any one of claims 1 to 7 in the preparation of pesticides for preventing and controlling sweet potato and taro diseases.

9. The use according to claim 8, characterized in that The taro disease is taro soft rot, and the sweet potato disease is sweet potato black spot.

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