Raw lacquer sensitization removal method based on rhodococcus degradation technology
Through the biodegradation technology of Rhodococcus strain CGMCC 4.1814 and raw paint, the sensitizing terpenes are directly degraded, solving the sensitization problem of raw paint, and achieving efficient desensitization and environmentally friendly raw paint treatment.
Patent Information
- Application Number
- CN202510669136.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-23
- Publication Date
- 2025-08-15
AI Technical Summary
The prior art has poor results when reducing the sensitization of raw paint, which will affect the performance of raw paint and will have environmental pollution problems.
Rheumatoidea strain CGMCC 4.1814 was mixed with raw paint, and after shaking culture and centrifugation, the cytochrome P450 enzyme was used to catalyze the degradation of terpenes, change its chemical structure, and degrade sensitized terpenes.
It achieves efficient desensitization, reduces the risk of sensitization, keeps the raw paint performance unchanged, and is environmentally friendly and pollution-free.
Smart Images
Figure CN120478921A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for removing sensitization of raw lacquer based on rhodococcus degradation technology, belonging to the technical field of biological desensitization. Background Art
[0002] Raw lacquer, also known as natural lacquer or lacquer, is a milky white, viscous liquid collected from the phloem of the lacquer tree. The main components of raw lacquer include urushiol, gum, water, other nitrogen-containing substances, and minerals. Because raw lacquer contains α / β-terpenes (sensitization rate >70%), their volatiles can penetrate the skin and activate immune responses, leading to severe contact dermatitis. Chemical oxidation, a common method for reducing the allergenicity of raw lacquer, has the following limitations: poor degradation and only targeting specific terpenes (such as limonene). Furthermore, degradation can affect the properties of the raw lacquer (e.g., a decrease in film hardness by approximately 40% and an increase in drying time by approximately three times), ultimately producing acidic wastewater and polluting the environment. Summary of the Invention
[0003] The technical problem to be solved by the present invention is to overcome the defects of the existing technology and provide a method for removing the allergenicity of raw lacquer based on Rhodococcus degradation technology, which can degrade multiple allergic components to achieve efficient desensitization without affecting the performance of the raw lacquer and is highly environmentally friendly.
[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is:
[0005] A method for removing allergenicity of raw lacquer based on Rhodococcus degradation technology comprises the following steps:
[0006] Step a, inoculating a frozen strain of Rhodococcus rubervillei with a strain number of CGMCC 4.1814 into an inorganic culture medium containing β-caryophyllene for shaking culture, then centrifuging the shaken cultured bacteria, and finally resuspending the collected bacteria with sterile physiological saline to obtain a bacterial suspension;
[0007] Step b, homogenizing the natural raw lacquer and then dispensing it into a sterile container, then adding the inorganic culture medium and the bacterial suspension into the sterile container, mixing them with a sterile magnetic stirrer to ensure that the bacteria are evenly dispersed, and obtaining an inoculated raw lacquer reaction liquid;
[0008] Step c: placing the sterile container containing the inoculated raw lacquer reaction solution into a constant temperature shaker for shaking culture under an aerobic environment.
[0009] In step a and step c, the inorganic culture medium is composed of: Na2HPO4 5~7g / L, KH2PO4 2~4g / L, NH4Cl 0.8~1.2 g / L and MgSO4 0.15~0.25 g / L.
[0010] In step a, the shaking culture temperature is 28-32° C., the shaking speed is 140-160 rpm, and the time is 22-26 h.
[0011] In step a, the centrifugal collection speed is 3500-4500 rpm and the time is 8-12 min.
[0012] In step a, the amount of β-caryophyllene added is 0.08-0.12% by volume of the inorganic culture medium.
[0013] In step b, the volume ratio of the raw lacquer to the sum of the bacterial suspension and the inorganic culture medium is 1:5-15.
[0014] In step b, the stirring temperature of the sterile magnetic stirrer is 28-32°C, the stirring speed is 140-160 rpm, and the stirring time is 8-12 min.
[0015] In step c, the shaking culture temperature is 28-32° C., the speed is 140-160 rpm, and the time is 70-74 h.
[0016] In step c, the sterile container is sealed with a PTFE breathable membrane.
[0017] The air permeability of PTFE breathable membrane is ≥500 mL / min·m².
