Multifunctional adjusting filter element for escherichia coli culture and preparation method of multifunctional adjusting filter element

Abstract

The invention provides a multifunctional adjusting filter element for escherichia coli culture and a preparation method of the multifunctional adjusting filter element, and relates to the technical field of microorganism culture. The multifunctional adjusting filter element for escherichia coli culture is of a composite sphere structure, the composite sphere structure comprises an inner core and an outer shell, the inner core is of a porous structure, holes are filled with an acid-base buffering agent, and the outer shell sequentially comprises a nutrition supplementing layer and a shell layer from inside to outside. The preparation raw materials of the inner core comprise an inorganic adsorption powder material and ultra-high molecular weight plastic powder, the preparation raw materials of the nutrition supplement layer comprise amino acid, vitamins, glucose, tryptone, a yeast extract and magnesium ions, and the preparation raw material of the shell layer comprises gelatin. The multifunctional adjusting filter element for escherichia coli culture can effectively adjust acid-base balance in the escherichia coli culture process, provide nutrient substances and reduce accumulation of bacteriocin generated in the proliferation and fermentation process, so that the growth speed and density of escherichia coli are increased.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial culture, and particularly relates to a multifunctional regulating filter element for Escherichia coli culture and a preparation method thereof. Background Art

[0002] As an important model organism and industrial production strain, Escherichia coli has a wide range of applications in the fields of biotechnology and medicine. Its clear genetic background, simple technical operation, relatively simple culture conditions, and the economy of large-scale fermentation make it one of the most commonly used hosts for expressing foreign genes. The applications of Escherichia coli in biotechnology include, but are not limited to, the production of drugs such as L-asparaginase, which has a good effect on treating leukemia, showing its great industrial value. However, in the actual culture process, the growth and density of Escherichia coli are often affected by various factors, thus limiting its application effect. Currently, the commonly used method for culturing Escherichia coli is mainly to use LB medium (Luria-Bertani medium), which is divided into two forms: liquid medium and solid medium, and can be used to culture the gene engineering recipient bacterium Escherichia coli. Although LB medium has been widely used in basic research and industrial applications, there are still some challenges in the culture process.

[0003] Escherichia coli has relatively strict requirements for the pH value, generally between 6.8 and 7.0. An overly acidic or alkaline environment will affect the growth and metabolism of Escherichia coli. Too high or too low pH value may damage the nutrients in the medium, affect the metabolic activities of Escherichia coli, and even inhibit its growth. During the fermentation process, Escherichia coli metabolizes glucose to produce acetic acid, resulting in a decrease in pH value. Therefore, it is necessary to regularly adjust the pH value to maintain an appropriate acid-base balance. At the same time, Escherichia coli also produces metabolic by-products such as bacteriocins during the growth and fermentation process, which have an inhibitory effect on the growth of the bacteria and the synthesis of products. When the concentration of bacteriocins reaches a certain level, it will significantly affect the lag phase, maximum specific growth rate, and bacterial concentration. Therefore, controlling the generation and accumulation of metabolic by-products such as bacteriocins is the key to improving the fermentation efficiency of Escherichia coli.

[0004] In summary, although Escherichia coli has broad application prospects in the fields of biotechnology and medicine, there are still problems such as difficulty in maintaining acid-base balance and accumulation of metabolites such as bacteriocins in the actual culture process, which limit the growth and density of Escherichia coli.

[0005] Therefore, there is an urgent need for a multifunctional regulating filter element for Escherichia coli culture and a preparation method thereof to solve the above problems. Summary of the Invention

[0006] In view of the above problems, the purpose of the present invention is to provide a multifunctional regulating filter element for Escherichia coli culture and a preparation method thereof. The multifunctional regulating filter element for Escherichia coli culture of the present invention can effectively regulate the acid-base balance in the process of Escherichia coli culture, provide nutrients and reduce the accumulation of bacteriocin metabolites, thereby improving the growth rate and density of Escherichia coli.

