Preparation method and application of a sugar-responsive cell adhesion material

Abstract

The present invention discloses a preparation method and application of a sugar-responsive cell adhesion material. A fibrous material is reacted in a co-deposition system containing a cross-linking agent, an adhesive, a reducing agent, and a compound containing a phenylboronic acid group to obtain a sugar-responsive fibrous material. The prepared sugar-responsive fibrous material can efficiently recover cells with the decrease of sugar concentration, and the immobilized cells have good reusability. The application scope of the compound containing a phenylboronic acid group is broadened. In addition to neutral or weakly alkaline conditions, the sugar-responsive fibrous material can also be applied to acidic fermentation processes.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biochemistry and microbial fermentation, and relates to a preparation method and application of a sugar-responsive cell adhesion material. Background Art

[0002] Immobilized cells have better environmental tolerance than free cells, making them have broad application prospects in industrial production and environmental protection. However, some commonly used immobilized cell carriers have certain toxicity to cells and low cell recovery rates of the carriers. Therefore, developing new types of highly efficient, green and non-toxic immobilization materials is the main direction of the development of the current immobilized cell technology.

[0003] Yuan et al. (ACS Applied Materials and Interfaces. 2016, 18, 11309 - 11317) prepared a nano - composite (ESKAP) capable of specifically killing Escherichia coli by grafting sugar polymers and quaternary ammonium salt polymers onto the surface of gold nanoparticles simultaneously. The recognition of sugar - lectin and the sugar - cluster effect increased the specific recognition of the material for Escherichia coli and the affinity between the two, enabling ESKAP to exhibit obvious antibacterial effects at lower doses. Liang et al. (ACS Nano. 2021, 4, 7078 - 7093) constructed a hydrogel sealant with antibacterial activity and self - healing properties by using trivalent iron, protocatechuic aldehyde, and quaternized chitosan to form a dual - dynamic cross - linked network through metal coordination and Schiff base. The dynamic bonds in this hydrogel will break and cross - link in different pH environments, thus endowing the hydrogel with antibacterial, self - healing, and adhesive properties, which can be used for the healing of skin wounds. Shi et al. (ACS Applied Materials Engineering and Interfaces. 2016, 38, 23523 - 23532) synthesized PNIPAM with adamantane (Ad) end - groups and quaternary ammonium salt polymers respectively by atom transfer radical polymerization (ATRP), and then self - assembled them onto the surface of β - cyclodextrin (β - CD) - modified silicon wafers. A thermoresponsive antibacterial surface was constructed through the host - guest interaction between β - CD and Ad. CN 113144205 B is based on the good biocompatibility and glucose responsiveness of a polymer with dihydroxy and hydrophobic groups and mesoporous silica modified with amino and phenylboronic acid groups. It is applied to the controlled release of the drug insulin for the treatment of diabetes, has good drug - loading capacity, and the cumulative drug release amount of insulin shows obvious differences in glucose solutions with different concentrations. CN112250978 A synthesized a polymer by free - radical copolymerization method, and then prepared a glucose - responsive sugar - sensitive microgel with polyvinyl alcohol by inverse microemulsion method. The obtained glucose - responsive sugar - sensitive microgel responds rapidly to changes in glucose concentration in the physiological environment, but its preparation process is relatively complex. CN 11098782 A prepared a glucose - responsive photonic crystal sensor for rapid detection of glucose concentration. Compared with traditional membrane - like sensors, it is thinner, which is beneficial to quickly reach the equilibrium state, and solves the problems of large size and long response time of glucose - responsive photonic crystals in the prior art.

[0004] It can be seen that various interface modification methods and stimulus-responsive technologies have been widely applied in various research fields. However, the modification process is generally cumbersome and mostly used in antibacterial, drug release, sensors, etc. Most researchers have ignored the application of this interface modification method in immobilized microbial fermentation. During the immobilized cell fermentation process, cells are in a continuous adsorption and desorption process, resulting in a decrease in recovery rate. Therefore, there is an urgent need to develop a sugar-responsive fiber material with good biocompatibility, a simple modification process, and sustainable function to achieve the efficient recovery of biofilms. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide a sugar-responsive fiber material with good biocompatibility, a simple modification process, and sustainable function in view of the deficiencies of the prior art, which is applied to the continuous fermentation process of cell immobilization and realizes the efficient recovery of biofilms.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A preparation method of a cell adhesion material with sugar responsiveness, characterized in that a fiber material is placed in a co-deposition system containing a cross-linking agent, an adhesive, a reducing agent, and a compound containing a phenylboronic acid group to react to obtain a sugar-responsive fiber material.