[0018] The beneficial effects of the present invention are as follows: the present invention provides a method for removing allergenicity from raw lacquer based on Rhodococcus degradation technology, in which the Rhodococcus agent is mixed with raw lacquer and cultured at a constant temperature to directly degrade allergenic terpenes. The cytochrome P450 enzyme in the Rhodococcus can catalyze the structural modification of terpene olefins and change its chemical structure through hydroxylation or epoxidation reactions, thereby reducing the total terpene content in the raw lacquer from 3.5% to 0.25% (the sensitization risk level is reduced from level IV to level I). The method has a good desensitization effect and can degrade multiple allergic components with a high desensitization spectrum. In addition, due to the use of biological methods, the method has the advantages of not affecting the performance of the raw lacquer and being highly environmentally friendly compared to the existing chemical oxidation method. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is a schematic flow chart of a method for removing allergenicity from raw lacquer based on Rhodococcus degradation technology of the present invention;
[0020] Figure 2 This is a GC-MS comparison chart of the raw lacquer before and after treatment by the present invention;
[0021] Figure 3 This is a schematic diagram of the principle of using Rhodococcus bacteria in the present invention to remove allergenicity of raw lacquer. DETAILED DESCRIPTION
[0022] The present invention will be further described below. The following examples are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.
[0023] Example 1
[0024] The strain of Rhodococcus in the present invention is from: Rhodococcus (preservation number: CGMCC 4.1814), which was purchased from the General Microbiology Center of China Culture Collection Administration.
[0025] like Figure 1 As shown, the present invention mixes the Rhodococcus bacterial agent with raw lacquer and then cultures at a constant temperature to directly degrade allergenic terpenes, comprising the following steps:
[0026] Step 1: strain treatment and activation. The frozen strain of Rhodococcus was inoculated into an inorganic culture medium containing β-caryophyllene for shaking culture. The bacteria after shaking culture were collected by centrifugation. Finally, the collected bacteria were resuspended in sterile physiological saline to induce the expression of terpene degradation enzymes to obtain a bacterial suspension (concentration of 10 9 CFU / mL).
[0027] The inorganic culture medium consisted of 5 g / L Na₂HPO₄, 4 g / L KH₂PO₄, 0.8 g / L NH₄Cl, and 0.25 g / L MgSO₄. The shaking culture was maintained at 28°C, 160 rpm, and 22 hours. Centrifugation was performed at 4500 rpm for 8 minutes. β-Caryophyllene was added as an inducer and the sole carbon source. The amount of β-Caryophyllene added was 0.12% by volume of the inorganic culture medium.
[0028] Step 2: Lacquer Treatment and Inoculation: Homogenize the natural lacquer (urushiol content ≥ 65%) and dispense it into sterile containers (50g / pack). Add the inorganic culture medium and bacterial suspension to the sterile container and mix using a sterile magnetic stirrer to ensure uniform dispersion of the bacteria. This will yield the inoculated lacquer reaction solution.
[0029] The inorganic culture medium is the same as that in step 1. 7 The volume ratio of the total volume of the culture medium (CFU / g) and the inorganic culture medium was 1:5. The stirring temperature of the sterile magnetic stirrer was 28°C, the stirring speed was 160 rpm, and the stirring time was 8 min.
[0030] Step 3: Warm degradation culture: Place the sterile container containing the inoculated raw lacquer reaction solution in a constant temperature shaker for shaking culture under an aerobic environment.
[0031] The shaking culture temperature is 28°C, the speed is 160 rpm, and the time is 70 hours. The sterile container is sealed with a PTFE breathable membrane with an air permeability of ≥500 mL / min·m².
[0032] Example 2
[0033] The strain of Rhodococcus in the present invention is from: Rhodococcus (preservation number: CGMCC 4.1814), which was purchased from the General Microbiology Center of China Culture Collection Administration.
[0034] like Figure 1 As shown, the present invention mixes the Rhodococcus bacterial agent with raw lacquer and then cultures at a constant temperature to directly degrade allergenic terpenes, comprising the following steps:
[0035] Step 1: strain treatment and activation. The frozen strain of Rhodococcus was inoculated into an inorganic culture medium containing β-caryophyllene for shaking culture. The bacteria after shaking culture were collected by centrifugation. Finally, the collected bacteria were resuspended in sterile physiological saline to induce the expression of terpene degradation enzymes to obtain a bacterial suspension (concentration of 10 9 CFU / mL).
[0036] The inorganic culture medium consisted of 7 g / L Na₂HPO₄, 2 g / L KH₂PO₄, 1.2 g / L NH₄Cl, and 0.15 g / L MgSO₄. The shaking culture was maintained at 32°C, 140 rpm, and 26 hours. Centrifugation was performed at 4500 rpm for 8 minutes. β-Caryophyllene was added as an inducer and the sole carbon source. The amount of β-Caryophyllene added was 0.08% by volume of the inorganic culture medium.
