Free living nematode extracting solution as well as preparation method and application thereof

Abstract

The invention provides a free living nematode extracting solution as well as a preparation method and application thereof, and relates to the technical field of nematode extracting solutions, the free living nematode extracting solution is obtained by mixing glycerol, dimethyl sulfoxide and water; wherein the concentration of the glycerol and the dimethyl sulfoxide in the water is 1.15 g / cm < 3 >. The free living nematode extracting solution can be used for extracting marine nematodes and freshwater nematodes from various nematodes and has good selectivity, the extraction process is simple, and the extraction efficiency is high. The free living nematode extracting solution can effectively separate nematodes from sand grains, meanwhile, the integrity of the nematodes is kept, and it is ensured that the number of the nematodes in a sample can be accurately calculated.

Description

Technical Field

[0001] The invention relates to the technical field of nematode extracts, and in particular to a free-living nematode extract and a preparation method and application thereof. Background Art

[0002] Small benthic organisms refer to a class of multicellular metazoans with a size between 0.420mm and 0.500mm. They are divided into two categories: temporary small benthic organisms and permanent small benthic organisms. Temporary small benthic organisms mainly include the larvae of certain large benthic organisms or planktonic organisms, such as polychaetes. Permanent small benthic organisms include free-living nematodes, benthic copepods, polychaetes, and ostracods.

[0003] Free-living nematodes are a very significant group of invertebrates in the marine environment. With their wide distribution, rich species diversity, strong adaptability and significant numerical advantage, they have become an important part of small benthic animals. Nematodes can pass through a mesh with a pore size of 0.500mm, but will be intercepted by a mesh with a pore size of 0.042mm, showing their unique body characteristics. The two ends are pointed, the body is not segmented, bilaterally symmetrical, and has no appendages. The body surface is covered with a layer of tough keratin. Free-living nematodes have a complete digestive system, nervous system, excretory system, and a pseudocoelom, all of which provide guarantees for their survival in the marine environment.

[0004] Nematodes usually adhere to the surface of sediments or to the surface of algae and organic matter with the secretions of the tail gland, and use the head end to swim freely to feed. Free-living nematodes are dioecious organisms. Males are usually slightly smaller than females and have a short life cycle. They reproduce mainly through sexual reproduction, mainly oviparous, and occasionally viviparous. In their life history, there is no planktonic stage, and the larvae need to go through four molting processes before they can reach sexual maturity. Free-living nematodes dominate small benthic animals, and their abundance can be as high as 70% to 90% of the total abundance of small benthic animals. Some research stations even exceed 95%. Their biomass is also among the best, fully demonstrating their important position in the marine ecosystem. The species diversity of free-living nematodes is of great significance to the marine benthic ecosystem. In addition, they play an important role in the complex benthic food web of the marine ecosystem and have potential monitoring value in marine environmental pollution, which makes them increasingly valued.

[0005] Marine nematodes are an important group of small benthic organisms in the ocean. They play an important role in the energy flow and material cycle of benthic ecosystems, and in the nutrient cycle, secondary production, sediment transport and mineralization of the water layer-benthic ecosystem, as well as in stimulating microbial production. Nematodes are divided into two categories: parasitic nematodes and free-living nematodes. Free-living nematodes can live in soil, freshwater and marine sediments. Free-living nematodes specifically refer to nematodes living in marine sediments and nematodes living in freshwater sediments. There are many types of small benthic animals, among which free-living nematodes have the highest dominance. They are not only numerous, diverse in species, but also widely distributed. Marine nematodes have the characteristics of short life cycle, easy sampling in the field, and no mayfly stage in their life history. The health of the marine benthic environment can be inferred based on the richness index, maturity index, biomass, species composition and community structure changes of marine nematodes.

[0006] In the delicate and diverse work of sorting small benthic organisms, the current methods include manual direct selection, panning, transfer, ice floating, oxygen consumption and density gradient methods. However, the above methods also have their limitations. For different types of small benthic organisms, the sorting effect will be different, and there are certain differences in the sorting rate. Most of the nematode extracts in the prior art are Ludox solutions, which are nano-scale silica sols. The advantages are high extraction efficiency, and the collection rate can reach 90% through three centrifugation. The disadvantages are that the cost of using nano-scale silica sols is high, and the subsequent manual screening is time-consuming and laborious. The main component of nano-scale silica sols is nano-scale silica, which will cause harm to human lungs when in excess. Ingestion will cause abdominal fibrosis, and itching and other symptoms will occur when it comes into contact with the skin. In addition, impurities will inevitably exist in the supernatant. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a free-living nematode extract and a preparation method and application thereof.