[0007] To achieve the above object, on the one hand, the present invention provides a multifunctional regulating filter element for Escherichia coli culture, which is a composite sphere structure. The composite sphere structure includes a core and a shell. The core has a porous structure and the pores are filled with an acid-base buffer. The shell includes a nutrient supplement layer and a shell layer from inside to outside in sequence. The preparation raw materials of the core include an inorganic adsorption powder material and a ultra-high molecular weight plastic powder. The preparation raw materials of the nutrient supplement layer include amino acids, vitamins, glucose, tryptone, yeast extract and magnesium ions. The preparation raw materials of the shell layer include gelatin.

[0008] Compared with the prior art, the preparation raw materials of the shell layer of the multifunctional regulating filter element of the present invention include gelatin, and the gelatin can be slowly decomposed in the LB culture solution, so that the nutrient supplement layer, the core inside the shell layer are in contact with and dissolved in the LB culture solution, thereby maintaining a good growth environment for the growth of Escherichia coli and supplementing nutrients. Specifically, the preparation raw materials of the nutrient supplement layer of the present invention include amino acids, vitamins, glucose, tryptone, yeast extract and magnesium ions, which can supplement new nutrients to the LB culture solution and provide sufficient energy for the rapid growth of Escherichia coli; the preparation raw materials of the core include an inorganic adsorption powder material and a ultra-high molecular weight plastic powder. The inorganic adsorption powder material has an interstitial or pore structure in its structure, and the ultra-high molecular weight plastic powder has the characteristics of high density and high strength. Therefore, the core prepared from the above two raw materials has a porous structure and has strong mechanical properties and durability, so that it can better fill the acid-base buffer, support and fix the nutrient supplement layer and the shell layer, so that the core of the present invention can effectively regulate the pH value in the process of Escherichia coli culture. At the same time, the adsorption material contained in the core can also effectively adsorb metabolites such as bacteriocin produced during the growth and fermentation of Escherichia coli, and further more effectively promote the proliferation and growth of Escherichia coli. Therefore, applying the multifunctional regulating filter element of the present invention to Escherichia coli culture can effectively regulate the acid-base balance, provide nutrients and reduce the accumulation of metabolites such as bacteriocin, thereby improving the growth rate and density of Escherichia coli.

[0009] As a preferred technical solution, the inorganic adsorption powder material of the present invention is selected from at least one of molecular sieve, activated carbon, kaolin, and diatomite.

[0010] As a preferred technical solution, the ultra-high molecular weight plastic powder of the present invention is selected from at least one of ultra-high molecular weight polyethylene powder and ultra-high molecular weight polypropylene powder.

[0011] As a preferred technical solution, the particle size of the inorganic adsorption powder material of the present invention is 1 μm to 10 μm.

[0012] As a preferred technical solution, the particle size of the ultra-high molecular weight plastic powder of the present invention is 5 μm to 100 μm.

[0013] As a preferred technical solution, the diameter of the core of the present invention is 8 mm to 15 mm.

[0014] As a preferred technical solution, the thickness of the nutrient supplement layer of the present invention is 1 mm to 4 mm.

[0015] As a preferred technical solution, the thickness of the housing layer of the present invention is 1 mm to 3 mm.

[0016] As a preferred technical solution, the acid-base buffer of the present invention is selected from at least one of sodium bicarbonate, HEPES, MES, and phosphates.