[0037] Step 2: Lacquer Treatment and Inoculation: Homogenize the natural lacquer (urushiol content ≥ 65%) and dispense it into sterile containers (50g / pack). Add the inorganic culture medium and bacterial suspension to the sterile container and mix using a sterile magnetic stirrer to ensure uniform dispersion of the bacteria. This will yield the inoculated lacquer reaction solution.
[0038] The inorganic culture medium is the same as that in step 1. 8 The volume ratio of the total volume of the culture medium (CFU / g) to the inorganic culture medium was 1:10. The stirring temperature of the sterile magnetic stirrer was 32°C, the stirring speed was 140 rpm, and the stirring time was 12 min.
[0039] Step 3: Warm degradation culture: Place the sterile container containing the inoculated raw lacquer reaction solution in a constant temperature shaker for shaking culture under an aerobic environment.
[0040] The shaking culture temperature is 32°C, the speed is 140 rpm, and the time is 74 hours. The sterile container is sealed with a PTFE breathable membrane with an air permeability of ≥500 mL / min·m².
[0041] Example 3
[0042] The strain of Rhodococcus in the present invention is from: Rhodococcus (preservation number: CGMCC 4.1814), which was purchased from the General Microbiology Center of China Culture Collection Administration.
[0043] like Figure 1 As shown, the present invention mixes the Rhodococcus bacterial agent with raw lacquer and then cultures at a constant temperature to directly degrade allergenic terpenes, comprising the following steps:
[0044] Step 1: strain treatment and activation. The frozen strain of Rhodococcus was inoculated into an inorganic culture medium containing β-caryophyllene for shaking culture. The bacteria after shaking culture were collected by centrifugation. Finally, the collected bacteria were resuspended in sterile physiological saline to induce the expression of terpene degradation enzymes to obtain a bacterial suspension (concentration of 10 9 CFU / mL).
[0045] The inorganic culture medium consisted of 6 g / L Na₂HPO₄, 3 g / L KH₂PO₄, 1.0 g / L NH₄Cl, and 0.20 g / L MgSO₄. The shaking culture was maintained at 30°C, 150 rpm, and 24 hours. The culture was centrifuged at 4000 rpm for 10 minutes. β-Caryophyllene was added as an inducer and the sole carbon source. The amount of β-Caryophyllene added was 0.10% by volume of the inorganic culture medium.
[0046] Step 2: Lacquer Treatment and Inoculation: Homogenize the natural lacquer (urushiol content ≥ 65%) and dispense it into sterile containers (50g / pack). Add the inorganic culture medium and bacterial suspension to the sterile container and mix using a sterile magnetic stirrer to ensure uniform dispersion of the bacteria. This will yield the inoculated lacquer reaction solution.
[0047] The inorganic culture medium is the same as that in step 1. 9 The volume ratio of the total volume of the culture medium (CFU / g) to the inorganic culture medium was 1:15. The stirring temperature of the sterile magnetic stirrer was 30°C, the stirring speed was 150 rpm, and the stirring time was 10 min.
[0048] Step 3: Warm degradation culture: Place the sterile container containing the inoculated raw lacquer reaction solution in a constant temperature shaker for shaking culture under an aerobic environment.
[0049] The shaking culture temperature is 30°C, the speed is 150 rpm, and the time is 72 hours. The sterile container is sealed with a PTFE breathable membrane with an air permeability of ≥500 mL / min·m².
[0050] In order to verify the desensitization effect of the raw lacquer based on the Rhodococcus degradation technology of the present invention, the components of the raw lacquer before and after treatment were tested. The methods for terpene content detection and product analysis are as follows:
[0051] 1. Pretreatment method:
[0052] Headspace sampling (high volatility terpenes): Take 1 g of sample into a headspace vial, add 0.1 g of anhydrous sodium sulfate, and equilibrate at 60°C for 30 minutes.
[0053] SPME adsorption (semivolatile terpenes): using a PDMS / DVB fiber (Supelco), adsorption at 50°C for 30 minutes.
[0054] 2. GC-MS analysis:
[0055] Chromatographic column: DB-5MS (30 m × 0.25 mm × 0.25 μm).
[0056] Heating program: 50°C (2 min) → 10°C / min → 250°C (5 min).
[0057] Mass spectrometry conditions: EI source, 70 eV, scan range m / z 35-450.
[0058] Quantification method: Terpene content was calculated using an internal standard method (naphthalene-d10, retention time 8.2 min) with a standard curve covering 0.1-100 μg / mL (R²>0.995).
