Responsive cell-adhesive material, immobilization carrier and preparation method and application thereof

Abstract

The application discloses a responsive cell adhesion material, an immobilized carrier and a preparation method and application thereof. Dopamine hydrochloride, 3-acrylamidophenylboronic acid, polyethyleneimine, a reducing agent and a cationic conjugated polymer are mixed to prepare the responsive cell adhesion material, and then a fiber material is added, so that the responsive cell adhesion material is subjected to a co-deposition reaction on the surface of the fiber material to obtain the immobilized carrier with sugar response. The prepared immobilized carrier can quickly and firmly adhere on the basis of stimulating microbial cells to form a biofilm, and provides help for the application of microbial cells in a continuous fermentation process.

Description

Technical Field

[0001] This invention belongs to the field of biochemical and microbial fermentation technology, specifically relating to a responsive cell adhesion material, an immobilization carrier, its preparation method, and its application. Background Technology

[0002] In microbial fermentation, microbial cells themselves act as biocatalysts to transform substrates into products. Compared to chemical catalysts, microbial cells have lower reusability, are prone to decay and autolysis, have lower environmental adaptability, and insufficient cell density, making it impossible to achieve high-concentration continuous production like chemical catalysis. Current biofermentation processes cannot utilize cells continuously for extended periods, necessitating intermittent fermentation.

[0003] To address the problems in microbial fermentation, immobilized cell technology has been developed based on immobilized enzymes and is widely used in industries such as food, energy, environmental protection, and fermentation. Liu Ping et al. (Study on the algae removal effect of immobilized algaecides [J]. Environmental Engineering, 2016, 34(12): 28-31) immobilized algaecides RZ14 in a dynamic membrane reactor with micro-mesh material as the membrane matrix for wastewater treatment, and the water quality improvement effect was significant. Huang et al. (Butyric acid production from oilseed rape straw by Clostridium tyrobutyricum immobilized in a fibrousbed bioreactor [J]. Process Biochemistry, 2016, 51(12): 1930-1934.) immobilized Clostridium butyricum to produce butyric acid, which improved the butyric acid concentration and yield compared with free cell fermentation. CN 113444717A describes a method for immobilizing Escherichia coli using Fe3O4@SiO2-NH2-alkyne as a carrier and applying it to the biotransformation of 1,3-dihydroxyacetone. This biotransformation process is characterized by mild reaction conditions, high specificity, and high substrate utilization.

[0004] Various immobilization methods have been widely used in various research fields, but the choice of immobilization method can have a certain impact on the normal biological functions of microbial cells. However, immobilization using the biofilm formed by the microorganisms themselves as a medium will largely ensure the normal physiological functions of microbial cells. Therefore, there is an urgent need to develop a carrier material based on microbial biofilms for immobilization. Summary of the Invention

[0005] The first technical problem that this invention aims to solve is to address the shortcomings of existing technologies by providing a responsive cell adhesion material that can stimulate microbial cells to produce biofilms, thereby achieving controllable microbial aggregation and dispersion.

[0006] The second technical problem solved by this invention is to provide an immobilized carrier that can stimulate the formation of cell biofilms and enable the adsorption and desorption of microorganisms on the carrier at different sugar concentrations.

[0007] The third technical problem solved by the present invention is to provide a method for preparing the above-mentioned immobilized carrier.

[0008] The fourth technical problem solved by this invention is to provide the application of the above-mentioned immobilized carrier in microbial fermentation.

[0009] To solve the first technical problem mentioned above, the present invention adopts the following technical solution:

[0010] A responsive cell adhesion material is obtained by mixing dopamine hydrochloride, 3-acrylamidophenylboronic acid, polyethyleneimine, a reducing agent, and a cationic conjugated polymer in a reaction solution and stirring the mixture at 12–60 °C.

[0011] Furthermore, in the responsive cell adhesion material of the present invention, the mass ratio of 3-acrylamidophenylboronic acid, dopamine hydrochloride, polyethyleneimine, reducing agent, and cationic conjugated polymer is (0.1-10):1:(0.3-5):(0.05-2):(0.05-5).

