Nematode model taking lactobacillus plantarum TWK10 as positive control and construction method of nematode model

Abstract

The invention discloses a nematode model taking lactobacillus plantarum TWK10 as a positive control and a construction method of the nematode model. The nematode model is used for evaluating the exercise improving effect of lactic acid bacteria. The TWK10 is used as a model strain of a positive control group, the strain is a biotechnology patent strain, and animal and human body experiments prove that the TWK10 has the effect of improving the movement performance of a host. By using the strain, the specificity and accuracy of the test can be ensured, the comprehensive evaluation method is beneficial to revealing the multi-dimensional mechanism of the lactic acid bacteria action from multiple aspects, powerful support is provided for deeply researching the efficacy of the lactic acid bacteria, and meanwhile application and development of the lactic acid bacteria in related fields are promoted.

Description

Technical Field

[0001] The present invention relates to a nematode model using Lactobacillus plantarum TWK10 as a positive control and a construction method thereof, belonging to the field of biotechnology. Background Art

[0002] Lactic acid bacteria are a type of probiotic bacteria found widely in the human intestine. They offer numerous health benefits, including regulating immunity, preventing intestinal diseases, and relieving stress. In recent years, with the continuous advancement of genomics and metabolic engineering, research on lactic acid bacteria has deepened, and their application areas have continued to expand. Lactic acid bacteria are widely used in food fermentation, medicine, and animal feed, demonstrating significant market potential.

[0003] However, evaluating the mechanisms of action and efficacy of lactic acid bacteria remains challenging. The complex intestinal flora of livestock and poultry, coupled with the high cost and technical requirements of sterile animals, limits research on the effects and mechanisms of probiotics such as lactic acid bacteria. This makes quality control of probiotic preparations (microecological preparations) difficult, significantly impacting their rational development and application. Therefore, an effective model is needed to evaluate the effects and mechanisms of lactic acid bacteria.

[0004] Caenorhabditis elegans (Caenorhabditis elegans) is a model organism with a simple structure and extensive research on gene regulation. It shares a high degree of similarity with mammals in many aspects, including pathogen activation mechanisms, infection processes, and immune responses. Its short life cycle, high reproduction rate, ease of maintenance, and ease of observation using fluorescent labeling make it an ideal model for studying the effects and mechanisms of probiotic action.

[0005] Caenorhabditis elegans has been widely used in genetics, developmental biology, and drug screening research. It was the first multicellular organism to have its entire genome sequenced. Its internal structure is highly homologous to human cells, containing 12 highly homologous signaling pathways and expressing numerous genes that characteristically change during aging. Therefore, the C. elegans model can be used to construct various gene knockout or transgenic models, offering unique advantages in screening and evaluating the efficacy of probiotics.

[0006] Although C. elegans has shown great potential as an experimental animal in the screening and evaluation of probiotic effects, existing technologies still have the following defects and shortcomings:

[0007] 1. Complex model construction: In existing technologies, the construction of the C. elegans model usually requires cumbersome operational steps, such as nematode culture, stimulation, screening and detection, etc., with low throughput and a lack of standardization of nematode lactic acid bacteria administration and treatment, labeling and tracking technology, and control verification. This makes it difficult to achieve precise stimulation delivery, manipulation and tracking of individual nematodes, affecting the accuracy of the results.

[0008] 2. Single evaluation method: Existing technologies usually only evaluate the antibacterial effect of lactic acid bacteria through in vitro antibacterial experiments or in vivo antibacterial experiments in nematodes. There is a lack of systematic evaluation methods and it is unable to fully reflect the effects and mechanisms of lactic acid bacteria.

[0009] 3. Lack of evaluation of exercise efficacy: Lactic acid bacteria have multiple health benefits, including regulating human immunity and preventing intestinal diseases. However, existing technologies lack an assessment of their ability to enhance exercise. Exercise, as an endogenous stimulus, can effectively increase the body's oxygen consumption, increase stress levels, and enhance the body's adaptability, significantly impacting the effects and mechanisms of probiotics. In particular, there is a lack of a model strain with an effective positive control group as a valid evaluation measure.

