CN113174387A - Hydrogel immobilized microorganism preservation method - Google Patents

Hydrogel immobilized microorganism preservation method Download PDF

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CN113174387A
CN113174387A CN202110522702.6A CN202110522702A CN113174387A CN 113174387 A CN113174387 A CN 113174387A CN 202110522702 A CN202110522702 A CN 202110522702A CN 113174387 A CN113174387 A CN 113174387A
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microorganism
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CN113174387B (en
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陈庆国
刘雨薇
刘梅
汪涛
竺柏康
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Zhejiang Ocean University ZJOU
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Abstract

The invention discloses a hydrogel immobilized microorganism preservation method, which relates to the field of immobilized biotechnology and comprises the following steps: the microorganism is fixed in the hydrogel immobilized carrier by an embedding method and stored and preserved at normal temperature. Wherein, the carrier is a composite gel ball, comprising: embedding filler, including N- (1, 8-dimethyl imidazo [1,2-a ] quinoxaline-4-yl) -1, 2-ethylenediamine modified chitosan microspheres; the framework material, including sodium alginate, forms an interpenetrating or semi-interpenetrating network structure with the embedding filler. The microorganism preservation method provided by the invention takes hydrogel as a carrier, and the hydrogel is embedded in the carrier through an immobilization technology, so that the long-term storage and transportation at normal temperature can be realized; the prepared composite gel ball carrier has good compressive strength, high porosity and good stability, and is more beneficial to microorganism attachment.

Description

Hydrogel immobilized microorganism preservation method
Technical Field
The invention belongs to the technical field of immobilized biology, and particularly relates to a hydrogel immobilized microorganism preservation method.
Background
The strain is an important biological resource of China and also a basic material for production, teaching and scientific research. In recent years, more and more microbial strains are researched and applied to various fields of food, medicine, agriculture and the like, the strains serve as production sources, and the quality of the strains is directly related to the yield and the quality of products. In the field of microorganisms, both basic research works and application research of biotechnology, correct strain preservation methods and techniques are required to ensure the quality and the activity of strains. China starts late in the field of research of strain preservation technology, has no systematic operation rules, and mainly has the following problems: the contrast test frequency is limited, the test types are few, the research technical means needs to be further updated, and the control research on test factors (such as the age of the strain, the size of inoculated mycelium blocks, the type of a culture medium, the speed of cooling, freezing and heating recovery, detection indexes, the type of an anti-freezing protective agent and the like) is insufficient.
The currently commonly used strain preservation methods are a liquid nitrogen ultralow temperature preservation method, a vacuum freeze drying preservation method, a periodic transplantation method, a mineral oil preservation method and the like. Partial research shows that the first two methods have good preservation effect and are suitable for long-term preservation of strains. However, there are still problems, such as even if the cordyceps militaris of the same species, the similar method is used by different researchers, and the conclusion is different. Meanwhile, the liquid nitrogen ultra-low temperature preservation method and the vacuum freeze-drying preservation method need good equipment and technology, have too high investment and can not be widely applied to production; the latter two methods are simple and cheap, but occupy a large storage space, and because the tube transfer times are too many, the possibility of mutation is increased, strain degeneration is easily caused, and loss is brought to production. Therefore, it is necessary to develop a culture preservation method which is efficient, inexpensive and easy to operate, thereby obtaining more scientific and practical results.