[0008] In order to achieve the above purpose, the specific technical solutions of the present invention are as follows.

[0009] The first aspect of the present invention provides a free-living nematode extract, wherein the free-living nematode extract is an aqueous solution of glycerol and dimethyl sulfoxide;

[0010] The concentrations of glycerol and dimethyl sulfoxide in water are both 1.15 g / cm 3 .

[0011] The free-living nematode extract of the present invention is prepared by mixing glycerol, dimethyl sulfoxide and water to obtain a density of 1.15 g / cm 3The extract can effectively separate the nematodes from the sediment while maintaining the integrity of the nematodes. Its density is 1.15g / cm 3 The density is the same as that of free nematodes, and nematodes of different sizes but the same density can be effectively separated, and then different nematodes can be extracted in a targeted manner. If the density is too small or too large, the extraction effect will be affected. In addition, the free-living nematode extract in the present invention does not contain nano-silicon dioxide, so it can effectively avoid the harm caused by nano-silicon dioxide and reduce impurities in the supernatant.

[0012] In another preferred embodiment, the free-living nematodes are marine nematodes and freshwater nematodes.

[0013] The second aspect of the present invention provides a method for preparing a free-living nematode extract, wherein the free-living nematode extract is provided by the first aspect of the present invention, comprising the following steps:

[0014] Mix glycerol and dimethyl sulfoxide with water to make the concentration of glycerol and dimethyl sulfoxide 1.15 g / cm 3 .

[0015] The third aspect of the present invention provides the use of the free-living nematode extract in extracting free-living nematodes.

[0016] The fourth aspect of the present invention provides a method for extracting free-living nematodes, comprising mixing the free-living nematode extract provided by the first aspect of the present invention with sediment, centrifuging, collecting the supernatant, and filtering to obtain free-living nematodes; the sediment is sieved to obtain mud and sand containing free-living nematodes.

[0017] In another preferred embodiment, the volume ratio of the sediment to the free-living nematode extract is 1:2 to 2.5. The volume ratio of the commonly used extract, such as Ludox solution, to the sediment is 3 to 4:1, and the present invention can still ensure the extraction effect of the free-living nematodes while reducing the amount of the extract.

[0018] In another preferred embodiment, the filtration uses a screen with a pore size of 0.042 mm to ensure that only nematodes of a desired size are retained.

[0019] In another preferred embodiment, the centrifugation is performed 3 to 4 times at a rotation speed of 1800 rpm to 2000 rpm.

[0020] In another preferred embodiment, the living nematodes are marine nematodes and freshwater nematodes.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The free-living nematode extract of the present invention is prepared by mixing glycerol and dimethyl sulfoxide with water, and the obtained free-living nematode extract has a good extraction effect on marine nematodes and freshwater nematodes, good selectivity, and a simple extraction process with high extraction efficiency. The free-living nematode extract can effectively separate the nematodes from the sand while maintaining the integrity of the nematodes, ensuring that the number of nematodes in the sample can be accurately counted.

[0023] The free-living nematode extract of the present invention does not contain nano-scale silicon dioxide, thereby avoiding the harm caused by nano-scale silicon dioxide and reducing impurities in the supernatant after centrifugation, thereby further improving the extraction effect.

[0024] (2) The free-living nematode extract of the present invention can extract 118±42 ind. of marine nematodes and 96±2 ind. of freshwater nematodes, indicating that the free-living nematode extract of the present invention can specifically extract different nematodes from mud and sand. DETAILED DESCRIPTION