[0008] Preferably, the compound containing a phenylboronic acid group is selected from at least one of 2-acrylamidophenylboronic acid, 3-acrylamidophenylboronic acid, 2-aminophenylboronic acid hydrochloride, 3-aminophenylboronic acid, phenyl-1,4-diboronic acid, and 4-vinylphenylboronic acid.

[0009] Preferably, the cross-linking agent is selected from at least one of 4-(2-aminoethyl)-1,2-benzenediol hydrochloride, acrylamide, silica, and polyvinyl alcohol;

[0010] Preferably, the adhesive is selected from at least one of hydroxytyrosol, polyvinylpyrrolidone, hypromellose, polyaziridine, and sodium carboxymethylcellulose;

[0011] Preferably, the reducing agent is selected from at least one of sodium borohydride, glucose, sodium bisulfite, and sodium citrate.

[0012] Preferably, the fiber material is selected from at least one of cotton, linen, nylon, polyester, polypropylene fiber, and polyethylene fiber. The fiber material is cut into small pieces of 2.5 cm * 2.5 cm, washed 3-5 times with distilled water, and dried in a vacuum drying oven for standby.

[0013] Preferably, the mass ratio of the crosslinking agent, binder, reducing agent and the compound containing a boric acid group is 1:(0.1-20):(0.01-10):(0.2-10).

[0014] Preferably, the addition amount of the fiber material in 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.

[0015] Furthermore, the present invention also claims the cell adhesion material with sugar responsiveness prepared by the above preparation method.

[0016] Furthermore, the present invention also provides the application of the above cell adhesion material with sugar responsiveness as a cell carrier in immobilized cell fermentation.

[0017] Preferably, in the above application, the dosage of the cell adhesion material with sugar responsiveness is 10-100 g / L of the fermentation medium.

[0018] Specifically, in the above application, when the sugar concentration of the cell adhesion material with sugar responsiveness is between 50-100 g / L, the desorption of the immobilized cells on the carrier can be realized; when the sugar concentration of the cell adhesion material with sugar responsiveness is between 0.05-10 g / L, the adsorption of the immobilized cells on the carrier can be realized.

[0019] Specifically, in the above application, the immobilized cells include but are not limited to Escherichia coli, Acetobacter, Lactobacillus, Clostridium acetobutylicum and Saccharomyces cerevisiae cells.

[0020] Specifically, in the above application, the immobilized fermentation method is multi-batch continuous fermentation.

[0021] Specifically, in the above application, the microbial seed liquid is transferred to the fermentation medium containing the fiber material for culture at an inoculation amount of 1%-10% of the volume of the fermentation medium.

[0022] Specifically, in the above application, the cell adhesion material with sugar responsiveness has responsiveness within the range of pH 2.0-8.5.

[0023] Specifically, in the above application, the immobilized cells can adhere to the surface of the cell adhesion material with sugar responsiveness within 6-10 h of fermentation.

[0024] Specifically, in the above application, the cell adhesion material with sugar responsiveness can efficiently recover the cells with the decrease of sugar concentration, and the OD of the fermentation broth can be reduced to 5%-15% of the original system within 6-10 h. 600 Drop to 5%-15% of the original system.

[0025] Beneficial effects:

[0026] (1) Without affecting the oxidative deposition of 4-(2-aminoethyl)-1,2-benzenediol hydrochloride (PDA-CF), the present invention effectively modifies the phenylboronic acid group and endows the immobilized carrier with sugar-responsive characteristics.

[0027] (2) The sugar-responsive fiber material prepared by the present invention can efficiently recover cells with the decrease of sugar concentration, and the immobilized cells have good reusability.

[0028] (3) The sugar-responsive fiber material prepared by the present invention broadens the application scope of compounds containing phenylboronic acid groups. In addition to neutral or weakly alkaline conditions, the sugar-responsive fiber material can also be applied to acidic fermentation processes.

