A method for extracting natural rubber from wild lettuce

Abstract

The application discloses a wild lettuce natural rubber extraction method. The natural rubber is extracted from wild lettuce by using wild lettuce latex as raw material, through elution, acetone elution and petroleum ether extraction processes, and the extraction liquid is non-toxic or low-toxic, and the cost is relatively low, which provides technical support for enriching natural rubber resources and accelerating the industrialization process of natural rubber new crops.

Description

Technical Field

[0001] This invention belongs to the field of natural rubber preparation, specifically relating to a method for extracting natural rubber from wild lettuce. Background Technology

[0002] Natural rubber is a natural high-molecular polymer with cis-1,4-polyisoprene as its main component. Due to its irreplaceable high elasticity, impact resistance, abrasion resistance, and unique thermal dispersibility and low-temperature ductility compared to synthetic rubber, it is widely used not only as an important industrial raw material in medical and health, and automobile tire manufacturing industries, but also as an irreplaceable strategic material in the defense sector. Currently, there are over 50,000 types of rubber industrial products. However, the Brazilian rubber tree (Hevea brasiliensis), the only commercial source of natural rubber, mainly grows in plantations in tropical and subtropical regions of Southeast Asia, and its production cannot meet the rapidly growing global industrial demand for natural rubber. my country is a major consumer of natural rubber, accounting for approximately 43% of the world's total consumption. However, my country's self-sufficiency rate for natural rubber is severely insufficient, with over 85% relying on imports. Therefore, there is an urgent need to develop new rubber-producing crops with broader environmental adaptability to ensure the security of strategic material self-sufficiency.

[0003] Wild lettuce (Lactuca serriola) is an annual herbaceous plant belonging to the genus Lactuca in the family Asteraceae. The entire plant can produce high-molecular-weight natural rubber, with the stem being the primary rubber-producing part. It grows rapidly, has a large biomass, wide environmental adaptability, and strong resistance to adverse conditions, making it a potential new crop for natural rubber. The biosynthesis of natural rubber in wild lettuce takes place in highly specialized latex cells (lactifers) with vascular bundles. Mature latex cells have perforations in their cell walls, and the cytoplasm connects to form a cross-linked network structure. The cytoplasm is milky white and called latex. The specific organelles for synthesizing natural rubber are the spherical rubber particles located within the latex.

[0004] In 1913, Charles P. Fox was the first to use benzene solution to extract natural rubber from wild lettuce latex and estimated that the natural rubber content in fresh wild lettuce latex was about 2% (Fox C P. 1913. Wild lettuce rubber. Journal of Industrial and Engineering Chemistry, 5, 477–478.). However, the rubber extracted by this method contained acidic compounds, and benzene solution was highly toxic and carcinogenic, making it unsuitable for large-scale rubber extraction (Fox 1913). In 2006, Bushman et al. extracted natural rubber from fresh lettuce latex using dichloromethane, which has low toxicity but an irritating odor, and determined that the molecular weight and distribution of lettuce rubber were comparable to those of rubber tree rubber (Bushman BS, Scholte AA, Cornish K, Scott DJ, Brichta JL, Vederas JC, Ochoa O, Michelmore RW, Shintani DK, Knapp S J. 2006. Identification and comparison of natural rubber from two Lactuca species. Phytochemistry, 67, 2590–2596). In 2015, Bell et al. further utilized the less toxic n-hexane to extract trace amounts of natural rubber from the dehydrated latex of wild lettuce (Bell JL, Burke IC, Neff MM. 2015. Genetic and biochemical evaluation of natural rubber from Eastern Washington prickly lettuce (Lactuca serriola L.). Journal of Agricultural and Food Chemistry, 63, 593–602). Dichloromethane is a polar molecule, and its solubility for nonpolar rubber hydrocarbons is not as high as that of the nonpolar molecule n-hexane.