[0010] In summary, the existing technology still has many defects and deficiencies in the evaluation of the effects and mechanisms of lactic acid bacteria. It is necessary to establish a more systematic, accurate and efficient evaluation method to comprehensively reflect the effects and mechanisms of lactic acid bacteria and provide strong support for the development and application of lactic acid bacteria. Summary of the Invention

[0011] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a nematode model using Lactobacillus plantarum TWK10 as a positive control and a method for constructing the same.

[0012] To achieve the above object, the present invention adopts the following technical solutions:

[0013] 1. The present invention provides a method for constructing a nematode model using Lactobacillus plantarum TWK10 as a positive control. The nematodes are thawed and cultured for more than three generations. The nematodes are crushed to extract eggs for synchronization to obtain L1 larvae. The larvae are fed with lactic acid bacteria until the larvae develop into young adults and mature adults. After the feeding is completed, the nematodes are washed multiple times until the worm bodies are as free of bacteria as possible and thoroughly cleaned. The exercise-enhancing effect of lactic acid bacteria is evaluated based on three indicators: crawling movement, swimming movement, and nematode muscle mass.

[0014] As one of the preferred technical solutions, the young adults are Day 1 and the mature adults are Day 5.

[0015] As one of the preferred technical solutions, a group fed only with the standard nematode food Escherichia coli OP50 was set as a blank control group.

[0016] As one of the preferred technical solutions, the nematodes are transferred to a sterile plate and allowed to crawl freely, and the crawling movement is analyzed by video recording;

[0017] As one of the preferred technical solutions, the nematodes were transferred to a sterile plate with a 1 cm layer of M9 buffer and allowed to swim freely, and the swimming movements were videotaped and analyzed;

[0018] As one of the preferred technical solutions, the cells were first rapidly frozen with liquid nitrogen and then permeabilized with acetone, followed by the use of Alexa Fluor TM The 488Phalloidin staining was performed overnight at room temperature, and the muscle mass of the nematodes was quantified by fluorescence photography. These three analyses evaluated the effectiveness of lactic acid bacteria in enhancing nematode motility.

[0019] 2. The present invention also provides a nematode model using Lactobacillus plantarum TWK10 as a positive control and a method for constructing the same, which is constructed using the aforementioned method.

[0020] Beneficial effects of the present invention:

[0021] In the present invention, Lactobacillus plantarum TWK10 was used as a positive control group to construct a nematode (Caenorhabditis elegans) model for evaluating the exercise-enhancing effect of lactic acid bacteria.

[0022] The present invention uses TWK10 as the model strain for the positive control group. This strain is a patented strain of Shenghe Biotechnology and has been proven to have the effect of improving the host's athletic performance through animal and human experiments.

[0023] By using this strain, the specificity and accuracy of the test can be ensured. The comprehensive evaluation method helps to reveal the multidimensional mechanism of action of lactic acid bacteria from multiple aspects, providing strong support for in-depth research on its efficacy, and also helps to promote the application and development of lactic acid bacteria in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 (A) Crawling speed, (B) swimming speed, and (C) body bending frequency during swimming of young adults on Day 1 and mature adults on Day 5 in each experimental group. Data are expressed as mean ± SD and compared using one-way analysis of variance with Tukey's post hoc test for parity comparisons or using Kruskal-Wallis test with Dunn's post hoc test for non-parity comparisons. Different letters (a, b) indicate significant differences between groups at p < 0.05; n ≥ 100.

[0025] Figure 2 Phalloidin staining shows the filamentous actin-positive areas in situ in (A) young adults on Day 1 and (B) mature adults on Day 5 of each experimental group, as well as (C) quantification of filamentous actin-positive areas in individual nematodes. Data are presented as mean ± standard deviation and were compared using the Kruskal-Wallis test with a Dunn post hoc test. Different letters (a, b) indicate significant differences between groups at p < 0.05; n = 30. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its contents.

[0027] 1. Brief description of the test target:

[0028] 1. TWK10: Lactiplantibacillus plantarum TWK10, a patented strain of Shenghe Biotechnology, has been proven to enhance athletic performance in animal and human studies.

[0029] 2.LGG: Lacticaseibacillus rhamnosus GG, ATCC No. 53103, BCRC No. 16000, the world's most well-known probiotic strain, with more than 30 years of accumulated scientific research.

[0030] 3. ATCC 14917: Lactiplantibacillus plantarum standard strain, ATCC No. 14917, BCRC No. 10069, experimental control.