Disclosure of Invention
The invention aims to provide a hydrogel immobilized microorganism preservation method, which takes hydrogel as a carrier and is embedded in the carrier through an immobilization technology, so that the long-term storage and transportation at normal temperature can be realized; the prepared composite gel ball carrier has good compressive strength, high porosity and good stability, and is more beneficial to microorganism attachment.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a composite gel pellet comprising:
embedding filler, including N- (1, 8-dimethyl imidazo [1,2-a ] quinoxaline-4-yl) -1, 2-ethylenediamine modified chitosan microspheres;
the framework material, including sodium alginate, forms an interpenetrating or semi-interpenetrating network structure with the embedding filler. The composite gel microspheres provided by the invention are spherical particles, have high porosity, enlarged specific surface area and no cytotoxicity; the network pores of the internal structure are favorable for the attachment of microorganisms; the molecular structures of the raw materials form an interpenetrating or semi-interpenetrating network structure through chemical or physical interaction, and the swelling behavior is controllable, so that the obtained microbial carrier material has good stability; the existence of N- (1, 8-dimethyl imidazo [1,2-a ] quinoxaline-4-yl) -1, 2-ethylenediamine can effectively improve the swelling degree of the composite gel sphere; meanwhile, in the presence of a large number of active groups, the microbial carrier material gives consideration to the stability of a microbial immobilized carrier to a certain extent and improves the diffusion behavior of microbes. The prepared composite gel ball has excellent mechanical property, and the compressive strength is obviously improved.
The embedding filler is prepared by preparing an active intermediate through a water/oil inverse emulsion crosslinking reaction and then carrying out chemical surface modification on N- (1, 8-dimethylimidazo [1,2-a ] quinoxaline-4-yl) -1, 2-ethylenediamine.
The embedding filler and the framework material form gel spheres through a crosslinking reaction under the action of a catalyst.
Further, the preparation method of the composite gel ball comprises the following steps:
s1: adding chitosan powder into an acetic acid aqueous solution (the concentration is 1.8-2.5%, w/w), and stirring until the chitosan powder is completely dissolved; CaCO is added under magnetic stirring3Stirring for 10-15 min, and then carrying out ultrasonic treatment for 15-20 min; then adding liquid paraffin, and violently stirring for 3-5 min; dripping span-80 under the water bath condition of 40-45 ℃, and stirring for 25-30 min; keeping the water bath stirring state, slowly dropwise adding a glutaraldehyde solution (the concentration is 23-27%, w/w), and carrying out a crosslinking reaction for 60-70 min; finally adding N- (1, 8-dimethyl imidazo [1, 2-a)]Stirring and reacting quinoxaline-4-yl) -1, 2-ethylenediamine for 65-70 min, adding a 5M NaOH solution to adjust the pH value to 10.0-10.5, adjusting the reaction temperature to 70-75 ℃, and continuing stirring and reacting for 100-110 min; sequentially rinsing the reaction product with petroleum ether, absolute ethyl alcohol and deionized water for multiple times, and drying at 50 ℃ to obtain an intermediate product M;
s2: taking the intermediate product M, rinsing with 0.1M HCl solution to remove CaCO in the gel spheres3Washing the particles with deionized water, then fully mixing the particles with sodium alginate in the deionized water, and continuously stirring for 22-24 hours to obtain a mixed solution A; under the condition of stirring, dripping the mixed solution A into a calcium chloride solution (with the concentration of 4-5%, w/v) by using an injector to form ion-crosslinked gel balls, and further hardening for 22-24 hours; and then, filtering and collecting the reacted gel spheres, washing the gel spheres with distilled water to remove redundant calcium ions on the surfaces of the gel spheres, sucking the redundant water on the surfaces, and freeze-drying for 20-24 hours to obtain the composite gel spheres.
CaCO in step S13The mass ratio of the chitosan powder to the chitosan powder is 0.75-0.82: 1; the solid-to-liquid ratio of the chitosan powder to the paraffin is 1 g: 150-160 mL; the solid-to-liquid ratio of chitosan powder to span-80 is 1 g: 1.8-2.2 mL; the solid-to-liquid ratio of the chitosan powder to the glutaraldehyde solution is 1 g: 2-2.3 mL; n- (1, 8-dimethylimidazo [1, 2-a)]The mass ratio of the quinoxaline-4-yl) -1, 2-ethylenediamine to the chitosan powder is 1.76-2.03: 1.
in step S2, the mass ratio of the intermediate M to sodium alginate is 1: 0.8 to 1.2; the solid-liquid ratio of the sodium alginate to the calcium chloride solution is 0.1 g: 18-24 mL.