[0005] Currently, there are no reports on the potential of wild lettuce for producing natural rubber in my country. Using low-toxicity or non-toxic, and inexpensive non-polar reagents to extract natural rubber from wild lettuce would help enrich the sources of natural rubber and accelerate the industrialization of wild lettuce as a new natural rubber crop. Summary of the Invention

[0006] The purpose of this invention is to provide a method for extracting natural rubber from wild lettuce.

[0007] The method for extracting natural rubber from wild lettuce provided by this invention includes the following steps:

[0008] 1) Add the eluent to the wild lettuce latex, elute, centrifuge and discard the eluent, retaining the white paste-like emulsion layer;

[0009] 2) Add the eluent to the obtained white paste-like emulsion layer, and repeat step 1) several times to obtain a milky white eluent;

[0010] 3) Add acetone to the obtained milky white eluent, heat and shake to elute the resin substances, centrifuge, discard the acetone solution, and retain the white gel.

[0011] 4) Repeat step 3) several times to obtain white micelles after acetone elution;

[0012] 5) Add petroleum ether to the white gel obtained after acetone elution, extract, centrifuge, collect the upper petroleum ether solution, dry, and obtain a light yellow elastic solid, which is wild lettuce natural rubber.

[0013] In step 1) of the above method, the wild lettuce latex is obtained by the following method: make an incision in the wild lettuce stem with a blade (specifically, a 45° incision), and the white latex flows out and is collected in a clean container.

[0014] The eluent consisted of 100 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 5 mM DTT and 1×cocktail protease inhibitor.

[0015] The ratio of the wild lettuce latex to the eluent can be: 300mg: 1-2mL;

[0016] When the ratio of eluent to wild lettuce latex is less than 1-2 mL: 300 mg, the rubber yield is about 1.2%, while when it is greater than 1-2 mL: 300 mg, the yield is basically about 2.5% and will not increase further.

[0017] The elution conditions are: elution by shaking at room temperature for 15-25 minutes, specifically 20 minutes;

[0018] The centrifugation conditions are 10,000×g at room temperature for 5-15 minutes, specifically 10 minutes;

[0019] In step 2), the repetition of step 1) multiple times can specifically be repeated 2-3 times, or more specifically, repeated 1) twice.

[0020] The ratio of acetone added in step 3) to the amount of lettuce latex added in step 1) can be: 1-2 mL : 300 mg;

[0021] The heating and oscillation conditions are: 45-55℃ (specifically 50℃), 600-900 rpm / min (specifically 750 rpm / min), 0.5-1.5 hours (specifically 1 hour);

[0022] The centrifugation conditions are 10,000×g at room temperature for 5-15 minutes, specifically 10 minutes;

[0023] In step 4), the repetition of step 3) multiple times can specifically be repeated 2-3 times, or more specifically, repeated 3) twice.

[0024] In step 5), the ratio of the amount of petroleum ether added to the amount of lettuce latex added in step 1) can be: 0.5-1.5 mL : 300 mg;

[0025] The extraction conditions are: 45-55℃ (specifically 50℃), 600-900 rpm / min (specifically 750 rpm / min), 1-3 hours (specifically 2 hours);

[0026] The centrifugation conditions are 10,000×g at room temperature for 5-15 minutes, specifically 10 minutes;

[0027] The drying temperature can specifically be 60°C.

[0028] The wild lettuce natural rubber is composed of cis-1,4-polyisoprene.

[0029] The yield of natural rubber from the wild lettuce was 1.93-3.84%.

[0030] Wild lettuce can produce high-quality natural rubber, and it grows rapidly, has a large biomass, wide environmental adaptability, and strong resistance, making it a potential new natural rubber crop. This invention uses wild lettuce latex as raw material and extracts natural rubber from it through processes such as aqueous solution elution, acetone elution, and petroleum ether extraction. The extract is non-toxic or low-toxic, and the cost is relatively low, providing technical support for enriching natural rubber resources and accelerating the industrialization of new natural rubber crops. Attached Figure Description

[0031] Figure 1 This is a diagram showing the wild lettuce natural rubber obtained in an embodiment of the present invention and its stretched morphology.

