Method for preparing alkyl glycoside by using caenorhabditis elegans
Through the metabolic regulation and multimodal separation process of C. elegans, the product diversity and environmental pollution problems in the preparation of traditional alkyl glycosides were solved, and efficient and green preparation of C7-C15 series alkyl glycosides was achieved.
Patent Information
- Application Number
- CN202510563423.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-08-12
AI Technical Summary
Traditional alkyl glycoside preparation methods have problems such as low product diversity, mechanical wall breaking can easily lead to thermal damage, low extraction efficiency and serious environmental pollution, and it is difficult to obtain alkyl glycosides with wide molecular weight range and different glycosidic bond configurations simultaneously.
The metabolic regulation and multimodal separation process of C. elegans were used to prepare alkyl glycosides by combining ultrasonic crushing, liquid nitrogen freeze-drying, green extractant and high-performance liquid chromatography-mass spectrometry detection.
The C7-C15 series alkyl glycosides were obtained simultaneously, which improved the extraction efficiency by 40%, and there were no toxic by-products throughout the process, which was green and environmentally friendly.
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Figure CN120463754A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for preparing alkyl glycoside by utilizing Caenorhabditis elegans, belonging to the technical field of biochemical engineering. Background Art
[0002] Glucosides are natural organic compounds with important biological activities, formed by glycosidic bonds linking a sugar molecule (usually glucose) to another molecule (such as an alcohol, phenol, or organic acid). A specific type of alkyl glycoside combines the advantages of both common nonionic and anionic green surfactants. These alkyl glycosides are formed by condensing starch or its hydrolyzed sugars with fatty alcohols, and are readily available from a wide range of sources. As early as 1893, Emil Fischer proposed a method for methylating glycoside molecules. Alkyl polyglycosides (APGs) were first synthesized in 1936. For more than 40 years thereafter, knowledge of APGs remained at the level of academic interest. It was not until the 1980s that scientists began to focus on the development of APG processes. For example, known methyl-β-D-glucopyranoside, butyl-β-D-glucopyranoside, hexyl-β-D-glucopyranoside, and nonyl-β-D-glucopyranoside can be synthesized by direct methods. Due to their excellent emulsifying, dispersing, and detergency properties, complete biodegradability, and non-toxicity, alkyl polyglycosides are often used as emulsifiers, dispersants, detergents, and skin nourishing cleansers. They have a wide range of applications and have attracted increasing attention in recent years. The increasing resistance of microorganisms to antibiotics is a major public health concern. Many pathogens utilize quorum sensing to regulate their pathogenicity, so combined therapy with quorum sensing inhibitors and antibiotics may represent a novel therapeutic option for infectious diseases in the future. Methyl-β-D-glucopyranoside, isolated from a metabolite of Scabiosa comosa, has shown no antimicrobial activity as a quorum sensing inhibitor. Furthermore, methyl-β-D-glucopyranoside has been reported to be an antitumor agent with low cytotoxicity (Davaapurev et al. 2022). Butyl-β-D-glucopyranoside exhibits antioxidant activity. Hexyl-β-D-glucopyranoside is a surfactant that protects tryptophan residues from potential photoinduced oxidation.
[0003] Traditional methods for preparing alkyl glycosides rely on chemical synthesis or a single microbial metabolic pathway, and have the following problems: low product diversity, making it difficult to simultaneously obtain alkyl glycosides with a wide molecular weight range (C7-C15) and different glycosidic bond configurations; mechanical wall breaking during the extraction process can easily lead to thermal damage and low extraction efficiency; the use of chemical synthetic raw materials or highly polluting solvents causes serious environmental pollution; and traditional column chromatography has insufficient resolution, making it difficult to distinguish alkyl glycosides with similar structures. Summary of the Invention
[0004] In order to solve the problems existing in the prior art, the present invention provides a method for preparing alkyl glycosides using Caenorhabditis elegans, the method specifically comprising the following steps:
[0005] (1) Cultivate Caenorhabditis elegans and stop culturing when the nematodes crawl all over the bottle wall.
[0006] (2) Cleaning and collecting the Caenorhabditis elegans cultured in step (1), ultrasonically disrupting the nematodes, and immediately freezing them with liquid nitrogen and then freeze-drying them at ultra-low temperatures to obtain dry powder.
[0007] (3) Extracting the dried powder with an extractant and filtering to obtain an extract.
[0008] (4) The extract obtained in step (3) is subjected to rotary evaporation to obtain a solid substance, the solid substance is redissolved with methanol and allowed to stand, and after the standing is completed, the supernatant is centrifuged to obtain the supernatant, the supernatant is allowed to stand again and then centrifuged, and the standing and centrifugation are repeated until no precipitate appears in the supernatant, thereby obtaining a precipitate-free supernatant.
[0009] (5) The precipitate-free supernatant obtained in step (4) was filtered and then subjected to LC-MS detection to obtain methyl-β-D-pyranoglucoside, butyl-β-D-pyranoglucoside, hexyl-β-D-pyranoglucoside and nonyl-β-D-pyranoglucoside.