Furthermore, in step S2, N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidineacetamide is added, and the mass ratio of the N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidineacetamide to the sodium alginate is 0.4-0.7: 1. adding N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidine acetamide, and crosslinking with other components to obtain composite gel spheres, which can effectively improve the compressive strength and mechanical properties of the gel spheres; and can obviously enhance the thermal stability of the gel ball; after the prepared composite gel balls are loaded with microorganisms, the activity of the microorganisms can be effectively maintained for a long time, and the storage period is further prolonged. Meanwhile, under the condition that N- (1, 8-dimethyl imidazo [1,2-a ] quinoxaline-4-yl) -1, 2-ethylenediamine modified chitosan microspheres exist at the same time, the gel bead compressive strength and the long-term maintenance of microbial activity are enhanced better.
It is still another object of the present invention to provide the use of N- (1, 8-dimethylimidazo [1,2-a ] quinoxalin-4-yl) -1, 2-ethylenediamine to enhance the porosity and compressive strength of gel beads.
The invention also aims to provide the application of the composite gel spheres in preparing microorganism immobilized carriers.
A hydrogel immobilized microorganism preservation method comprises fixing microorganism in hydrogel immobilized carrier by embedding method, and storing at room temperature; the hydrogel immobilization carrier comprises the composite gel ball. The invention takes the composite gel ball as the immobilized carrier to fix the microorganism in the hydrogel, and the prepared hydrogel containing the microorganism can be stored at normal temperature. When in use, the hydrogel containing the microorganisms is put into water, can be rapidly degraded to release the microorganisms, and can also be rapidly revived and proliferated. The composite gel ball provided by the invention is spherical particles, has high porosity and enlarged specific surface area; network pores formed by the internal structure of the biological filter are beneficial to the attachment of microorganisms; the configuration of the cell components can be stabilized by the affinity of hydrogen bonds or ionic bonds to water and cells to prevent or reduce the damage of the surrounding environment to the cells, so that the gel-ball-conforming carrier can maintain the microbial activity for a long time, and the preservation period is greatly prolonged. The strain preservation method is simple and convenient to operate, convenient to transport, long in microbial life, capable of being stored at normal temperature, and very suitable for daily preservation and transport of bacteria, fungi and other microorganisms separated in a clinical microbial strain laboratory.
Furthermore, the microorganism preservation method specifically comprises the following steps:
in the process of preparing the composite gel ball, mixing the microorganism strain concentrated solution with the mixed solution A, dripping the mixed solution A into a calcium chloride solution (with the concentration of 4-5%, w/v) by using a syringe under the condition of stirring to form an ion-crosslinked gel ball, and further hardening for 22-24 hours; and then, filtering and collecting the reacted gel spheres, washing the gel spheres with distilled water to remove redundant calcium ions on the surfaces of the gel spheres, absorbing the redundant water on the surfaces, and freeze-drying for 20-24 hours to obtain the microorganism-loaded composite gel spheres.
It is noted that the microorganism is one or more of an aerobic microorganism, a facultative microorganism, or a microorganism that can hibernate for more than 2 hours or can survive for more than 2 hours under anaerobic conditions.
The concentration of the microorganism strain concentrate is 1.2-2.6 × 104mg/L; the adding amount of the microbial strain concentrated solution is 0.8-10% of the mass of the mixed solution A.
The composite gel ball is used for prolonging the preservation time of microorganisms.