[0029] (4) Different from common temperature-sensitive immobilized carriers, the sugar-responsive fiber material prepared by the present invention can rapidly respond to changes in sugar concentration, and the response mode is milder, with less impact on the loss of cell metabolic activity.

[0030] (5) The sugar-responsive fiber material prepared by the present invention has good biocompatibility and helps the formation of biofilms, effectively improving the fermentation performance of immobilized cells in continuous catalytic processes. Description of the drawings

[0031] The following further describes the present invention in detail with reference to the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0032] Figure 1 It is the FTIR spectra of unmodified cotton fibers and modified cotton fibers.

[0033] Figure 2 It is the sugar response of sugar-responsive cotton fibers at different sugar concentrations.

[0034] Figure 3 It is the influence of cotton fiber co-deposition before and after on fermentation parameters.

[0035] Figure 4 It is the result graph of continuous multi-batch fermentation. Specific embodiments

[0036] The present invention can be better understood according to the following embodiments.

[0037] Example 1: The pretreatment and responsive modification of the fiber material were carried out as follows:

[0038] (1) Take commercially available cotton fibers, wash them 3 times with an appropriate amount of clear water, dry them in an oven at 55 °C, and cut them into square pieces of 2.5 cm * 2.5 cm.

[0039] (2) Prepare 1 L of 50 mM Tris-HCl solution, then adjust the pH to about 8.5 with dilute hydrochloric acid. Add 1 g each of 4-(2-aminoethyl)-1,2-benzenediol hydrochloride and polyaziridine with a relative molecular weight of 600, with final concentrations of 1 g / L respectively. Add 0.1 g of ascorbic acid as a reducing agent.

[0040] (3) Weigh 30 g of the cotton fibers prepared in step (1) and soak them in the solution prepared in step (2). Then add 1 g of 3-acrylamidophenylboronic acid to make its concentration 1 g / L, and modify at 30 °C and 120 rpm for 8 h. Rinse the modified cotton fibers with pure water 3 - 5 times and dry them in an oven at 55 °C. The obtained cotton fibers are sugar-responsive cotton fibers, denoted as APBA-1.

[0041] The results of the infrared spectrum are as Figure 1 shown. Spectrum a is the cotton fiber modified only with 4-(2-aminoethyl)-1,2-benzenediol hydrochloride, and spectrum b is the sugar-responsive cotton fiber. There is a vibration peak of C=O in 3-acrylamidophenylboronic acid at 1640 cm -1 There is a vibration peak of C-B at 1430 cm -1 There is a vibration peak of B-O at 1340 cm -1 All of these indicate that 3-acrylamidophenylboronic acid is successfully modified on the surface of the cotton fiber.

[0042] Example 2: Application of the sugar-responsive fiber material in the sugar response effect at different sugar concentrations:

[0043] (1) The microbial cells selected in the experiment are Saccharomyces cerevisiae CICC1308, and the seed medium is: glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L.

[0044] (2) The microbial cells selected in the experiment are Saccharomyces cerevisiae CICC1308, and the fermentation medium is: peptone 4 g / L, yeast extract 3 g / L, (NH4)2SO4 4 g / L, MgSO4 0.5 g / L, KH2PO4 3 g / L, ZnSO4·7H2O 0.05 g / L, FeSO4·7H2O 0.05 g / L, and different concentrations of glucose, which are: 0.1 g / L, 0.5 g / L, 1 g / L, 2 g / L, 5 g / L, 10 g / L.

[0045] (3) In the above several media with different sugar concentrations, test the response effect of the sugar-responsive material in the immobilized cells. Transfer the activated Saccharomyces cerevisiae CICC1308 seed liquid to the fermentation medium containing the sugar-responsive cotton fibers prepared in Example 1 at an inoculation amount of 10 vt%, so that the initial OD600 For immobilized culture at 1.0, fermentation was carried out at 35 °C and 200 rpm for 16 h. Keeping the inoculum size unchanged, samples were taken every 1 h to measure OD 600 The change was used to illustrate the response of the glucose-responsive cotton fiber prepared in Example 1 to the change in glucose concentration. The specific results are as Figure 2 shown.