[0025] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0026] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0027] Abroad, while conducting marine sediment research in the United States, scientist Couch developed a method called the Baerman funnel method, which is specifically used to concentrate and collect nematodes in the ocean. The Baerman funnel method mainly includes the following steps: the researchers will initially concentrate the collected marine sediments to reduce the volume and water content of the samples, and the concentrated sediments will be poured into a special funnel, which is covered with a layer of 63μm nylon mesh, which allows particles smaller than 63μm, including nematodes, to pass through, while intercepting larger particles. On top of the nylon mesh, researchers will place a layer of glass beads or sand with a particle size greater than 125μm. The main function of this filter layer is to serve as a support layer and prevent the nylon mesh from being crushed or blocked by large particles when pouring sediments. In this way, nematodes and other microorganisms smaller than 63μm can pass through the nylon mesh smoothly into the bottom of the funnel, while larger particles are intercepted on the net. The design capacity of each funnel can collect approximately 5,800 nematodes, which provides a rich sample for subsequent nematode research and analysis. The Baermann funnel method not only improves the efficiency of nematode collection, but also effectively removes impurities in the sediment through screening and filtration, providing a purer sample for subsequent laboratory analysis, with a collection rate of up to 80%. Each device can screen at least 1,000 nematodes. Screening is the simplest method. During the screening process, the selection of the sieve requires that most particles be screened out while retaining a large number of nematodes. Usually, a sieve with an aperture of 20 μm to 75 μm is used. The smaller the aperture, the more nematodes are retained, but the more impurities are retained. This method is simple and easy to operate and can be used for a variety of materials and substrates. The main disadvantages are large differences in extraction efficiency, lack of quantifiable results, low extraction rate, and slow experimental efficiency. The advantages are low labor requirements and simple extraction methods.

[0028] The extracting liquid in the prior art is mostly nano-scale silica sol, which has a high collection rate, but is expensive and has certain risks during operation.

[0029] Therefore, the free-living nematode extract obtained by mixing glycerol, dimethyl sulfoxide and water in the present invention can not only screen out marine nematodes and freshwater nematodes from nematode samples, but also has an extraction rate comparable to that of nano-scale silica sol. In addition, the free-living nematode extract in the embodiment of the present invention has low cost and does not cause harm to the human body.

[0030] A free-living nematode extract is described in detail below.

[0031] 1. Methodology and sampling

[0032] 1.1 Sampling location and experimental materials

[0033] 1.1.1 Sampling location

[0034] In March and April 2024, sample collection was carried out in the main stream area of ​​Tanghe River in Qinhuangdao and Houhai Bathing Beach in Qinhuangdao. To ensure the accuracy of the collection process, a columnar sampler with a diameter of 2.8 cm was used to accurately collect 5 cm nematode samples, which were then stained with eosin for subsequent nematode counting using a dissecting microscope.

[0035] 1.1.2 Experimental Materials

[0036] Experimental supplies: density meter; measuring cylinder; 0.042mm and 0.500mm aperture sieves; rubber-tipped dropper; centrifuge tube; culture dish; dissecting needle; 2.8cm diameter cylindrical sampler.

[0037] Experimental reagent: Ludox solution (Sigma), density 1.15 g / cm 3 , distilled water, glycerol, dimethyl sulfoxide (Analytical Grade, Chinese Medicine) density 1.02g / cm 3 , 75% concentration alcohol, eosin stain.

[0038] Experimental instruments: centrifuge, dissecting microscope, electronic balance.

[0039] 3 kinds of extract solution configuration:

[0040] Glycerol is mixed with distilled water to obtain a density of 1.15 g / cm 3 of glycerol solution.

[0041] Glycerol, dimethyl sulfoxide and distilled water were mixed to obtain a free-living nematode extract, wherein the concentration of glycerol and dimethyl sulfoxide in water was 1.15 g / cm 3 .

[0042] Dimethyl sulfoxide and distilled water were mixed to obtain a concentration of 1.15 g / cm 3 of dimethyl sulfoxide solution.

[0043] 1.2 Screening of free-living nematode extracts

[0044] Free-living nematode extraction experiments

[0045] (1) Extraction of free-living nematodes using Ludox solution:

[0046] The sample was preliminarily processed by sieving with a sieve with an upper mesh size of 0.500 mm and a lower mesh size of 0.042 mm to remove large silt impurities and obtain sediment. The screened sample was transferred to a centrifuge tube and 2 to 2.5 times the volume of the sediment was added with Ludox solution. Then it was placed in a centrifuge tube and centrifuged at a level of no more than two-thirds of the liquid level. This centrifugation process needed to be repeated three times. After each centrifugation, the sample supernatant was collected and filtered through a 0.042 mm mesh to ensure that only nematodes of the required size were retained. After filtering, the nematodes were collected in a culture dish for further observation and counting.