[0017] On the other hand, the present invention provides a method for preparing the aforementioned multifunctional regulating filter element for Escherichia coli culture, including the steps: (1) Preparing the core Mix the inorganic adsorption powder material and the ultra-high molecular weight plastic powder, place them in a spherical mold, and heat and mold to obtain a spherical structure; Prepare an acid-base buffer solution by mixing the acid-base buffer and water; Immerse the spherical structure in the acid-base buffer solution, and then dry it to obtain the core; (2) Preparing the outer shell (2.1) Preparing the nutrient supplement layer Mix amino acids, vitamins, glucose, tryptone, yeast extract, and magnesium ions in water, and coat the surface of the core to form a nutrient supplement layer; (2.2) Preparing the housing layer Dissolve gelatin in water to form a gelatin solution; Immerse the spherical structure with the nutrient supplement layer in the gelatin solution to form a housing layer.

[0018] As a preferred technical solution, the mass ratio of the inorganic adsorption powder material and the ultra-high molecular weight plastic powder of the present invention is 0.01 to 0.3:1.

[0019] As a preferred technical solution, the temperature of the heat molding of the present invention is 120 °C to 170 °C, and the time is 20 min to 40 min.

[0020] As a preferred technical solution, the concentration of the acid-base buffer solution of the present invention is 0.1 mol / L to 0.5 mol / L.

[0021] As a preferred technical solution, the drying temperature of the present invention is 40°C to 60°C, and the drying time is 6h to 12h.

[0022] As a preferred technical solution, the mass ratio of amino acid, vitamin, glucose, tryptone, yeast extract, magnesium ion, and water in the present invention is 3:0.05 - 0.2:0.05 - 0.2:5 - 10:10 - 20:5 - 10:0.1 - 0.5:10 - 20.

[0023] As a preferred technical solution, the coating method of the present invention is selected from at least one of spraying and dipping.

[0024] As a preferred technical solution, the concentration of the gelatin solution of the present invention is 5wt% - 10wt%.

[0025] As a preferred technical solution, after step (2) of the present invention, it further includes drying the spherical structure containing the shell layer to obtain a multifunctional regulating filter element. Description of the Drawings

[0026] Figure 1 It is a schematic structural diagram of the multifunctional regulating filter element for Escherichia coli culture of the present invention. Detailed Embodiments

[0027] The present invention provides a multifunctional regulating filter element for Escherichia coli culture. As Figure 1 shown, the multifunctional regulating filter element of the present invention is a composite spherical structure 100. The composite spherical structure 100 includes a core 10 and a shell 20. The core 10 has a porous structure 11 and the pores are filled with an acid-base buffer 12. The shell 20 includes a nutrient supplement layer 21 and a shell layer 22 in sequence from inside to outside.

[0028] The preparation raw materials of the core 10 include an inorganic adsorption powder material and a ultra-high molecular weight plastic powder. The inorganic adsorption powder material has interstitial or pore structures in its structure, which can effectively adsorb the metabolites produced during the growth of Escherichia coli. The ultra-high molecular weight plastic powder has the characteristics of high density and high strength, which helps to enhance the mechanical properties and durability of the composite spherical structure. The core 10 prepared from the above two raw materials has a porous structure, and its porous structure can provide a storage space for the acid-base buffer, can effectively regulate the pH value during the culture of Escherichia coli, can effectively adsorb metabolites such as bacteriocins produced during the fermentation of Escherichia coli, and can better support and fix the nutrient supplement layer 21 and the shell layer 22, thereby more effectively promoting the proliferation and growth of Escherichia coli.

[0029] Among them, the inorganic adsorbent powder material can be selected from at least one of molecular sieve, activated carbon, kaolinite, and diatomite. The ultra-high molecular weight plastic powder can be selected from at least one of ultra-high molecular weight polyethylene powder and ultra-high molecular weight polypropylene powder. Preferably, the inorganic adsorbent powder material of the present invention is selected from molecular sieve, and the ultra-high molecular weight plastic powder is selected from ultra-high molecular weight polyethylene powder. Using these two as the raw materials for the core has good adsorption performance and molding stability. The molecular sieve can effectively adsorb metabolites such as bacteriocin, and the ultra-high molecular weight polyethylene powder can endow the core with better physical strength, which is beneficial to maintaining the structural integrity in the culture environment. The particle size of the inorganic adsorbent powder material is 1 μm to 10 μm. As an example, the particle size of the inorganic adsorbent powder material can be, but is not limited to, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm. The particle size of the ultra-high molecular weight plastic powder is 5 μm to 100 μm. As an example, the particle size of the inorganic adsorbent powder material can be, but is not limited to, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm. The acid-base buffer is selected from at least one of sodium bicarbonate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), and phosphate. Preferably, the acid-base buffer of the present invention can be selected from HEPES, which has good biocompatibility and buffering capacity and can more effectively maintain the acid-base balance during the cultivation of Escherichia coli.