[0010] Preferably, the culture temperature in step (1) is 24-28° C., and the culture medium used is oatmeal culture medium.
[0011] Preferably, the method for washing the C. elegans in step (2) is filtering and rinsing with sterile water.
[0012] Preferably, the operating parameters of the ultrasonic cell disruptor in step (2) are: power 70-80 Hz, ultrasonic time 5 s, rest time 10 s, and a total ultrasonic time of 25-30 min.
[0013] Preferably, in step (3), 20 to 30 ml of an extractant is added to every 2 g of dry powder, and the extractant used is methanol.
[0014] Preferably, the extraction conditions in step (3) are: extraction at 20-22° C. and 170-200 rpm for 23-25 hours, and extraction at least 3 times; after each extraction, the extract is filtered and collected with filter paper, and the filter residue obtained is used for the next extraction.
[0015] Preferably, the standing time in step (4) is 12 to 16 hours, the standing temperature is 4 to 6° C., and the centrifugation condition is 5000 to 6000 rpm for 3 to 4 minutes.
[0016] Preferably, the LC conditions in the LC-MS detection in step (5) are: the chromatographic column is Hypersil GOLD™ 100×2.1, the column temperature is 40° C., the mobile phase A is 0.5% formic acid water by volume, the mobile phase B is methanol, the gradient elution conditions are 0-2 min: 5% B, 12-15 min: 5%-95% B, 16-20 min 5% B, and the flow rate is 0.3 mL / min.
[0017] Technical effects of the present invention:
[0018] (1) For the first time, methyl-β-D-pyranoside, butyl-β-D-pyranoside, hexyl-β-D-pyranoside, and nonyl-β-D-pyranoside were obtained simultaneously through nematode metabolic regulation and multimodal separation technology.
[0019] (2) Ultrasonic crushing + liquid nitrogen quick freezing combined technology increases extraction efficiency by 40%.
[0020] (3) The entire process uses nematode metabolites and green extractants, without any toxic by-products. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is the experimental flow chart of the present invention.
[0022] Figure 2 is the structural formula of methyl-β-D-pyranoside prepared in Example 1.
[0023] Figure 3 is the structural formula of butyl-β-D-pyranoside prepared in Example 1.
[0024] Figure 4 is the structural formula of hexyl-β-D-pyranoside prepared in Example 1.
[0025] Figure 5 is the structural formula of nonyl-β-D-glucopyranoside prepared in Example 1. DETAILED DESCRIPTION
[0026] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to the contents described above.
[0027] Preparation of culture medium used in the examples
[0028] Oatmeal culture medium: Thoroughly mix coarse and fine oats in a 1:1 mass ratio. Accurately weigh 30g and evenly spread it on the bottom of a 250ml Erlenmeyer flask. Then, add purified water to the flask, adding enough water to completely submerge the oats and extend 2cm above the surface. After completing the above steps, sterilize the prepared culture medium using moist heat sterilization (121°C, 20 minutes, 103.4kPa pressure). After sterilization, store the culture medium in a sterile environment until use to prevent contamination by other bacteria.
[0029] Example 1
[0030] A method for preparing alkyl glycosides using Caenorhabditis elegans, the process flow is as follows Figure 1 The specific steps are as follows:
[0031] (1) Use a 22 cm stainless steel spoon to scoop a spoonful of Caenorhabditis elegans into a 250 ml conical flask containing 100 ml of oatmeal culture medium and incubate at 28°C until the nematodes crawl all over the flask wall.
[0032] (2) Rinse the nematodes on the bottle wall with sterile double-distilled water (ddH2O), filter the oat residue through a sieve to retain the nematodes, and dispense the nematodes into 50ml centrifuge tubes. Add ddH2O to 45ml again to allow natural sedimentation and wash the nematodes. Repeat the washing process until the supernatant is clear and collect the cleaned nematodes.
[0033] (3) Crushing: The collected worms were crushed using an ultrasonic cell disruptor with a working time of 5 s, an intermission time of 10 s, and a power of 80 Hz. The nematodes were crushed for 30 min. The crushed nematode homogenate was poured into a freeze-drying bottle, quickly frozen with liquid nitrogen, and then placed in an ultra-low temperature freeze dryer for 3 days. To ensure the freeze-drying effect, the volume of nematodes in a 100 mL drying bottle should not exceed 30 mL.
[0034] (4) Gradient dynamic extraction: After drying the nematode powder, add 40 ml of methanol for every 2 g of powder, seal the container and place it in a shaker. Extract at 20°C and 180 rpm for 24 h, then filter with filter paper and collect the filtrate. Add the extractant to the residue and extract it again. Extract for a total of 3 times. Combine the filtrates obtained each time, and the combined filtrates are the extracts.
[0035] (5) Rotary evaporation: The combined extracts were subjected to rotary evaporation to obtain a solid substance. The solid substance was re-dissolved with 1 ml of methanol and then added to a 1.5 ml centrifuge tube. The solid substance was precipitated in a 4°C refrigerator overnight. The supernatant was centrifuged at 5000 rpm for 3 min. The supernatant was placed in a 4°C refrigerator overnight and precipitated. The supernatant was centrifuged at 5000 rpm for 3 min. The precipitation and centrifugation operations were repeated at 4°C until no precipitate appeared in the supernatant to obtain a precipitate-free supernatant.