[0047] (2) Extraction of free-living nematodes using three kinds of extracts:

[0048] Sediments were centrifuged by adding 2 to 2.5 times the volume of the three extracts to the sediments, and the remaining steps were the same as in (1). In order to ensure the accuracy of the results, repeated experiments were set up. Marine nematodes were collected by the supernatant pouring method, and the supernatant was filtered through a 0.042 mm pore size sieve to ensure that only nematodes of the required size were retained. The filtered samples were collected in a culture dish for further observation and counting.

[0049] (3) In order to more comprehensively explore the effect of different solutions with the same density on the centrifugal effect of the sample, the density of each solution was 1.15 g / cm 3 The sediment was centrifuged using the three extracts, glycerol solution, free-living nematode extract, and dimethyl sulfoxide solution. The remaining steps were the same as the above method. In order to ensure the accuracy of the results, parallel experiments were set up. The entire experimental process not only ensured the purity and accuracy of the freshwater nematode samples, but also provided reliable data support for this study.

[0050] 2. Experimental results and analysis

[0051] 2.1 Data and analysis of nematode extract screening experiments

[0052] Table 1 shows a series of extract comparison experiments conducted in April 2024. Ludox solution showed stable extraction performance, and its average extraction amount reached 139.5±1.5ind.. However, among the freshwater nematode extracts, the free-living nematode extract showed a higher extraction efficiency, with the average extraction amount of the solution reaching 96±2ind., equivalent to 68.81% of the average extraction amount of Ludox solution, becoming the choice with the highest average extraction amount except Ludox extract. In contrast, the extraction effect of glycerol solution was slightly inferior, with an average extraction amount of 78.5±1.5ind., which was only 56.27% of the average extraction amount of Ludox solution, becoming the extract with the lowest average extraction amount.

[0053] Table 1 Quantity of freshwater nematodes extracted

[0054]

[0055] Table 2 shows a comparison experiment of various nematode extracts in May 2024. In the comparison experiment of various extracts conducted in April 2024, the extraction effects of different extracts on marine nematodes were compared. The results showed that the average extraction amount of Ludox solution was 155±56ind., which was a higher level among various extracts. However, the average extraction amount of free-living nematode extracts was higher, reaching 118±42ind., which was equivalent to 76.13% of the average extraction amount of Ludox solution. In contrast, the average extraction amount of dimethyl sulfoxide solution was the lowest, at 71±15ind., accounting for only 45.81% of the average extraction amount of Ludox solution.

[0056] Table 2 Extraction quantity of marine nematodes

[0057]

[0058] As can be seen from the results in Tables 1 and 2, freshwater nematode analysis: After careful analysis of the experimental data, it was found that the free-living nematode extract had the closest performance to the Ludox solution in terms of the number of nematodes extracted. The average number of Ludox solution extracted was as high as 139.5±1.5ind., while the number of free-living nematode extracts extracted was 68.81% of that of Ludox solution, with an average number of 96±2ind. In contrast, when dimethyl sulfoxide or glycerol was used alone as the extract, the number of nematodes extracted was significantly reduced. The average extraction amount of dimethyl sulfoxide solution was 87.5±4.5ind., while the average extraction amount of glycerol solution was only 78.5±1.5ind., accounting for only 62.72% and 56.27% of the number of Ludox solution extractions, respectively.

[0059] Marine nematode analysis: After careful analysis of the experimental data and comparative experiments, it was observed that the free-living nematode extract showed similar performance to the Ludox solution in terms of the number of nematode extractions. Specifically, the average extraction number of the Ludox solution was as high as 155±56ind., while the extraction number of the free-living nematode extract reached 76.13% of that of the Ludox solution, showing its significant potential in nematode extraction. The average extraction number was 118±42ind. In contrast, when dimethyl sulfoxide or glycerol was used alone as an extraction solution, the number of nematodes extracted was significantly reduced. The average extraction amount of the dimethyl sulfoxide solution was 71±15ind., while the average extraction amount of the glycerol solution was only 101±39ind., accounting for only 65.16% and 15.81% of the extraction amount of the Ludox solution, respectively. As described in the experiment, the free-living nematode extract became the extract with the closest extraction rate to the Ludox solution.

[0060] 2.2 Comparative experiment on nematode extraction rate

[0061] Table 3 is a single-factor ANOVA table. The p value of the type of extract is 0.000. It can be seen that the type of extract has a significant effect on the extraction rate of small benthic animals.