[0059] like Figure 2 As shown, the present invention has significant lacquer sensitization removal ability and can simultaneously degrade caryophyllene, humulene, A-bergamotene, and α-longifolia pinene, among which, caryophyllene Area % 10.82→0; humulene Area % 26.16→1.49; α-longifolia pinene Area % 30.21→1.72; A-bergamotene Area % 0.63→0.
[0060] like Figure 3 As shown, in the present invention, the cytochrome P450 enzyme in the cocci can catalyze the structural modification of terpenoid olefins and change their chemical structure through hydroxylation or epoxidation reactions.
[0061] The method for removing allergenicity of raw lacquer based on Rhodococcus degradation technology of the present invention has the following advantages:
[0062] 1. Precise desensitization: Targeted degradation of the most allergenic β-caryophyllene (IC 50 =0.1 μg / mL), reducing the risk of allergy by 98%. The total terpene content in raw lacquer was reduced from 3.5% to 0.25% within 72 hours (the allergy risk level was reduced from IV to I).
[0063] 2. Preservation of paint properties: After treatment, the polymerization degree of urushiol in the raw lacquer remains >95%, and the paint film hardness (2H→2H), adhesion (level 1→level 1), and water resistance (no blistering for 24 hours) of the raw lacquer are not deteriorated.
[0064] 3. Environmentally friendly and low cost: The degradation products are CO2, H2O and trace amounts of terpene alcohols (non-toxic, EC50>100 mg / L), which comply with the GB 8978-1996 wastewater discharge standard; the cost of the microbial agent is only ￥3 / kg of raw lacquer.
[0065] 4. Industrial potential: The bacterial agent can be immobilized and reused (efficiency >80% after 5 times) and is suitable for continuous bioreactors.
[0066] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for removing sensitization of raw lacquer based on Rhodococcus degradation technology, characterized by: The following steps are involved: Step a, inoculating a frozen strain of Rhodococcus rubervillei with a strain number of CGMCC 4.1814 into an inorganic culture medium containing β-caryophyllene for shaking culture, then centrifuging the shaken cultured bacteria, and finally resuspending the collected bacteria with sterile physiological saline to obtain a bacterial suspension; Step b, homogenizing the natural raw lacquer and then dispensing it into a sterile container, then adding the inorganic culture medium and the bacterial suspension into the sterile container, mixing them with a sterile magnetic stirrer to ensure that the bacteria are evenly dispersed, and obtaining an inoculated raw lacquer reaction liquid; Step c: placing the sterile container containing the inoculated raw lacquer reaction solution into a constant temperature shaker for shaking culture under an aerobic environment.
2. The method for removing sensitization of raw lacquer based on Rhodococcus degradation technology according to claim 1, characterized in that: In step a and step c, the inorganic culture medium is composed of: Na2HPO4 5~7g / L, KH2PO4 2~4g / L, NH4Cl 0.8~1.2 g / L and MgSO4 0.15~0.25 g / L.
3. The method for removing sensitization of raw lacquer based on Rhodococcus degradation technology according to claim 1, characterized in that: In step a, the shaking culture temperature is 28-32° C., the shaking speed is 140-160 rpm, and the time is 22-26 h.
4. The method for removing sensitization of raw lacquer based on Rhodococcus degradation technology according to claim 1, characterized in that: In step a, the centrifugal collection speed is 3500-4500 rpm and the time is 8-12 min.
5. The method for removing sensitization of raw lacquer based on Rhodococcus degradation technology according to claim 1, characterized in that: In step a, the amount of β-caryophyllene added is 0.08-0.12% by volume of the inorganic culture medium.
6. The method for removing sensitization of raw lacquer based on Rhodococcus degradation technology according to claim 1, characterized in that: In step b, the volume ratio of the raw lacquer to the sum of the bacterial suspension and the inorganic culture medium is 1:5-15.
7. The method for removing sensitization of raw lacquer based on Rhodococcus degradation technology according to claim 1, characterized in that: In step b, the stirring temperature of the sterile magnetic stirrer is 28-32°C, the stirring speed is 140-160 rpm, and the stirring time is 8-12 min.
8. The method for removing sensitization of raw lacquer based on Rhodococcus degradation technology according to claim 1, characterized in that: In step c, the shaking culture temperature is 28-32° C., the speed is 140-160 rpm, and the time is 70-74 h.
9. The method for removing sensitization of raw lacquer based on Rhodococcus degradation technology according to claim 1, characterized in that: In step c, the sterile container is sealed with a PTFE breathable membrane.
10. The method for removing sensitization of raw lacquer based on Rhodococcus degradation technology according to claim 9, characterized in that: The air permeability of PTFE breathable membrane is ≥500 mL / min·m².