[0030] The diameter of the core 10 is 8 mm to 15 mm. Preferably, the diameter of the core is 10 mm to 13 mm. More preferably, the diameter of the core 10 is 12 mm. As an example, the diameter of the core 10 can be, but is not limited to, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 10.5 mm, 11 mm, 11.5 mm, 12 mm, 12.5 mm, 13 mm, 13.5 mm, 14 mm, 14.5 mm, 15 mm.

[0031] The raw materials for preparing the nutrient supplement layer 21 include amino acids, vitamins, glucose, tryptone, yeast extract, and magnesium ions. Among them, the amino acids may specifically include serine, glutamic acid, and histidine with a mass ratio of 1:1:1, but are not limited thereto. The vitamins may specifically include vitamin B1, vitamin B2, vitamin B5, and vitamin B7 with a mass ratio of 1:1:1:1, but are not limited thereto. The magnesium ions may specifically be magnesium sulfate (MgSO4) or magnesium chloride (MgCl2). The foregoing nutrients can provide sufficient nutrition for the growth of Escherichia coli and promote its rapid multiplication. The thickness of the nutrient supplement layer 21 is 1 mm to 4 mm. Preferably, the thickness of the nutrient supplement layer 21 is 1 mm to 3 mm. More preferably, the thickness of the nutrient supplement layer 21 is 2 mm. As an example, the thickness of the nutrient supplement layer may be, but is not limited to, 1 mm, 1.2 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.8 mm, 2 mm, 2.2 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.8 mm, 3 mm, 3.2 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.8 mm, 4 mm.

[0032] The raw materials for preparing the housing layer 22 include gelatin. Gelatin can be slowly decomposed in the LB culture medium, so that the nutrient supplement layer, the inner core inside the housing layer 22, and the culture medium come into contact and dissolve. The nutrient supplement layer can supplement new nutrients in the LB culture medium to provide sufficient energy for the rapid growth of Escherichia coli. The acid-base buffer filled in the pores of the inner core can maintain the acid-base balance in the LB medium, and the adsorption material contained in the inner core can adsorb metabolites such as bacteriocins produced by the growth and fermentation of Escherichia coli in the culture medium to reduce the adverse effects on its growth. The thickness of the housing layer 22 is 1 mm to 3 mm. As an example, the thickness of the housing layer 22 may be, but is not limited to, 1 mm, 1.2 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.8 mm, 2 mm, 2.2 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.8 mm, 3 mm. On the other hand, the present invention discloses a preparation method of a multifunctional regulating filter element for culturing Escherichia coli, including the steps: (1) Preparing the inner core Mix the inorganic adsorption powder material and the ultra-high molecular weight plastic powder, place them in a spherical mold, and heat and mold to obtain a spherical structure; Prepare an acid-base buffer solution by mixing an acid-base buffer and water; Immerse the spherical structure in the acid-base buffer solution, and then dry it to obtain the inner core; (2) Preparing the outer shell (2.1) Preparing the nutrient supplement layer Mix amino acids, vitamins, glucose, tryptone, yeast extract, and magnesium ions in water, and coat the surface of the inner core to form a nutrient supplement layer; (2.2) Preparation of the shell layer Dissolve gelatin in water to form a gelatin solution; Immerse the spherical structure containing the nutritional supplement layer into the gelatin solution to form the shell layer.