[0062] Table 3 One-way ANOVA

[0063]

[0064] Note: * indicates significant correlation (p<0.05); ** indicates extremely significant correlation (p<0.01)

[0065] Table 4 shows the results of the comparative experiment of the number of centrifugal extractions of freshwater nematodes by various extracts in April 2024. In this experiment, the average centrifugal extraction number of Ludox solution reached 155±5.6ind., which accounted for 89.19% of the total extraction number, showing the significant advantage of Ludox solution in centrifugal extraction of nematodes. Among the many extracts, the extraction effect of free-living nematode extract is particularly obvious. The average centrifugal extraction number of this extract is 76±1.5ind., accounting for 79.17% of the total number, which is close to the extraction efficiency of Ludox solution. This data shows that the free-living nematode extract has a good performance in centrifugal extraction of nematodes and has the potential to be a substitute for Ludox solution. In contrast, the centrifugal extraction effect of glycerol solution is relatively inferior. Its average centrifugal extraction number is 62±2.5ind., accounting for only 44.29% of the total number. This data shows that the effect of glycerol solution in centrifugal extraction of freshwater nematodes is not as significant as other extracts, and it is not suitable for use as the main extract.

[0066] Table 4 Extraction rate of freshwater nematodes

[0067] Extract Extraction rate Ludox 89.19％ glycerin 44.29％ Dimethyl sulfoxide 65.12％ Free-living nematode extract 79.17％

[0068] Table 5 shows the significant differences in the number of centrifugal extractions of nematodes observed in various extracts in the comparison experiment of the number of centrifugal extractions of marine nematodes conducted in April 2024. The specific data are as follows: Ludox solution stood out in the experiment with its stable performance, and its average centrifugal extraction number reached 155±56ind., and the number of centrifugal extractions accounted for 77.55% of the total number, fully demonstrating the excellent performance of Ludox solution in centrifugal extraction of nematodes. Among the many extracts, the number of centrifugal extractions of free-living nematode extracts is more significant. Its average centrifugal extraction number is 118±42ind., accounting for 64.96% of the total number, which is second only to Ludox solution, showing the strong potential of glycerol and dimethyl sulfoxide mixed solution in centrifugal extraction of nematodes, and is expected to become a substitute for Ludox solution. However, the performance of glycerol solution in centrifugal extraction of nematodes is relatively weak. Its average centrifugal extraction number is 101±39ind., accounting for only 46.92% of the total number, and its extraction efficiency is significantly lower than that of other extracts.

[0069] Table 5 Extraction rate of marine nematodes

[0070]

[0071]

[0072] Through the above experimental data, we can clearly see the experimental results. The free-living nematode extract showed good performance in both extraction number and extraction rate. This result shows that the extract has potential application value in the extraction of freshwater nematodes, especially in cost control and large-scale application. It has significant advantages. The extraction rate of the free-living nematode extract is 64.96%, and the price of the agent is more suitable after the two solutions are mixed.

[0073] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A free-living nematode extract, characterized in that: The free-living nematode extract is an aqueous solution of glycerol and dimethyl sulfoxide; The concentrations of glycerol and dimethyl sulfoxide in water are both 1.15 g / cm 3 .

2. The free-living nematode extract according to claim 1, characterized in that The free-living nematodes are marine nematodes and freshwater nematodes.

3. A method for preparing the free-living nematode extract according to claim 1, characterized in that: The following steps are involved: Mix glycerol, dimethyl sulfoxide and water so that the concentration of glycerol and dimethyl sulfoxide is 1.15 g / cm 3 .

4. Use of the free-living nematode extract according to claim 1 in extracting free-living nematodes.

5. A method for extracting free-living nematodes, characterized in that: The free-living nematode extract of claim 1 is mixed with sediment, centrifuged, the supernatant is collected, and filtered to obtain the free-living nematodes; The sediment is sifted to obtain the mud and sand containing free-living nematodes.

6. The method for extracting free-living nematodes according to claim 5, characterized in that: The volume ratio of the sediment to the free-living nematode extract is 1:2 to 2.

5.

7. The method for extracting free-living nematodes according to claim 5, characterized in that: The filtration uses a sieve with a pore size of 0.042 mm.

8. The method for extracting free-living nematodes according to claim 5, characterized in that: The number of centrifugation is 3 to 4 times, and the rotation speed is 1800 rpm to 2000 rpm.

9. The method for extracting free-living nematodes according to claim 5, characterized in that: The living nematodes are marine nematodes and freshwater nematodes.

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