[0012] Dopamine can spontaneously polymerize into polydopamine under aerobic and alkaline conditions, which can be deposited on various organic or inorganic materials to form functional coatings. However, dopamine deposition often results in large particle sizes. The addition of polyethyleneimine can disrupt the non-covalent bond effect during dopamine deposition, making the co-deposited coating more uniform. During the oxidative self-polymerization of dopamine hydrochloride, it can covalently crosslink with polyethyleneimine through the Schiff base reaction and Michael addition reaction of the amino group. Due to the covalent reaction, polyethyleneimine greatly promotes the deposition process of polydopamine. However, the addition of polyethyleneimine may lead to co-deposition with dopamine due to the Schiff base reaction, which will occupy the exposed diol structure of the dopamine molecule, thus affecting the covalent bonding between the phenylboronic acid group and dopamine. Ultimately, this results in a low modification level of phenylboronic acid. Therefore, a reducing agent is added to inhibit its oxidation, allowing it to co-deposit with polyethyleneimine only through the Michael addition reaction.

[0013] 3-Acrylamidophenylboronic acid dehydrates to form borate ester bonds under alkaline conditions. In acidic environments, this dehydration can reversibly hydrolyze, breaking the borate ester bonds. Furthermore, the phenylboronic acid group has a stronger affinity for glucose molecules. Other compounds containing cis-diols in the solution, besides glucose, lack competitiveness. At higher glucose concentrations, the phenylboronic acid group preferentially reacts with glucose. During the fermentation of *Saccharomyces cerevisiae*, the pH of the fermentation broth changes from neutral or weakly alkaline to acidic as fermentation progresses, and the sugar concentration decreases until fermentation ends. At this point, the borate ester bonds formed by the phenylboronic acid group and glucose at high sugar concentrations break, and the phenylboronic acid group binds to glycoproteins in the microbial biofilm, beginning to adsorb onto microbial cells. Through microbial proliferation and the continuous fusion and accumulation of the biofilm, an aggregation effect is formed.

[0014] The addition of cationic conjugated polymers can improve the biofilm-forming ability of microbial cells.

[0015] Furthermore, the concentration of dopamine hydrochloride in the reaction solution is 0.2–5 g / L, preferably 0.8–3.5 g / L.

[0016] Furthermore, the concentration of the polyethyleneimine in the reaction solution is 0.5–10 g / L, preferably 1–5 g / L.

[0017] Furthermore, the cationic conjugated polymer is selected from any one or a combination of two or more of PFP-G2, PMNT, PMI, and PFP, and the amount of cationic conjugated polymer added is not higher than 10 g / L.

[0018] Furthermore, the reducing agent is any one or a combination of two or more of sodium borohydride, glucose, sodium bisulfite, and sodium citrate.

[0019] To solve the second technical problem mentioned above, the present invention adopts the following technical solution:

[0020] An immobilization carrier includes a fibrous material and a responsive cell adhesion material as described above, modified on the surface of the fibrous material.

[0021] The immobilized carrier prepared in this invention exhibits stimulus-responsive characteristics, capable of stimulating the formation of microbial cell membranes and effectively matching the fermentation process of microorganisms to respond to stimuli, achieving high-sugar desorption and low-sugar adsorption. This has a positive effect on improving the tolerance of microbial cells and facilitating continuous, high-density fermentation processes.

[0022] To solve the third technical problem mentioned above, the present invention adopts the following technical solution:

[0023] The above-mentioned method for preparing an immobilized carrier with stimuli-responsive characteristics involves mixing dopamine hydrochloride, 3-acrylamidophenylboronic acid, polyethyleneimine, a reducing agent, and a cationic conjugated polymer to obtain a responsive cell adhesion material. Subsequently, a fibrous material is added to the obtained responsive cell adhesion material, and the responsive cell adhesion material is modified on the surface of the fibrous material through a co-deposition modification reaction.

[0024] Furthermore, the specific steps include the following:

[0025] (1) Cut the fiber material into blocks or sheets that are suitable for the size of the reaction vessel;

[0026] (2) Prepare a tris(hydroxymethyl)aminomethane solution;

[0027] (3) Add dopamine hydrochloride, polyethyleneimine, reducing agent and cationic conjugated polymer to the tris(2) tris(hydroxymethyl)aminomethane solution and stir thoroughly to react;

[0028] (4) Immerse the fiber material in the solution of step (3), then add 3-acrylamidophenylboronic acid, and carry out a co-deposition modification reaction in a shaker to obtain the product.