[0033] Among them, the mass ratio of the inorganic adsorption powder material to the ultra-high molecular weight plastic powder is 0.01~0.3:1. As an example, the mass ratio of the inorganic adsorption powder material to the ultra-high molecular weight plastic powder can be but is not limited to 0.01:1, 0.05:1, 0.1:1, 0.12:1, 0.14:1, 0.16:1, 0.18:1, 1.2:1, 0.21:1, 0.22:1, 0.23:1, 0.24:1, 0.25:1, 0.26:1, 0.27:1, 0.28:1, 0.29:1, 0.3:1. The temperature for heat forming is 120°C~170°C. As an example, the temperature for heat forming can be but is not limited to 120°C, 125°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, 170°C. The time for heat forming is 20 min~40 min. As an example, the time for heat forming can be but is not limited to 20 min, 25 min, 30 min, 35 min, 40 min.

[0034] The concentration of the acid-base buffer solution is 0.1 mol / L~0.5 mol / L. As an example, the concentration of the acid-base buffer solution can be but is not limited to 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L.

[0035] The temperature for drying is 40°C~60°C. As an example, the temperature for drying can be but is not limited to 40°C, 45°C, 50°C, 55°C, 60°C. The time for drying is 6 h~12 h. As an example, the time for drying is 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h. Slow drying at this temperature can make the acid-base buffer better filled and loaded into the pores of the inner core.

[0036] The mass ratio of amino acid, vitamin, glucose, tryptone, yeast extract, magnesium ion, and water is 3:0.05~0.2:5~10:10~20:5~10:0.1~0.5:10~20. The coating method is selected from at least one of spraying and dipping.

[0037] The concentration of the gelatin solution is 5 wt%~10 wt%. As an example, the concentration of the gelatin solution is 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%.

[0038] After step (2), it further includes drying the sphere structure containing the shell layer to obtain a multi-functional regulating filter element.

[0039] To better illustrate the purpose, technical solution and beneficial effects of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be noted that the following described method of the embodiments is a further explanatory illustration of the present invention and should not be regarded as a limitation of the present invention.

[0040] Example 1 This embodiment provides a preparation method of a multi-functional regulating filter element for Escherichia coli culture, including the steps: (1) Prepare the inner core Put 0.3 g of activated carbon with a particle size D V 50 of 5 μm and 1.0 g of ultra-high molecular weight polyethylene powder with a particle size D V 50 of 50 μm are mixed and placed in a spherical mold and heated at 160 °C for 40 min to obtain a sphere structure; Prepare a 0.1 mol / L sodium bicarbonate buffer solution by dissolving 1.68 g of sodium bicarbonate in 200 g of water; Immerse the sphere structure in the sodium bicarbonate buffer solution for adsorption and filling, and then dry it at 50 °C for 8 h to obtain an inner core with a diameter of 12 mm; (2) Prepare the outer shell (2.1) Prepare the nutrient supplement layer Spray 1 g of serine, 1 g of glutamine, 1 g of histidine, 0.1 g of vitamins (vitamins B1, B2, B5 and B7 with a mass ratio of 1:1:1:1), 10 g of glucose, 15 g of tryptone, 7.5 g of yeast extract, and 0.3 g of magnesium sulfate dissolved in 15 g of water on the surface of the inner core to form a nutrient supplement layer with a thickness of 2 mm; (2.2) Prepare the shell layer Put 10 g of gelatin in 100 g of water and heat it to 80 °C to dissolve to form a 10 wt% gelatin solution; Immerse the sphere structure containing the nutrient supplement layer in the gelatin solution to form a shell layer with a thickness of 2 mm, and then dry it at 40 °C for 10 h to obtain a multi-functional regulating filter element.