[0032] Figure 2 This is the nuclear magnetic resonance image of the wild lettuce natural rubber obtained in the embodiments of the present invention. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0035] Example 1: Extraction of natural rubber from wild lettuce

[0036] 1. Add 1.5 mL (w / v = 1:5) of elution buffer (100 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 5 mM DTT and 1×cocktail protease inhibitor) to each 310 mg of fresh wild lettuce latex (wild lettuce plants are grown on the farm of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The inventor cuts the wild lettuce stems with a blade and collects the latex (makes a 45° cut on the wild lettuce stems with a blade, and the white latex flows out and is collected in a clean container) for subsequent rubber extraction) and elute at room temperature with shaking for 20 minutes.

[0037] 2. Centrifuge at room temperature, 10,000×g for 10 minutes, discard the eluent, and retain the white paste-like emulsion layer;

[0038] 3. Add 1.5 mL of elution buffer to the white paste-like emulsion layer again, and repeat steps “1”-“2” twice;

[0039] 4. Add 1.5 mL of acetone to the obtained milky white eluent, heat and shake at 50°C and 750 rpm / min for 1 hour to elute the resinous substances;

[0040] 5. Centrifuge at room temperature, 10,000×g for 10 minutes, discard the acetone solution, and retain the white gel.

[0041] 6. Add 1.5 mL of acetone to the obtained white micelle, and repeat steps “4” and “5” twice;

[0042] 7. Add 1 mL of petroleum ether to the white micelles after elution with acetone, and extract at 50°C and 750 rpm / min for 2 hours.

[0043] 8. Centrifuge at room temperature, 10,000×g for 10 minutes, transfer the upper petroleum ether solution to a clean container, and dry at 60℃ to obtain 9.4mg of pale yellow elastic solid, which is wild lettuce natural rubber.

[0044] Figure 1 The obtained wild lettuce natural rubber and its stretched morphology are shown in the figure.

[0045] Figure 2 The nuclear magnetic resonance image of the obtained wild lettuce natural rubber.

[0046] Depend on Figure 2 It can be seen that the rubber hydrocarbon structure of wild lettuce is cis-1,4-polyisoprene.

[0047] Example 2: Extraction of natural rubber from wild lettuce

[0048] 1. Add 1.5 mL (w / v = 1:5) of elution buffer (100 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 5 mM DTT and 1×cocktail protease inhibitor) to 300 mg of fresh wild lettuce latex (wild lettuce plants are grown in the artificial climate chamber of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The inventor cuts the wild lettuce stem with a blade and collects the latex (makes a 45° cut on the wild lettuce stem with a blade, and the white latex flows out and is collected in a clean container) for subsequent rubber extraction) and elute at room temperature with shaking for 20 minutes.

[0049] 2. Centrifuge at room temperature, 10,000×g for 10 minutes, discard the eluent, and retain the white paste-like emulsion layer;

[0050] 3. Add 1.5 mL of elution buffer to the white paste-like emulsion layer again, and repeat steps “1”-“2” twice;

[0051] 4. Add 1.5 mL of acetone to the obtained milky white eluent, heat and shake at 50°C and 750 rpm / min for 1 hour to elute the resinous substances;

[0052] 5. Centrifuge at room temperature, 10,000×g for 10 minutes, discard the acetone solution, and retain the white gel.

[0053] 6. Add 1.5 mL of acetone to the obtained white micelle, and repeat steps “4” and “5” twice;

[0054] 7. Add 1 mL of petroleum ether to the white micelles after elution with acetone, and extract at 50°C and 750 rpm / min for 2 hours.

[0055] 8. Centrifuge at room temperature, 10,000×g for 10 minutes, transfer the upper petroleum ether solution to a clean container, and dry at 60℃ to obtain 8.0 mg of pale yellow elastic solid, which is wild lettuce natural rubber.

[0056] Example 3: Extraction of natural rubber from wild lettuce

[0057] 1. Add 1.5 mL (w / v = 1:5) of elution buffer (100 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 5 mM DTT and 1×cocktail protease inhibitor) to 320 mg of fresh wild lettuce latex (wild lettuce plants are grown in greenhouses in Changping, Beijing. The inventor cuts the wild lettuce stems with a blade and collects the latex (makes a 45° cut on the wild lettuce stem with a blade, and the white latex flows out and is collected in a clean container) for subsequent rubber extraction) and elute at room temperature with shaking for 20 minutes.

