Crude enzyme preparation, preparation method thereof and culture medium for production
By using cheap carbon sources and optimizing the medium formula, the operating process is simplified, and the problem of high cost of polycyclic aromatic hydrocarbon degradation method is solved, and a low-cost and environmentally friendly preparation of crude enzyme liquid is achieved, which is suitable for large-scale industrial production.
Patent Information
- Application Number
- CN202411658823.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-19
- Publication Date
- 2025-01-03
AI Technical Summary
In the prior art, the degradation method of polycyclic aromatic hydrocarbons is high cost and is not suitable for large-scale production. The existing Pseudomonas MPDS culture medium is high cost and complex in composition, making it difficult to achieve low-cost and stable preparation of crude enzyme solution.
Using an optimized medium formula based on cheap carbon sources (molasses) and appropriate amounts of peptone and yeast extract, crude enzyme liquid is prepared through inclined surface culture of test tubes and liquid expansion culture steps, simplifying the operation process and reducing production costs.
It significantly reduces production costs, is suitable for large-scale industrial production, realizes low-cost and environmentally friendly crude enzyme liquid preparation, reduces chemical residues, meets the needs of industrial promotion, and provides an economical and applicable biodegradable solution.
Smart Images
Figure CN119242598A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biological fermentation, and in particular to a crude enzyme preparation and a preparation method thereof and a production culture medium. Background Art
[0002] Polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic hydrocarbons are a class of organic pollutants that pose a serious threat to human health and the ecological environment. They pollute the soil and atmosphere, threaten human health, and cause deformities, mutations, and cancer. These pollutants come from incomplete combustion of organic fuels, high-temperature industrial processes, and refinery wastewater. With the development of industry, the application of polycyclic aromatic hydrocarbons (PAHs) is becoming more and more widespread, and the content in the environment is becoming more and more serious.
[0003] At present, the methods for degrading PAHs are mainly divided into three categories: physical, chemical and microbial degradation:
[0004] Physical method: including water washing, steam treatment, etc., using physical means to remove some PAHs. Such methods usually require high energy consumption or a large amount of water resources, and the processing cost is high, which is difficult to meet the needs of large-scale production.
[0005] Chemical method: degrade or adsorb PAHs by adding specific chemicals. However, chemical treatment may cause residue problems, affect product safety, and the treatment cost is also high, which is not conducive to large-scale promotion.
[0006] Microbial degradation method: Microbial degradation method has gradually attracted attention due to its simple operation and environmental friendliness. Many microorganisms can degrade PAHs through metabolic pathways, but in current industrial production, the preparation of low-cost and stable crude enzyme solution for PAH degradation is still a key problem.
[0007] Pseudomonas spp. has metabolic diversity in degrading complex organic matter, making it one of the potential strains for biodegradation of PAHs. Specific Pseudomonas strains (such as MPDS) have good PAH degradation capabilities. However, the existing culture medium is expensive, has complex ingredients, and has low efficiency in producing crude enzyme solution, which is not suitable for large-scale, low-cost production needs.
[0008] To this end, technicians in this field are committed to developing optimized culture medium formulas and culture methods for Pseudomonas MPDS fermentation to produce low-cost crude enzyme solution, hoping to promote its application in industry to meet the needs of industrial PAH reduction and meet environmental protection requirements. Summary of the invention
[0009] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a method for producing crude Pseudomonas MPDS enzyme solution which is low-cost, simple to operate, capable of industrial production and environmentally friendly and a culture medium formula thereof.
[0010] To achieve the above object, the present invention provides a method for preparing a crude enzyme preparation, characterized in that the method comprises the following steps:
[0011] Step 1, culturing Pseudomonas MPDS in a test tube slant;
[0012] Step 2, liquid expansion culture of the cultured Pseudomonas in step 1;
[0013] Step 3: Prepare crude enzyme solution using the product of step 2.