[0041] Example 2 Put 0.2 g of activated carbon with a particle size D V 50 of 3 μm and 1 g of ultra-high molecular weight polyethylene powder with a particle size D V 50 of 80 μm are mixed and placed in a spherical mold and heated at 165 °C for 35 min to obtain a sphere structure; Prepare a 0.15 mol / L sodium bicarbonate buffer solution by dissolving 2.52 g of sodium bicarbonate in 200 g of water; Immerse the spherical structure in a sodium bicarbonate buffer solution for adsorption and filling, and then dry it at 55 °C for 7 h to obtain a core with a diameter of 13 mm; (2)Prepare the outer shell (2.1)Prepare the nutrient supplement layer Dissolve 1 g of serine, 1 g of glutamine, 1 g of histidine, 0.15 g of vitamins (vitamins B1, B2, B5, and B7 with a mass ratio of 1:1:1:1), 10 g of glucose, 15 g of tryptone, 7.5 g of yeast extract, and 0.3 g of magnesium sulfate in 18 g of water, mix them, and spray them on the surface of the core to form a nutrient supplement layer with a thickness of 3 mm (2.2)Prepare the shell layer Place 12 g of gelatin in 120 g of water and heat it to 80 °C to dissolve it to form a 10 wt% gelatin solution; Immerse the spherical structure containing the nutrient supplement layer in the gelatin solution to form a shell layer with a thickness of 2.5 mm, and then dry it at 45 °C for 9 h to obtain a multifunctional regulating filter element.

[0042] Example 3 Put 0.1 g of diatomaceous earth with a particle size D V 50 of 2 μm and 1 g of ultra-high molecular weight polyethylene powder with a particle size D V 50 of 60 μm are mixed and placed in a spherical mold, and heated and molded at 160 °C for 40 min to obtain a spherical structure; Prepare a 0.1 mol / L HEPES buffer solution by dissolving 4.776 g of HEPES in 200 g of water; Immerse the spherical structure in the HEPES buffer solution for adsorption and filling, and then dry it at 45 °C for 10 h to obtain a core with a diameter of 12.4 mm; (2)Prepare the outer shell (2.1)Prepare the nutrient supplement layer Dissolve 1 g of serine, 1 g of glutamine, 1 g of histidine, 0.08 g of vitamins (vitamins B1, B2, B5, and B7 with a mass ratio of 1:1:1:1), 8 g of glucose, 12 g of tryptone, 6 g of yeast extract, and 0.2 g of magnesium sulfate in 12 g of water, mix them, and spray them on the surface of the core to form a nutrient supplement layer with a thickness of 1.5 mm; (2.2)Prepare the shell layer Place 8 g of gelatin in 80 g of water and heat it to 80 °C to dissolve it to form a 10 wt% gelatin solution; Immerse the spherical structure containing the nutrient supplement layer in the gelatin solution to form a shell layer with a thickness of 1.8 mm, and then dry it at 38 °C for 12 h to obtain a multifunctional regulating filter element.

[0043] The following is the application of the multifunctional regulating filter element for Escherichia coli culture in the present invention in the culture of Escherichia coli. In the application of the multifunctional regulating filter element for Escherichia coli culture in the present invention, the LB medium is from GF1181 of Shenzhen Biocomma Biotechnology Co., Ltd.; the Escherichia coli is from the China Center of Industrial Culture Collection (CICC, strain number: CICC 24121, E. coli DH5α).

[0044] Application Example 1 (1)Prepare the LB medium. Place 250 ml of the LB medium in a culture container, and then sterilize it at 120 °C for 0.5 h for later use.

[0045] (2)Under sterile conditions, use a sterile pipette to aspirate 2 mL of the Escherichia coli bacterial solution (colony density is 1×10 6 CFU / mL) and inoculate it into the culture container containing the LB medium, and gently shake the culture container to make the Escherichia coli evenly distributed in the medium.