[0031] 4.PCC: Limosilactobacillusfermentum Bioxyne's patented strain has immune-modulating properties, but clinical studies in athletes have shown that it does not enhance athletic performance.

[0032] 2. Experimental Design

[0033] The nematodes were thawed and cultured for more than three generations. The worm bodies were broken and the eggs were extracted for synchronization to obtain L1 larvae. The larvae were fed with TWK10, LGG, ATCC 14917 and PCC until they developed into young adults (Day 1) and mature adults (Day 5). The group fed only with the standard nematode food Escherichia coli OP50 (OP50) served as the blank control group. After the end of the experimental feeding, the nematodes were washed several times until the worm bodies were free of bacteria as much as possible. The thoroughly washed nematodes in each group were (1) transferred to a sterile plate and allowed to crawl freely. The crawling movement was videotaped and analyzed; (2) transferred to a sterile plate with a 1 cm M9 buffer water layer and allowed to swim freely. The swimming movement was videotaped and analyzed; (3) first quickly frozen with liquid nitrogen and then permeabilized with acetone. Then, Alexa Fluor was used to analyze the nematodes. TM The 488Phalloidin staining was performed overnight at room temperature, and the muscle mass of the nematodes was quantified by fluorescence photography. These three analyses evaluated the effectiveness of lactic acid bacteria in enhancing nematode motility.

[0034] 3. Test results

[0035] 1. Based on the analysis of motor behavior, the crawling and swimming speeds of young and mature nematodes in the TWK10 group were significantly improved compared with the OP50 control group, and the body swing frequency during swimming was also significantly increased. However, there was no significant difference between the LGG, ATCC 14917, and PCC groups and the OP50 control group, indicating that TWK10 feeding significantly and continuously improves the motor performance of nematodes. Figure 1 )

[0036] 2. Based on quantitative analysis of muscle fiber staining, the muscle mass of young and mature nematodes in the TWK10 group was significantly increased compared to the OP50 control group, while there was no significant difference between the LGG, ATCC 14917, and PCC groups and the OP50 control group, indicating that TWK10 feeding significantly and continuously increases the muscle mass of nematodes. Figure 2 )

[0037] Taken together, these results indicate that TWK10 feeding significantly improved athletic performance and muscle mass in both young and mature nematodes, consistent with previous animal and clinical trial results. The other three control lactic acid bacteria strains did not exhibit similar effects. Since LGG and PCC have been shown in previous clinical trials not to enhance host athletic performance, the nematode model can be used to evaluate the effectiveness of specific strains in enhancing host muscle mass and athletic performance.

[0038] Although the above describes the specific embodiments of the present invention in conjunction with the accompanying drawings, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A method for constructing a nematode model using Lactobacillus plantarum TWK10 as a positive control, characterized in that: The nematodes were thawed and cultured for more than three generations. The worm bodies were broken and the eggs were extracted for synchronization to obtain L1 larvae. The larvae were fed lactic acid bacteria until they developed into young adults and mature adults. After the feeding period, the nematodes were washed multiple times until the worm bodies were as free of bacteria as possible. After being thoroughly cleaned, the exercise-enhancing effect of lactic acid bacteria was evaluated using three indicators: crawling movement, swimming movement, and muscle mass of the nematodes.

2. The method according to claim 1, characterized in that Young adults are Day 1, and mature adults are Day 5.

3. The method according to claim 1, characterized in that Set up feeding only nematode standard food Escherichia coli The OP50 group served as the blank control group.

4. The method according to claim 1, wherein The nematodes were transferred to a sterile plate and allowed to crawl freely, and the crawling movements were recorded and analyzed.

5. The method according to claim 1, wherein The nematodes were transferred to a sterile plate with a 1 cm layer of M9 buffer and allowed to swim freely, and the swimming movements were videotaped and analyzed.

6. The method according to claim 1, characterized in that The cells were first snap-frozen in liquid nitrogen and then permeabilized with acetone. They were then stained with Alexa Fluor™ 488 Phalloidin overnight at room temperature, and fluorescence photography was used to quantify muscle mass. These three analyses evaluated the effectiveness of lactic acid bacteria in enhancing nematode motility.

7. A nematode model using Lactobacillus plantarum TWK10 as a positive control, characterized in that: It is obtained by the method according to any one of claims 1 to 6.

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