[0029] Preferably, in step (1), the fiber material is a square shape that has been cut to fit the size of the fermentation bottle.

[0030] Furthermore, in step (2), the concentration of the tris(hydroxymethyl)aminomethane solution is 2.5–10 g / L, and the pH value is adjusted to be between 3.5 and 9.0 using dilute hydrochloric acid.

[0031] Furthermore, in step (3), the amount of dopamine hydrochloride used is 0.2-5 g / L of tris(hydroxymethyl)aminomethane solution, preferably 0.8-3.5 g / L; the amount of polyethyleneimine used is 0.5-10 g / L of tris(hydroxymethyl)aminomethane solution, preferably 1-5 g / L, more preferably 1-2 g / L; the amount of reducing agent used is 0.1-3.0 g / L of tris(hydroxymethyl)aminomethane solution, preferably 0.6-1.5 g / L; and the amount of cationic conjugated polymer used is not higher than 10 g / L.

[0032] Furthermore, in step (4), the mass ratio of 3-acrylamidophenylboronic acid, dopamine hydrochloride, polyethyleneimine, and reducing agent is (0.1-10):1:(0.3-5):(0.05-2):(0.05-5).

[0033] Furthermore, in step (4), the temperature of the co-deposition modification reaction is 12-60℃, preferably 20-48℃; the reaction time is 0.5-30h, preferably 2-15h; and the shaking speed is 100-200r / min.

[0034] Preferably, the amount of fiber material added to the co-deposition system is 10-50 g / L; the reaction time of the fiber material in the co-deposition system is 0.5-30 h, the temperature is 25-80 °C, and the rotation speed is 100-220 rpm.

[0035] To address the fourth technical problem mentioned above, the present invention also provides the aforementioned responsive cell adhesion material and the application of the aforementioned immobilized carrier in immobilized cell fermentation.

[0036] The immobilized carrier with a surface modified with responsive cell adhesion material can stimulate the formation of microbial cell biofilms during the fermentation process of immobilized cells and work well with the fermentation process to achieve the effect of high sugar desorption and low sugar adsorption.

[0037] Furthermore, when using the above-mentioned immobilized carrier for fermentation of immobilized cells, the amount of the immobilized carrier used is 20-60 g / L of fermentation medium.

[0038] Preferably, the microbial cells used for immobilized cell fermentation are Saccharomyces cerevisiae cells, and the immobilized cell fermentation method is multi-batch continuous fermentation.

[0039] Furthermore, before the immobilized cell fermentation, the immobilization carrier is sterilized together with the fermentation reaction vessel; the microbial seed liquid is inoculated into the fermentation medium containing the immobilized carrier at a volume of 1% to 10% of the fermentation medium volume.

[0040] Furthermore, this invention enables continuous multi-batch fermentation by controlling the sugar concentration in the fermentation broth, allowing immobilized cells to adsorb and desorb onto the immobilized carrier. Immobilized cells can desorb at high sugar concentrations, with both free cell catalysis and cell catalysis adsorbed onto the fibrous material. Immobilized cells can adsorb at low sugar concentrations and aggregate on the fibrous material surface to form a film at the end of fermentation, facilitating continuous multi-batch fermentation.

[0041] Furthermore, the present invention achieves the desorption of immobilized cells from the immobilization carrier by controlling the sugar concentration in the fermentation broth between 50 and 100 g / L.

[0042] Furthermore, the present invention achieves the adsorption of immobilized cells on the immobilized carrier by controlling the sugar concentration in the fermentation broth between 0.05 and 10 g / L.

[0043] Beneficial effects:

[0044] (1) Based on the glucose and pH responsive properties of phenylboronic acid and its derivatives, the present invention prepares a responsive immobilized carrier that can be well integrated with the fermentation process of microorganisms.

[0045] (2) Compared with the traditional immobilization method, the present invention can control the adsorption and desorption of cells by changing the fermentation environment, and can effectively recycle and utilize cells.

[0046] (3) The modification method disclosed in this invention is relatively simple and does not require stringent modification conditions. It also has a good modification effect.

[0047] (4) The responsive cell adhesion material modified by the present invention can stimulate the formation of biofilms in microbial cells and has good biocompatibility and cell adhesion ability. The microbial cells are more tolerant and can increase ethanol production to a certain extent. Attached Figure Description

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0049] Figure 1 SEM images of microbial aggregation in ordinary cotton fibers and responsive cotton fibers after fermentation.