[0046] (3)Under sterile conditions, add the multifunctional regulating filter element prepared by the preparation method of Example 1 to the culture container containing Escherichia coli and the LB medium, and gently shake the culture container to make the multifunctional regulating filter element evenly dispersed in the LB medium, where the addition ratio of the multifunctional regulating filter element is 1 g / L.

[0047] (4)Place the culture container in a constant temperature shaker for culture, control the culture temperature at 37 °C, and the rotation speed at 200 r / min.

[0048] Application Example 2 The difference from Application Example 1 is that the multifunctional regulating filter element used is the multifunctional regulating filter element prepared by the preparation method of Example 2.

[0049] Application Example 3 The difference from Application Example 1 is that the multifunctional regulating filter element used is the multifunctional regulating filter element prepared by the preparation method of Example 3.

[0050] Application Example 4 The difference from Application Example 1 is that the addition ratio of the multifunctional regulating filter element is 1.5 g / L.

[0051] Control Example 1 (1)Prepare the LB medium. Place 250 ml of the LB medium in a culture container, and then sterilize it at 120 °C for 0.5 h for later use.

[0052] (2)Under sterile conditions, use a sterile pipette to aspirate 2 mL of the Escherichia coli bacterial solution and inoculate it into the culture container containing the LB medium, and gently shake the culture container to make the Escherichia coli evenly distributed in the medium.

[0053] (3) Place the culture vessel in a constant temperature shaker for cultivation, and control the cultivation temperature at 37 °C and the rotation speed at 200 r / min.

[0054] During the cultivation of Escherichia coli in Application Examples 1 to 4 and Control Example 1, a pH meter was used to regularly monitor the pH value of the culture system, and at the same time, when Escherichia coli was cultured for 20 h, a high-performance liquid chromatography analyzer was used to measure the bacteriocin content in the LB medium. The test results are shown in Table 1. The OD600 value in the Escherichia coli culture system was regularly measured by spectrophotometry, and the results are shown in Table 2.

[0055] Table 1 pH value test results during the cultivation of Escherichia coli in Application Examples 1 to 4 and Control Example 1

[0056] Table 2 OD600 value test results during the cultivation of Escherichia coli in Application Examples 1 to 4 and Control Example 1

[0057] Specifically, please refer to the test results in Table 1 and Table 2. During the growth process of Escherichia coli, first, it can be seen from the pH value test results of Application Examples 1 to 4 and Control Example 1 in Table 1 that the addition of the multifunctional regulating filter element of the present invention can effectively regulate the pH value of the Escherichia coli culture system, keep it within a suitable range, and avoid adverse effects on the growth of Escherichia coli due to acid-base imbalance. And as can be seen from Table 2, Escherichia coli can grow rapidly in the early stage, indicating that the nutrient supplement layer plays a role in enriching nutrients. In the later stage, as the colony density increases, the amount of metabolic toxic substances increases. Combining the test results of the bacteriocin content at 20 h of cultivation in Table 1, it can be known that the bacteriocin content in Application Examples 1 to 4 is less than that in Control Example 1, indicating that the multifunctional regulating filter element of the present invention can effectively adsorb bacteriocin, reduce the accumulation of bacteriocin, and thus reduce the inhibitory effect of bacteriocin on the growth and proliferation of Escherichia coli, which is beneficial to increasing the density of Escherichia coli. Further, from the test results of Application Example 1 and Control Example 1 in Table 2, it can be known that the OD600 value of Escherichia coli in Application Example 1 increases faster, indicating that the growth rate of Escherichia coli is faster and the density is higher, proving the significant advancement of the multifunctional regulating filter element of the present invention. In summary, the multifunctional regulating filter element for Escherichia coli culture of the present invention can effectively solve the problems of difficult maintenance of acid-base balance and accumulation of bacteriocin metabolites in the existing LB culture method, improve the growth rate and density of Escherichia coli, and has broad application prospects.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, it is not limited to the embodiments listed. Those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A multifunctional regulating filter element for Escherichia coli culture, characterized in that: The composite spherical structure comprises an inner core and an outer shell, wherein the inner core has a porous structure and the pores are filled with an acid-base buffer, and the outer shell comprises a nutrient supplement layer and a shell layer from the inside to the outside, and the raw materials for preparing the inner core comprise inorganic adsorption powder material and ultra-high molecular weight plastic powder, the raw materials for preparing the nutrient supplement layer comprise amino acids, vitamins, glucose, trypsin, yeast extract and magnesium ions, and the raw materials for preparing the shell layer comprise gelatin.