[0014] In a preferred embodiment of the present invention, step 1 specifically includes:
[0015] Prepare Luria-Bertani (LB) medium, sterilize at 121°C for 20 minutes, cool and place into a slant, inoculate Pseudomonas MPDS on the slant medium, culture at 30°C for 18-24 hours to obtain test tube cultures.
[0016] In another preferred embodiment of the present invention, the formula of the LB medium is 1% peptone, 1% sodium chloride, 0.5% yeast extract, and 1.8% agar.
[0017] In another preferred embodiment of the present invention, the step 2 includes seed culture and fermentation culture.
[0018] In another preferred embodiment of the present invention, the seed culture specifically includes: using MPDS to improve molasses culture medium, sterilizing at 115 degrees for 15 minutes, inoculating Pseudomonas MPDS, and culturing at 30 degrees C for 24 hours to obtain liquid strains.
[0019] In another preferred embodiment of the present invention, the formula of the MPDS improved molasses culture medium is 30 g / L industrial molasses and 5 g / L industrial yeast extract powder dissolved in 1 L of tap water.
[0020] In another preferred embodiment of the present invention, the fermentation culture specifically includes: adding the liquid strain obtained from the seed culture to the following conditions for fermentation culture for 48 hours, 1g KH2PO4, 0.5g MgSO4, 1mL 0.1% MnSO4, 3mL 0.1% FeSO4, 3mL 1% KCl, and adding 30g industrial molasses, 5g industrial yeast extract powder, and then adding water to 1000mL, the initial pH is set to 7.0, the overall pH is adjusted to 6-7, the inoculation amount is 3-5%, the rotation speed is 200rpm, and the temperature is 30°C.
[0021] In another preferred embodiment of the present invention, the step 3 specifically includes:
[0022] After fermentation, 1000 mL of fermentation broth was centrifuged at 8000 rpm for 10 minutes to collect the bacteria, and the bacteria were suspended in a sterile buffer, i.e., 50 mM phosphate buffer, pH 7.0, and then centrifuged at 8000 rpm for 15 minutes. This was repeated twice to ensure purity. The finally collected bacteria were suspended in 50 mL of buffer and disrupted by ultrasonic treatment. After treatment, they were centrifuged at 8000 rpm for 15 minutes to remove the disrupted bacterial cells. The supernatant, i.e., the crude enzyme solution, was collected and stored at -20°C for later use.
[0023] The present invention also includes a crude enzyme preparation prepared by the above-mentioned method for preparing the crude enzyme preparation.
[0024] The present invention also includes a production culture medium used in the preparation method of the crude enzyme preparation, which has a formula of 1g KH2PO4, 0.5g MgSO4, 1mL 0.1% MnSO4, 3mL 0.1% FeSO4, 3mL 1% KCl, and 30g industrial molasses and 5g industrial yeast extract powder are added, and then water is added to 1000mL.
[0025] Technical Effects
[0026] 1. The existing culture medium has high cost and complex formula in the process of producing crude enzyme solution, which is difficult to be applied to large-scale industrial production. Traditional culture medium components usually use expensive carbon sources and nitrogen sources, which are acceptable in small-scale experiments, but difficult to maintain cost-effectiveness in industrial applications. The present invention proposes an optimized culture medium formula based on a cheap carbon source (molasses) and an appropriate amount of peptone and yeast extract, which significantly reduces the production cost, is suitable for large-scale crude enzyme solution production, and is suitable for industrial production needs. The culture medium formula of the present invention is optimized to stably support the fermentation of Pseudomonas MPDS to produce crude enzyme solution, and replaces part of the traditional carbon source with low-cost molasses, and scientifically adjusts the ratio with peptone and yeast extract to provide sufficient nutritional support for Pseudomonas MPDS to meet its production of crude enzyme solution. At the same time, the cost of the culture medium is greatly reduced, and the use of cheap raw materials effectively reduces the production cost, making the large-scale production of crude enzyme solution more economical, and the culture medium components are cheap and easy to purchase, suitable for direct application of existing fermentation equipment, without the need for additional equipment modification, making it easier to implement in large-scale industrial production. The scheme shows good stability and consistency in small tests and has significant potential for industrial promotion.