Compared with the prior art, the invention has the following beneficial effects:
the invention takes the composite gel ball as the immobilized carrier to fix the microorganism in the hydrogel, and the prepared hydrogel containing the microorganism can be stored at normal temperature. The composite gel ball provided by the invention is spherical particles, has high porosity, enlarged specific surface area and higher swelling degree; the prepared composite gel ball has excellent mechanical property, and the compressive strength is obviously improved. Meanwhile, the network pores formed by the internal structure of the gel-compliant carrier are beneficial to the attachment of microorganisms, and can effectively prevent or reduce the damage of the surrounding environment to cells, so that the gel-compliant carrier can maintain the activity of the microorganisms for a long time, and the storage period is greatly prolonged. In addition, the addition of N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidine acetamide in the preparation process of the gel sphere carrier can effectively improve the compressive strength of the gel sphere, improve the mechanical property of the gel sphere and obviously enhance the thermal stability of the gel sphere; after the microorganism is loaded, the microorganism activity can be effectively maintained for a long time, and the preservation period is further prolonged; under the condition that N- (1, 8-dimethyl imidazo [1,2-a ] quinoxaline-4-yl) -1, 2-ethylenediamine modified chitosan microspheres coexist, the gel sphere compressive strength and microbial activity maintaining time duration enhancement effect is better. The strain preservation method is simple and convenient to operate, convenient to transport, long in microbial life, capable of being stored at normal temperature, and very suitable for daily preservation and transport of bacteria, fungi and other microorganisms separated in a clinical microbial strain laboratory.
Therefore, the invention provides a hydrogel immobilized microorganism preservation method, which takes hydrogel as a carrier and is embedded in the carrier through an immobilization technology, so that the long-time storage and transportation at normal temperature can be realized; the prepared composite gel ball carrier has good compressive strength, high porosity and good stability, and is more beneficial to microorganism attachment.
Drawings
FIG. 1 is a SEM test result of intermediate M obtained in comparative example 1 of Experimental example 1 of the present invention;
FIG. 2 shows SEM test results of intermediate M obtained in example 1 of Experimental example 1 of the present invention;
FIG. 3 shows results of the thermogravimetric analysis test in test example 1 of the present invention.
Detailed Description
The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings:
example 1:
preparation of a composite gel ball:
s1: adding chitosan powder into acetic acid aqueous solution (concentration is 2.1%, w/w), and stirring until completely dissolving; CaCO is added under magnetic stirring3(the mass ratio of chitosan powder is 0.79: 1), stirring for 12min, and then carrying out ultrasonic treatment for 15 min; followed by the addition of liquid paraffin (chitosan powder fixed with paraffin)The liquid ratio is 1 g: 156mL), stirring vigorously for 5 min; dripping span-80 (the solid-to-liquid ratio of chitosan powder to span-80 is 1 g: 2.1mL) in water bath at 43 ℃, and stirring for 30 min; keeping the water bath stirring state, slowly dropwise adding a glutaraldehyde solution (the concentration is 24.5%, w/w) (the solid-to-liquid ratio of the chitosan powder to the glutaraldehyde solution is 1 g: 2.2mL), and carrying out crosslinking reaction for 65 min; finally adding N- (1, 8-dimethyl imidazo [1, 2-a)]Quinoxaline-4-yl) -1, 2-ethylenediamine (mass ratio of chitosan powder to quinoxaline-4-yl) -1, 2-ethylenediamine (1.87: 1) after stirring and reacting for 70min, adding 5M NaOH solution to adjust the pH value to 10.2, adjusting the reaction temperature to 74 ℃, and continuing stirring and reacting for 110 min; sequentially rinsing the reaction product with petroleum ether, absolute ethyl alcohol and deionized water for multiple times, and drying at 50 ℃ to obtain an intermediate product M;
s2: taking the intermediate product M, rinsing with 0.1M HCl solution to remove CaCO in the gel spheres3Washing the particles with deionized water, then fully mixing the particles with sodium alginate (the mass ratio of the two is 1: 0.96) in the deionized water, and continuously stirring for 22 hours to obtain a mixed solution A; dropping the mixed solution A into a calcium chloride solution (with the concentration of 4.3 percent and the w/v) (the solid-to-liquid ratio of sodium alginate to the calcium chloride solution is 0.1 g: 21.3mL) by using a syringe to form ion-crosslinked gel spheres under the stirring condition, and further hardening for 24 hours; and then, filtering and collecting the reacted gel spheres, washing the gel spheres with distilled water to remove redundant calcium ions on the surfaces of the gel spheres, sucking the redundant water on the surfaces, and freeze-drying for 24 hours to obtain the composite gel spheres.