[0046] (4) The immobilized cells with glucose-responsive cotton fiber showed glucose-responsive characteristics at a sugar concentration of 2 g / L. When the sugar concentration was 0.1 - 1 g / L, the OD of the fermentation broth 600 showed fluctuations, but the maximum value was always stable below 0.2. When the sugar concentration was 2 g / L, the fermentation OD 600 would increase slightly within the first 8 h, but as glucose was consumed, the OD 600 decreased below 0.5. When the sugar concentration was greater than 2 g / L, the OD of the fermentation broth 600 showed an upward trend from beginning to end until the sugar concentration decreased and the OD 600 tended to level off.

[0047] Example 3: Application of glucose-responsive fiber material in immobilized cell fermentation:

[0048] (1) The microbial cells selected in the experiment were Saccharomyces cerevisiae CICC1308. The seed medium was the same as that in Example 1, and the fermentation medium was: glucose 60 g / L, peptone 4 g / L, yeast extract 3 g / L, KH2PO4 3 g / L, (NH4)2SO4 4 g / L, MgSO4 0.5 g / L, ZnSO4·7H2O 0.05 g / L, FeSO4·7H2O 0.05 g / L.

[0049] (2) Free cell fermentation: The activated Saccharomyces cerevisiae CICC1308 seed solution was transferred to the fermentation medium at an inoculum size of 10 vt%, and free cell fermentation was carried out at 35 °C and 200 rpm. During the free cell fermentation process, the fermentation cycle was 12 h, the consumption rate of glucose was 5.0 g / L / h, the ethanol yield was 19.69 g / L, and the ethanol productivity was 1.64 g / L / h.

[0050] (3) Immobilized cell fermentation: The activated Saccharomyces cerevisiae CICC1308 seed solution was transferred to the fermentation medium containing the glucose-responsive cotton fiber prepared in Example 1 at an inoculum size of 10 vt% for immobilized fermentation. The addition amount of the glucose-responsive cotton fiber was 50 g / L, and immobilized cell fermentation was carried out at 35 °C and 200 rpm. At 8 h of fermentation, the OD of the fermentation broth 600The maximum value reached is 3.250. As the glucose in the fermentation broth is gradually consumed, Saccharomyces cerevisiae cells form borate ester bonds with the boric acid groups on the surface of the sugar-responsive cotton fibers, and the cells gradually adhere to the surface of the cotton fibers. At 12 h, the OD600 drops to 1.003. When the fermentation time is further extended to 24 h, the OD 600 can drop to 0.121. The specific results are as Figure 3 shown.

[0051] (4) Multi-batch continuous fermentation: During multi-batch continuous fermentation, for free cells, 10 vt% of the fermentation broth from the previous batch is left as the seed broth for the next batch. For immobilized fermentation, only the immobilized carrier needs to be left in the fermentation flask, all the fermentation broth is poured out, and then new fermentation broth is added for the second batch of fermentation. After the glucose is consumed, the fermentation broth is replaced and the third batch of fermentation is carried out according to the fermentation steps of the second batch, and so on for 7 consecutive batches of fermentation. Fermentation is considered to be over when the glucose concentration in the fermentation medium is lower than 1 g / L. The corresponding results of multi-batch fermentation are as Figure 4 shown.

[0052] During multi-batch fermentation, the cycle of continuous fermentation of free cells is shortened from 12 h to 9 h, and the glucose consumption rate is increased from 5 g / L / h to 6.67 g / L / h. However, the ethanol production is 15.65 g / L in the seventh batch, which is significantly lower than that of immobilized cell fermentation. The fermentation cycle of Saccharomyces cerevisiae immobilized on sugar-responsive cotton fibers (APBA-1) is shortened from 12 h to 4 h, and the glucose consumption rate is increased from 5 g / L / h to 15 g / L / h. Its ethanol production is 24.99 g / L in the seventh batch.

[0053] Comparative Example 1:

[0054] The preparation method is the same as that of Example 1, except that 3-acrylamidophenylboronic acid is not added to the co-deposition system, and the cotton fibers modified only with 4-(2-aminoethyl)-1,2-benzenediol hydrochloride are obtained, denoted as PDA-CF.

[0055] Comparative Example 2:

[0056] The preparation method is the same as that of Example 1, except that the concentration of 3-acrylamidophenylboronic acid in the co-deposition system is 0.5 g / L, that is, the ratio of 3-acrylamidophenylboronic acid to 4-(2-aminoethyl)-1,2-benzenediol hydrochloride is 1:2. The obtained sugar-responsive cotton fibers are denoted as APBA-0.5.