[0036] (6) The precipitate-free supernatant obtained in step (5) was filtered through a 0.22 μm filter and placed in a sample injection bottle for LC-MS detection.
[0037] (7) LC-MS detection: The instrument model for LC-MS detection is Orbitrap Exploris 120, and the chromatographic column is Hypersil GOLD tm 100×2.1. The elution gradient was 95% formic acid in water for 0-12 min, 95% methanol for 12-15 min, and then 95% formic acid in water for 16-20 min. The flow rate was 0.3 ml / min and the injection volume was 5 μl.
[0038] LC conditions: The instrument model for LC-MS detection is Orbitrap Exploris 120, the chromatographic column is Hypersil GOLD™ 100×2.1, the column temperature is 40°C, the mobile phases are A (0.5% formic acid in water) and B (methanol), the gradient elution is (0-12 min: 5% B→12-15 min: 5%-95% B, 16-20 min 5% B), the flow rate is 0.3 mL / min, and the injection volume is 5 μL.
[0039] MS conditions: ion source H-ESI, positive ion (+3500 V) and negative ion (-2500 V) modes, gas settings: sheath gas 35 Arb, auxiliary gas 10 Arb, sweep gas 1 Arb, ion transfer tube temperature 325°C, evaporator temperature 350°C, full scan, resolution 60,000, m / z 100→550, RF Lens 70%, expected LC peak width 6 seconds, internal mass calibration EASY-IC TM (Calibration starts during runtime).
[0040] (8) Result analysis: The result analysis is as follows Figures 2 to 5 As shown in the figure, it can be seen that methyl-β-D-glucopyranoside, butyl-β-D-glucopyranoside, hexyl-β-D-glucopyranoside and nonyl-β-D-glucopyranoside can be obtained simultaneously through the above steps.
Claims
1. A method for preparing alkyl glycosides using Caenorhabditis elegans, characterized in that: The following steps are involved: (1) Cultivating Caenorhabditis elegans until the nematodes crawl all over the bottle wall; (2) washing and collecting the C. elegans cultured in step (1), performing ultrasonic disruption, immediately freezing with liquid nitrogen, and then freeze-drying to obtain a dry powder; (3) extracting the dried powder with an extractant and filtering to obtain an extract; (4) The extract obtained in step (3) is subjected to rotary evaporation to obtain a solid substance, the solid substance is redissolved in methanol and allowed to stand, and after the standing is completed, the supernatant is centrifuged to obtain the supernatant, the supernatant is allowed to stand again and then centrifuged, and the standing and centrifugation are repeated until no precipitate appears in the supernatant, thereby obtaining a precipitate-free supernatant; (5) The precipitate-free supernatant obtained in step (4) was filtered and then subjected to LC-MS detection to obtain methyl-β-D-pyranoglucoside, butyl-β-D-pyranoglucoside, hexyl-β-D-pyranoglucoside and nonyl-β-D-pyranoglucoside.
2. The method for preparing alkyl glycosides using Caenorhabditis elegans according to claim 1, wherein: The culture temperature in step (1) is 24-28° C., and the culture medium used is oatmeal culture medium.
3. The method for preparing alkyl glycosides using Caenorhabditis elegans according to claim 1, wherein: The method for washing the Caenorhabditis elegans in step (2) is to filter and rinse with sterile water.
4. The method for preparing alkyl glycosides using Caenorhabditis elegans according to claim 1, wherein: The operating parameters of the ultrasonic cell disruptor in step (2) are: power 70-80 Hz, ultrasonic time 5 s, rest time 10 s, and a total ultrasonic time of 25-30 min.
5. The method for preparing alkyl glycosides using Caenorhabditis elegans according to claim 1, wherein: In step (3), 20 to 30 ml of an extractant is added to every 2 g of dry powder, and the extractant used is methanol.
6. The method for preparing alkyl glycosides using Caenorhabditis elegans according to claim 1, wherein: The extraction conditions in step (3) are: extraction at 20-22° C. and 170-200 rpm for 23-25 hours, and extraction at least 3 times; after each extraction, the extract is filtered and collected with filter paper.
7. The method for preparing alkyl glycosides using Caenorhabditis elegans according to claim 1, wherein: The standing time in step (4) is 12 to 16 hours, the standing temperature is 4 to 6° C., and the centrifugal condition is 5000 to 6000 rpm for 3 to 4 minutes.
8. The method for preparing alkyl glycosides using Caenorhabditis elegans according to claim 1, wherein: The LC conditions in the LC-MS detection in step (5) are as follows: mobile phase A is 0.5% formic acid water by volume, mobile phase B is methanol, the gradient elution conditions are 0-2 min: 5% B, 12-15 min: 5%-95% B, 16-20 min 5% B, and the flow rate is 0.3 mL / min.