Example 2:
a composite gel pellet was prepared as in example 1 except that:
CaCO in step S13The mass ratio of the chitosan powder to the chitosan powder is 0.76: 1; the solid-to-liquid ratio of the chitosan powder to the glutaraldehyde solution is 1 g: 2 mL; n- (1, 8-dimethylimidazo [1, 2-a)]The mass ratio of the quinoxaline-4-yl) -1, 2-ethylenediamine to the chitosan powder is 1.80: 1;
the mass ratio of the intermediate product M to the sodium alginate in the step S2 is 1: 0.8 to 1.2; the solid-liquid ratio of the sodium alginate to the calcium chloride solution is 0.1 g: 18.4 mL.
Example 3:
a composite gel pellet was prepared as in example 1 except that:
CaCO in step S13The mass ratio of the chitosan powder to the chitosan powder is 0.81: 1; the solid-to-liquid ratio of the chitosan powder to the glutaraldehyde solution is 1 g: 2.3 mL; n- (1, 8-dimethylimidazo [1, 2-a)]The mass ratio of the quinoxaline-4-yl) -1, 2-ethylenediamine to the chitosan powder is 2.01: 1;
the mass ratio of the intermediate product M to the sodium alginate in the step S2 is 1: 1.16; the solid-liquid ratio of the sodium alginate to the calcium chloride solution is 0.1 g: 23.6 mL.
Example 4:
a composite gel pellet was prepared as in example 1 except that:
step S2, adding N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidine acetamide, wherein the mass ratio of N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidine acetamide to sodium alginate is 0.54: 1.
example 5:
a composite gel pellet was prepared as in example 4 except that:
in step S1, N- (1, 8-dimethylimidazo [1,2-a ] quinoxalin-4-yl) -1, 2-ethylenediamine is not added.
Example 6:
a method for hydrogel-immobilized microbial preservation, comprising: and (3) screening, purifying and enriching microbial strains from the raw materials, adding the microbial strains into the process of preparing the composite gel spheres in the embodiment 1 in the logarithmic phase of growth to obtain the composite gel spheres loaded with the microorganisms, and storing at normal temperature. Wherein the concentration of the microorganism strain concentrated solution is 2.1 × 104mg/L; the adding amount of the microbial strain concentrated solution is 7.8 percent of the mass of the mixed solution A.
Example 7:
a method for preserving hydrogel-immobilized microorganisms, which is different from that of example 6, is: composite gel spheres were prepared as in example 4.
Example 8:
a method for preserving hydrogel-immobilized microorganisms, which is different from that of example 6, is: composite gel spheres were prepared as in example 5.
Comparative example 1:
a composite gel pellet was prepared as in example 1 except that:
in step S1, N- (1, 8-dimethylimidazo [1,2-a ] quinoxalin-4-yl) -1, 2-ethylenediamine is not added.
Comparative example 2:
a method for preserving hydrogel-immobilized microorganisms, which is different from that of example 6, is: the composite gel beads were prepared as in comparative example 1.
Test example 1:
1. SEM test
Drying the sample at normal temperature, carrying out gold spraying treatment, and placing the sample under a field emission scanning electron microscope to observe the surface appearance of the sample.