[0058] 2. Centrifuge at room temperature, 10,000×g for 10 minutes, discard the eluent, and retain the white paste-like emulsion layer;

[0059] 3. Add 1.5 mL of elution buffer to the white paste-like emulsion layer again, and repeat steps “1”-“2” twice;

[0060] 4. Add 1.5 mL of acetone to the obtained milky white eluent, heat and shake at 50°C and 750 rpm / min for 1 hour to elute the resinous substances;

[0061] 5. Centrifuge at room temperature, 10,000×g for 10 minutes, discard the acetone solution, and retain the white gel.

[0062] 6. Add 1.5 mL of acetone to the obtained white micelle, and repeat steps “4” and “5” twice;

[0063] 7. Add 1 mL of petroleum ether to the white micelles after elution with acetone, and extract at 50°C and 750 rpm / min for 2 hours.

[0064] 8. Centrifuge at room temperature, 10,000×g for 10 minutes, transfer the upper petroleum ether solution to a clean container, and dry at 60℃ to obtain 9.7mg of pale yellow elastic solid, which is wild lettuce natural rubber.

[0065] Parallel extraction experiments were conducted following the methods of Bushman and Bell to examine the experimental time and rubber yield. Compared with the methods of Bushman in 2006 and Bell in 2015, the method of this invention saves nearly 50 hours of time and reduces experimental toxicity. The average rubber yield of this method is 2.91%, while the average rubber yield of the method reported by Bushman is 2.21% and the average rubber yield of the method reported by Bell is 2.19%. The rubber yield of this method is increased by 30%, and the rubber elasticity is significantly increased.

[0066] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for extracting natural rubber from wild lettuce, comprising the following steps: 1) Add the eluent to the wild lettuce latex, elute, centrifuge and discard the eluent, retaining the white paste-like emulsion layer; In step 1), the eluent consists of 100 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 5 mM DTT and 1×cocktail protease inhibitor. The ratio of the wild lettuce latex to the eluent is 300 mg: 1-2 mL; 2) Add the eluent to the obtained white paste-like emulsion layer, and repeat step 1) several times to obtain a milky white eluent; 3) Add acetone to the obtained milky white eluent, heat and shake to elute the resinous substances, centrifuge, discard the acetone solution, and retain the white gel. 4) Repeat step 3) several times to obtain white micelles after acetone elution; 5) Add petroleum ether to the white gel obtained after acetone elution, extract, centrifuge, collect the upper petroleum ether solution, dry, and obtain a light yellow elastic solid, which is wild lettuce natural rubber.

2. The method according to claim 1, characterized in that: In step 1), the elution conditions are: elution by shaking at room temperature for 15-25 minutes; The centrifugation conditions were 10,000 × g for 5-15 min at room temperature.

3. The method according to claim 1, characterized in that: In step 2), step 1) is repeated 2-3 times.

4. The method according to claim 1, characterized in that: The ratio of acetone added in step 3) to the amount of lettuce latex added in step 1) is 1-2 mL: 300 mg; The heating and oscillation conditions are: 45-55 °C, 600-900 rpm, 0.5-1.5 hours; The centrifugation conditions were 10,000 × g for 5-15 min at room temperature.

5. The method according to claim 1, characterized in that: In step 4), step 3) is repeated 2-3 times.

6. The method according to claim 1, characterized in that: In step 5), the ratio of petroleum ether added to the lettuce latex in step 1) is 0.5-1.5 mL: 300 mg. The extraction conditions are: 45-55 °C, 600-900 rpm, 1-3 hours; The centrifugation conditions were 10,000 × g for 5-15 min at room temperature.

7. The method according to claim 1, characterized in that: The resulting wild lettuce natural rubber is composed of cis-1,4-polyisoprene.

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