[0050] Figure 2 This refers to the changes in microbial concentration parameters of responsive cotton fibers during fermentation.

[0051] Figure 3 This is a graph showing the results of multiple consecutive batches of fermentation. Detailed Implementation

[0052] The present invention can be better understood from the following embodiments.

[0053] Example 1: The pretreatment and responsive modification of the fiber material were carried out according to the following method:

[0054] (1) Take commercially available cotton fibers, wash them three times with an appropriate amount of water, dry them in a 55℃ oven, and cut them into small square pieces of 2.5cm*2.5cm.

[0055] (2) Prepare 1L of 50mM Tris-HCl solution, then adjust the pH to about 8.5 with dilute hydrochloric acid, add 1g each of dopamine hydrochloride and polyethyleneimine with a relative molecular weight of 600, the final concentration is 1g / L, add 0.5g of ascorbic acid as a reducing agent, and add 4g of cationic conjugated polymer.

[0056] (3) Weigh an appropriate amount of the cotton fibers prepared in step (1) and soak them in the solution prepared in step (2). Then add 1g of 3-acrylamidophenylboronic acid to make the concentration 1g / L, and modify them at 30℃ and 120r / min for 8h. Rinse the modified cotton fibers with pure water 3 to 5 times and dry them in an oven at 55℃ for later use.

[0057] Figure 1 These are SEM images of unmodified cotton fiber and responsive modified cotton fiber after fermentation, showing their effects on microbial aggregation. It can be seen that the responsive modified cotton fiber has a significant advantage in microbial cell aggregation and film formation. As the fermentation process ends and the sugar concentration decreases to between 0.05 and 10 g / L, microbial cells begin to rapidly and extensively adsorb onto the immobilized carrier surface. Comparatively, the modified cotton fiber promotes microbial cell film formation and aggregation, exhibiting higher immobilization capacity and efficiency than the unmodified cotton fiber.

[0058] Example 2: Application of responsive fiber materials in immobilized cell fermentation:

[0059] (1) The microbial cells used in the experiment were brewer's yeast cells, and the fermentation culture medium was: glucose 60g / L, peptone 4g / L, yeast extract 3g / L, (NH4)2SO4 4g / L, MgSO4 0.5g / L, ZnSO4·7H2O 0.05g / L, FeSO4·7H2O 0.05g / L.

[0060] (2) Free cell fermentation: The activated Saccharomyces cerevisiae CICC1308 seed culture was transferred to the fermentation medium at an inoculation rate of 10 vt%, and free cell fermentation was carried out at 35℃ and 200 rpm. During the free cell fermentation, the fermentation cycle was 12 h, the glucose consumption rate was 5.0 g / L, and the ethanol yield was 18.96 g / L.

[0061] (3) Immobilized cell fermentation: The activated Saccharomyces cerevisiae CICC1308 seed culture was transferred to the fermentation medium at an inoculum of 10 vt%, and immobilized cell fermentation was carried out at 35℃ and 200 rpm. The amount of responsive cotton fiber added was 50 g / L. At 6 h of fermentation, the OD of the fermentation broth was... 600 Reaching a maximum value of 2.855, as glucose was consumed during fermentation, the adhesion rate of microbial cells in the fermentation broth exceeded the growth rate, leading to rapid adhesion to the cotton fiber surface. At 12 hours, the OD... 600 It dropped to 0.725. Specific results are as follows: Figure 2 As shown.

[0062] Example 3: Application of responsive fiber materials in continuous multi-batch immobilized fermentation:

[0063] (1) The microbial cells used in the experiment were brewer's yeast cells, and the fermentation culture medium was: glucose 60g / L, peptone 4g / L, yeast extract 3g / L, (NH4)2SO4 4g / L, MgSO4 0.5g / L, ZnSO4·7H2O 0.05g / L, FeSO4·7H2O 0.05g / L.

[0064] In multi-batch continuous fermentation, for free-cell fermentation, 10 vt% of the fermentation broth from the previous batch was reserved as the seed culture for the next batch. For immobilized fermentation, only the immobilized carrier was left in the fermentation flask, all the fermentation broth was poured out, and then fresh fermentation broth was added for the second batch of fermentation. This process was repeated for seven consecutive batches. Fermentation was considered complete when the glucose concentration in the fermentation medium fell below 1 g / L. Results are as follows: Figure 3 As shown.