2. The multifunctional regulating filter element for Escherichia coli culture according to claim 1, characterized in that: The inorganic adsorption powder material is selected from at least one of molecular sieves, activated carbon, kaolinite, and diatomaceous earth, and the ultra-high molecular weight plastic powder is selected from at least one of ultra-high molecular weight polyethylene powder and ultra-high molecular weight polypropylene powder.

3. The multifunctional regulating filter element for Escherichia coli culture according to claim 1, characterized in that: The particle size of the inorganic adsorption powder material is 1 μm to 10 μm, and the particle size of the ultra-high molecular weight plastic powder is 5 μm to 100 μm.

4. The multifunctional regulating filter element for Escherichia coli culture according to claim 1, characterized in that: The diameter of the inner core is 8 mm to 15 mm, the thickness of the nutritional supplement layer is 1 mm to 4 mm, and the thickness of the shell layer is 1 mm to 3 mm.

5. The multifunctional regulating filter element for Escherichia coli culture according to claim 1, characterized in that: The acid-base buffer is selected from at least one of sodium bicarbonate, HEPES, MES and phosphate.

6. The method for preparing a multifunctional regulating filter element for Escherichia coli culture according to any one of claims 1 to 5, characterized in that: Includes steps: (1) Preparation of kernel The inorganic adsorption powder material and the ultra-high molecular weight plastic powder are mixed and placed in a spherical mold and heated to form a spherical structure; The acid-base buffer and water are configured into an acid-base buffer solution; Immersing the spherical structure in the acid-base buffer solution and then drying it to obtain the inner core; (2) Preparation of shell (2.1) Preparation of nutrient supplement layer The amino acids, the vitamins, the glucose, the tryptone, the yeast extract and the magnesium ions are mixed in water and coated on the surface of the inner core to form the nutrient supplement layer; (2.1) Preparation of shell layer Dissolving the gelatin in water to form a gelatin solution; The spherical structure containing the nutritional supplement layer is immersed in the gelatin solution to form the shell layer.

7. The method for preparing the multifunctional regulating filter element for Escherichia coli culture according to claim 6, characterized in that: The mass ratio of the inorganic adsorption powder material to the ultra-high molecular weight plastic powder is 0.01-0.3:1, the temperature of the heating molding is 120° C.-170° C., and the time is 20 min-40 min.

8. The method for preparing the multifunctional regulating filter element for Escherichia coli culture according to claim 6, characterized in that: The concentration of the acid-base buffer solution is 0.1 mol / L to 0.5 mol / L, the drying temperature is 40° C. to 60° C., and the drying time is 6 h to 12 h.

9. The method for preparing a multifunctional regulating filter element for culturing Escherichia coli according to claim 6, characterized in that: The mass ratio of the amino acid, the vitamin, the glucose, the tryptone, the yeast extract, the magnesium ion and the water is 3:0.05~0.2:5~10:10~20:5~10:0.1~0.5:10~20, and the coating method is selected from at least one of spraying and dipping.

10. The method for preparing the multifunctional regulating filter element for Escherichia coli culture according to claim 6, characterized in that: The concentration of the gelatin solution is 5wt%-10wt%. After step (2), the method further includes drying the spherical structure containing the shell layer to obtain the multifunctional regulating filter element.

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