The results of the above tests on intermediate M prepared in comparative example 1 and example 1 are shown in FIGS. 1-2. As can be seen from the analysis in the figure, the surface of the intermediate M prepared in example 1 is rougher compared with that of comparative example 1, and the success of surface modification of the chitosan microspheres by N- (1, 8-dimethylimidazo [1,2-a ] quinoxalin-4-yl) -1, 2-ethylenediamine is laterally demonstrated.
2. Thermogravimetric analysis (TGA)
Freeze-drying the gel ball sample, and performing thermal analysis on the sample by using STA 7300 thermal analyzer in N2The temperature rise rate of 10 ℃/min is within the range of 0-800 ℃, and the thermal stability of the sample is tested.
The above tests were performed on the gel samples prepared in comparative example 1, example 1 and example 4, and the results are shown in fig. 3. From the analysis in the figure, the thermal weight loss of the composite gel ball prepared by the invention is mainly divided into three stages. Compared with comparative example 1, the decomposition temperature of each stage of the composite gel ball prepared in example 1 is not obviously different from that of the composite gel ball prepared in example 4, the decomposition temperature of each stage of the sample prepared in example 1 is higher, and the weight loss amount of the sample prepared in example 4 is less than that of example 1, which shows that the thermal stability of the gel ball can be effectively improved by adding N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidine acetamide and crosslinking the N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidine acetamide with other components to prepare the composite gel ball.
3. Test for compressive Strength
And placing the gel ball sample between two glass slides, vertically extruding the gel ball by using a portable pressure gauge, and according to the P ═ F/S, wherein F is the pressure recorded by the system when the gel ball is broken, and S is the contact area of the pressure sensor.
The results of the above tests on the gel beads prepared in comparative example 1 and examples 1 to 5 are shown in Table 1:
TABLE 1 compressive Strength test results
Figure BDA0003064592000000061
Figure BDA0003064592000000071
As can be seen from the analysis in Table 1, the compressive strength of the gel beads prepared in example 1 is significantly higher than that of comparative example 1, which shows that the compressive strength of the gel beads can be effectively enhanced by performing surface modification on chitosan microspheres by using N- (1, 8-dimethylimidazo [1,2-a ] quinoxalin-4-yl) -1, 2-ethylenediamine and performing crosslinking with sodium alginate to prepare the composite gel beads. The compressive strength of the composite gel ball prepared in example 5 is higher than that of comparative example 1, which shows that the compressive strength of the gel ball can be remarkably improved and the mechanical properties of the gel ball can be improved by adding N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidine acetamide and crosslinking the N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidine acetamide with other components. In addition, example 4 is better than examples 1 and 5, showing that the enhancement effect of the compressive strength of the gel beads is better in the presence of N- (1, 8-dimethylimidazo [1,2-a ] quinoxalin-4-yl) -1, 2-ethylenediamine modified chitosan microspheres and N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidineacetamide.
4. Hydrogel swelling test
Weighing the freeze-dried gel balls with the mass of (W)d) Soaking and swelling at 20 deg.C under neutral pH condition, taking out at interval of 60 hr, and drying surface water with filter paper to obtain a mass WtAnd measuring for many times until the swelling balance is reached, and calculating the swelling degree SR according to the following formula:
SR＝(Wt－Wd)/Wd×100％
in the formula, WdInitial dry gel mass, g; wtThe mass of sol gel at time t, g; SR is the swelling degree,%.
The results of the above tests on the gel beads prepared in comparative example 1 and examples 1 to 5 are shown in Table 2:
TABLE 2 swelling characteristics test results
Sample (I) Degree of swelling (%)
Comparative example 1 392
Example 1 495
Example 2 501
Example 3 490
Example 4 505
Example 5 403
As can be seen from the analysis in Table 2, the swelling degree of the gel beads prepared in example 1 is significantly higher than that of comparative example 1, which shows that the swelling capacity of the gel beads can be effectively improved by performing surface modification on chitosan microspheres by using N- (1, 8-dimethylimidazo [1,2-a ] quinoxalin-4-yl) -1, 2-ethylenediamine, and then performing crosslinking with sodium alginate to prepare the composite gel beads. The swelling degree of the composite gel ball prepared in example 4 is equivalent to that of example 1, and the effect of example 5 is equivalent to that of comparative example 1, which shows that the swelling performance of the gel ball is not negatively influenced by the addition of N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidine acetamide and cross-linking with other components.