[0065] In the continuous fermentation process of free cells, the fermentation cycle was shortened from 12 hours to 9 hours, and the glucose consumption rate increased from 5 g / L / h to 6.67 g / L / h. However, the ethanol yield in the seventh batch was only 14.21 g / L, significantly lower than that of immobilized cell fermentation. In the immobilized fermentation process, the fermentation cycle of responsive cotton fibers was shortened from 12 hours to 4 hours (a 75% reduction), and the glucose consumption rate increased from 5 g / L / h to 15 g / L / h. The ethanol yield in the seventh batch was 25.11 g / L.

[0066] Comparative Example 1: The preparation method was the same as in Example 1, except that no cationic conjugated polymer and 3-acrylamidophenylboronic acid were added to the co-deposition system. The cotton fibers modified only with dopamine hydrochloride were recorded as Control Group 1.

[0067] Comparative Example 2: The preparation method is the same as in Example 1, except that no cationic conjugated polymer is added to the co-deposition system. The modified cotton fibers obtained are recorded as control group 2.

[0068] Comparative Examples 1 and 2 underwent single-batch and continuous fermentation of immobilized cells according to the methods described in Examples 2 and 3. During the single-batch fermentation, the carrier immobilization effect in Control Group 1 was poor; fermentation ended after 12 hours, and OD... 600 The OD value reached a maximum of 6.202 at the end of 12 hours of fermentation in control group 2. 600 The value was 0.988, slightly higher than the responsive carrier in Example 2. Specific results are as follows... Figure 2As shown in the figure. During multiple batches of fermentation, the fermentation cycle of immobilized Saccharomyces cerevisiae in control group 1 was shortened from 12h to 6h, the glucose consumption rate increased from 5g / L / h to 10g / L / h, and the ethanol yield was 20.02g / L in the seventh batch. In contrast, the fermentation cycle of immobilized Saccharomyces cerevisiae in control group 2 was shortened from 12h to 4h, the glucose consumption rate increased from 5g / L / h to 15g / L / h, and the ethanol yield was 23.49g / L in the seventh batch.

[0069] This invention provides a responsive cell adhesion material, an immobilization carrier, and its preparation and application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A responsive cell adhesion material, characterized in that, The mixture of dopamine hydrochloride, 3-acrylamidophenylboronic acid, polyethyleneimine, a reducing agent, and a cationic conjugated polymer is stirred and reacted at 12–60 °C to obtain the product. The mass ratio of 3-acrylamidophenylboronic acid, dopamine hydrochloride, polyethyleneimine, reducing agent, and cationic conjugated polymer is (0.1-10): 1: (0.3-5): (0.05-2): (0.05-5). The reducing agent is ascorbic acid.

2. An immobilized carrier, characterized in that, It includes fibrous materials, and a responsive cell adhesion material as described in claim 1, which is modified on the surface of the fibrous material.

3. The method for preparing the immobilized carrier according to claim 2, characterized in that, Dopamine hydrochloride, 3-acrylamidophenylboronic acid, polyethyleneimine, a reducing agent, and a cationic conjugated polymer were mixed and reacted to obtain a responsive cell adhesion material. Subsequently, a fibrous material was added to the obtained responsive cell adhesion material, and the responsive cell adhesion material was modified on the surface of the fibrous material through a co-deposition modification reaction.

4. The method for preparing the immobilized carrier according to claim 3, characterized in that, The fiber material is added to the co-deposition system at a rate of 10–50 g / L; the reaction time of the fiber material in the co-deposition system is 0.5–30 h, the temperature is 25–80 ℃, and the rotation speed is 100–220 rpm.

5. The application of the responsive cell adhesion material according to claim 1 in immobilized cell fermentation.

6. The application according to claim 5, characterized in that, The responsive cell adhesion material described above can achieve the detachment of immobilized cells from the carrier when the sugar concentration is between 50 and 100 g / L.

7. The application according to claim 5, characterized in that, The responsive cell adhesion material described above can achieve the adsorption of immobilized cells on a carrier when the sugar concentration is between 0.05 and 10 g / L.

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