[0057] Both Comparative Example 1 and Comparative Example 2 were carried out for single-batch and continuous fermentation of immobilized cells according to the method in Example 3. In the single-batch fermentation of PDA-CF immobilized cells, at 12 h, the OD of the fermentation broth 600The maximum value reached 7.749, and the immobilization effect of the carrier on the Saccharomyces cerevisiae cells was poor. When the fermentation time was further extended to 24 h, the OD 600 In a single batch fermentation of APBA-0.5 immobilized cells, the OD value of the fermentation liquid at 8 h was 600 The maximum value reached 3.879. When the fermentation time was further extended to 24h, the OD 600 The results show that the addition of 3-acrylamidophenylboronic acid will affect the immobilization efficiency and recovery efficiency of sugar-responsive cotton fiber for Saccharomyces cerevisiae cells. In the multi-batch fermentation process, for PDA-CF immobilized Saccharomyces cerevisiae fermentation, the fermentation cycle was shortened from 12h to 6h, the glucose consumption rate was increased from 5g / L / h to 10g / L / h, and the ethanol yield was 21.36g / L in the seventh batch, which was 14.5% lower than the ethanol yield of the seventh batch of APBA-1 immobilized fermentation. For APBA-0.5 immobilized Saccharomyces cerevisiae fermentation, the fermentation cycle was shortened from 12h to 4h, the glucose consumption rate was increased from 5g / L / h to 15g / L / h, and the ethanol yield was 22.38g / L in the seventh batch, second only to APBA-1 immobilized fermentation. This further shows that the addition of 3-acrylamidophenylboronic acid will affect the application effect of sugar-responsive cotton fiber in immobilized fermentation.

[0058] The present invention provides a method for preparing and applying a sugar-responsive cell adhesion material. Numerous methods and approaches exist for implementing this technical solution. The foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are considered within the scope of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A method for preparing a sugar-responsive cell adhesion material, characterized in that, A fibrous material is placed in a co-deposition system containing a cross-linking agent, a binder, a reducing agent, and a compound containing a phenylboronic acid group to react to obtain a sugar-responsive fibrous material; The cross-linking agent is 4-(2-aminoethyl)-1,2-benzenediol hydrochloride.

2. The preparation method of the sugar-responsive cell adhesion material according to claim 1, characterized in that, The compound containing a phenylboronic acid group is selected from at least one of 2-acrylamidophenylboronic acid, 3-acrylamidophenylboronic acid, 2-aminophenylboronic acid hydrochloride, 3-aminophenylboronic acid, phenyl-1,4-diboronic acid, and 4-vinylphenylboronic acid.

3. The preparation method of the sugar-responsive cell adhesion material according to claim 1, characterized in that The binder is selected from at least one of hydroxytyrosol, povidone, hypromellose, polyaziridine, and sodium carboxymethylcellulose; The reducing agent is selected from at least one of sodium borohydride, glucose, sodium bisulfite, and sodium citrate.

4. The preparation method of the sugar-responsive cell adhesion material according to claim 1, wherein, The fibrous material is selected from at least one of cotton, linen, nylon, polyester, polypropylene fiber, and polyethylene fiber.

5. The preparation method of the sugar-responsive cell adhesion material according to claim 1, characterized in that, The mass ratio of the cross-linking agent, the binder, the reducing agent, and the compound containing a boronic acid group is 1:(0.1-20):(0.01-10):(0.2-10).

6. The preparation method of the sugar-responsive cell adhesion material according to claim 1, characterized in that, The addition amount of the fibrous material in the co-deposition system is 10-50 g / L; the reaction time of the fibrous 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.

7. The sugar-responsive cell adhesion material prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the sugar-responsive cell adhesion material according to claim 7 as a cell carrier in immobilized cell fermentation.

9. The application according to claim 8, wherein The dosage of the sugar-responsive cell adhesion material is 10-100 g / L of the fermentation medium.

10. The application according to claim 8, wherein When the sugar concentration of the sugar-responsive cell adhesion material is between 50 and 100 g / L, the desorption of the immobilized cells on the carrier can be realized; when the sugar concentration of the sugar-responsive cell adhesion material is between 0.05 and 10 g / L, the adsorption of the immobilized cells on the carrier can be realized.

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