[0027] 2. The entire preparation process of the present invention does not require complicated formula adjustment, is suitable for production with conventional fermentation equipment, and simplifies the operation process.
[0028] 3. Existing physical and chemical PAH degradation methods may cause chemical residues or secondary pollution, which do not meet environmental protection requirements. The microbial method is used to produce crude enzyme liquid through Pseudomonas MPDS fermentation for the biodegradation of PAHs, which reduces the residues and pollution in chemical treatment. The PAH-degrading enzymes produced in the metabolic process of Pseudomonas MPDS are used to achieve the degradation of PAHs through biological means, thereby avoiding the use of chemical reagents, reducing the impact on the environment, and reducing dependence on expensive chemical reagents. There is no chemical residue in the production process, and the biodegradation is green and environmentally friendly, suitable for long-term promotion and application, meeting the social needs of environmental protection and sustainable development, and providing an economical and applicable solution for green industrial production. In addition, the biological method does not require additional chemical treatment equipment, simplifies the production process, and further reduces production costs.
[0029] The culture medium and crude enzyme solution preparation method of the present invention is a low-cost, easy-to-operate, suitable for large-scale production and environmentally friendly solution, which is suitable for the preparation of crude enzyme solution and polycyclic aromatic hydrocarbon treatment in the industrial field. Its low cost and environmental protection characteristics are in line with the current market trend of industrialized production and help to enhance the competitiveness of the industry. In the future, this technical solution can also be applied to the production of other environmentally friendly biological preparations, further expanding its application prospects.
[0030] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 This is a preferred embodiment of the present invention wherein the culture medium of the present invention is used to culture Pseudomonas;
[0032] Figure 2 It is a schematic diagram of the change of OD value after culturing Pseudomonas using the culture medium of the present invention in a preferred embodiment of the present invention. DETAILED DESCRIPTION
[0033] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.
[0034] Example 1: Cultivation of Pseudomonas MPDS
[0035] 1. Test tube slant culture
[0036] Culture medium: Luria-Bertani (LB) medium, formula: 1% peptone, 1% sodium chloride, 0.5% yeast extract, 1.8% agar.
[0037] Sterilization conditions: sterilize at 121℃ for 20 minutes, and place it into an inclined surface after cooling.
[0038] Inoculation and culture: Pseudomonas MPDS was inoculated on a slant culture medium and cultured at 30°C for 18-24 hours to obtain a test tube culture.
[0039] 2. Liquid expansion culture
[0040] Seed culture: Use MPDS modified molasses medium, the formula of which is: 30g / L industrial molasses + 5g / L industrial yeast extract powder dissolved in 1L tap water, sterilize at 115 degrees for 15 minutes, inoculate Pseudomonas MPDS, and culture at 30°C for 24 hours to obtain liquid strains.
[0041] Fermentation medium: add 1g KH2PO4, 0.5g MgSO4, 1mL MnSO4 (0.1%), 3mL FeSO4 (0.1%), 3mL KCl (1%), add 30g industrial molasses, 5g industrial yeast extract powder, then add water to 1000mL, and adjust the overall pH to 6-7. The initial pH is set to 7.0, the inoculation amount is 3-5%, the rotation speed is 200rpm, and the temperature is 30°C. Cultivate under the above fermentation conditions for 48 hours.
[0042] Example 2: Preparation of crude enzyme solution
[0043] After fermentation, 1000 mL of fermentation broth prepared from the culture medium was centrifuged at 8000 rpm for 10 minutes to collect the bacterial cells.
[0044] Bacterial suspension: The bacteria were suspended in a sterile buffer (50 mM phosphate buffer, pH 7.0), and then centrifuged at 8000 rpm for 15 minutes. This was repeated twice to ensure purity.
[0045] Disruption treatment: The collected bacteria were suspended in 50 mL of buffer and disrupted by ultrasonic treatment. After treatment, the cells were centrifuged at 8000 rpm for 15 minutes to remove the disrupted bacterial cells. The supernatant (crude enzyme solution) was collected and stored at -20°C for later use.
[0046] The culture medium and production process of the present invention can obtain a greater yield than that of the conventional Luria-Bertani culture medium, which is about twice that of the LB culture medium. After culturing for 30 hours, the OD 600nm is about 3.1, such as Figure 2 As shown, Figure 1 It is the state of bacterial liquid cultured using the culture medium and production process of the present invention.
[0047] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.
Claims
1. A method for preparing a crude enzyme preparation, characterized in that: The method comprises the following steps: Step 1, culturing Pseudomonas MPDS in a test tube slant; Step 2, liquid expansion culture of the cultured Pseudomonas in step 1; Step 3: Prepare crude enzyme solution using the product of step 2.
2. The method according to claim 1, characterized in that The step 1 specifically includes: Prepare Luria-Bertani (LB) medium, sterilize at 121°C for 20 minutes, cool and place into a slant, inoculate Pseudomonas MPDS on the slant medium, culture at 30°C for 18-24 hours to obtain test tube cultures.
3. The method according to claim 2, characterized in that The formula of the LB culture medium is 1% peptone, 1% sodium chloride, 0.5% yeast extract, and 1.8% agar.
4. The method according to claim 1, characterized in that The step 2 includes seed culture and fermentation culture.
5. The method according to claim 4, characterized in that The seed culture specifically includes: using MPDS to improve molasses culture medium, sterilizing at 115 degrees for 15 minutes, inoculating Pseudomonas MPDS, and culturing at 30 degrees C for 24 hours to obtain liquid strains.
6. The method according to claim 5, characterized in that The formula of the MPDS improved molasses culture medium is 30 g / L of industrial molasses and 5 g / L of industrial yeast extract powder dissolved in 1 L of tap water.
7. The method according to claim 4, characterized in that The fermentation culture specifically includes: adding the liquid strain obtained from the seed culture to the following conditions for fermentation culture for 48 hours: 1g KH2PO4, 0.5g MgSO4, 1mL 0.1% MnSO4, 3mL 0.1% FeSO4, 3mL 1% KCl, and adding 30g industrial molasses and 5g industrial yeast extract powder, and then adding water to 1000mL, the initial pH is set to 7.0, the overall pH is adjusted to 6-7, the inoculation amount is 3-5%, the rotation speed is 200rpm, and the temperature is 30°C.
8. The method according to claim 7, characterized in that The step 3 specifically includes: After fermentation, 1000 mL of fermentation broth was centrifuged at 8000 rpm for 10 minutes to collect the bacteria, and the bacteria were suspended in a sterile buffer, i.e., 50 mM phosphate buffer, pH 7.0, and then centrifuged at 8000 rpm for 15 minutes. This was repeated twice to ensure purity. The finally collected bacteria were suspended in 50 mL of buffer and disrupted by ultrasonic treatment. After treatment, they were centrifuged at 8000 rpm for 15 minutes to remove the disrupted bacterial cells. The supernatant, i.e., the crude enzyme solution, was collected and stored at -20°C for later use.
9. A crude enzyme preparation prepared by the method for preparing a crude enzyme preparation according to any one of claims 1 to 8.
10. A production medium used in the method for preparing a crude enzyme preparation according to any one of claims 1 to 8, characterized in that: The formula is 1g KH2PO4, 0.5g MgSO4, 1mL 0.1% MnSO4, 3mL 0.1% FeSO4, 3mL 1% KCl, and add 30g industrial molasses, 5g industrial yeast extract powder, and then add water to 1000mL.