5. Porosity testing
Measuring porosity of the dried gel ball by using a solvent displacement method, taking absolute ethyl alcohol as a medium, and weighing dry weight M of the hydrogel after freeze drying0(ii) a Measuring the height and diameter of the gel ball, and calculating the volume of the gel ball as V; after the sample is swelled and balanced by absolute ethyl alcohol, absorbing the liquid on the surface of the gel ball by using filter paper, and measuring the weight M1(ii) a The density of the absolute ethyl alcohol is rho, and the porosity P of the gel ball is calculated according to the following formular：
Pr＝(M1－M0)/(ρV)×100％
The results of the above tests on the gel beads prepared in comparative example 1 and examples 1 to 5 are shown in Table 3:
table 3 porosity test results
Sample (I) Porosity (%)
Comparative example 1 62.9
Example 1 76.2
Example 2 74.5
Example 3 75.7
Example 4 78.1
Example 5 64.3
As can be seen from the analysis in Table 3, the porosity of the gel beads prepared in example 1 is significantly higher than that of comparative example 1, which shows that the porosity of the gel beads can be effectively increased by performing surface modification on the chitosan microspheres by using N- (1, 8-dimethylimidazo [1,2-a ] quinoxalin-4-yl) -1, 2-ethylenediamine, and then performing crosslinking with sodium alginate to prepare the composite gel beads. Example 4 the porosity of the prepared composite gel sphere is equivalent to that of example 1, and the effect of example 5 is equivalent to that of comparative example 1, indicating that the addition of N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidineacetamide, which is cross-linked with other components, has no negative influence on the porosity of the gel sphere.
Test example 2:
characterization of microorganism-loaded composite gel sphere carrier
The experimental microorganism is nitrobacteria
1. Microbiological activity assay
Selecting free nitrobacteria concentrated solution with the same bacterial quantity and composite gel balls embedding nitrobacteria at room temperature of 25 ℃, respectively placing the free nitrobacteria concentrated solution and the nitrobacteria embedded composite gel balls into an oxygen dissolving bottle, adding culture solution containing ammonia nitrogen (water distribution composition and proportion are shown in table 4) into the oxygen dissolving bottle, controlling the water bath temperature to be 25-35 ℃, pre-aerating for 10min to enable the dissolved oxygen in the oxygen dissolving bottle to be saturated, inserting an oxygen dissolving instrument probe, rapidly adding water for sealing, slowly stirring by using a magnetic stirrer, measuring the concentration of the dissolved oxygen in the oxygen dissolving bottle at regular intervals, and mapping the obtained dissolved oxygen data and corresponding time so as to calculate the respiratory activity.
Figure BDA0003064592000000091
In the formula, Rr is the respiratory activity of free or immobilized nitrifying bacteria, mg-O2L.min; the dissolved oxygen concentration in the dissolved oxygen bottle at DO-t moment is mg/L; t-test time, min; k-slope of dissolved oxygen concentration versus time.
Rrr＝Rri/Rrf×100％
In the formula, Rrr-relative respiratory activity of the entrapped nitrobacteria,%; rriRespiratory activity of entrapping immobilized Nitrobacter mg-O2/L·min；RrfRespiratory activity of free nitrifying bacteria, mg-O2/L·min。
TABLE 4 nitrobacteria culture fluid composition (NH)4 +-N：40mg/L)
Components Concentration (mg/L)
NH4Cl 146
NaHCO3 457
Na2HPO4·12H2O 45.1
NaCl 21.6
KCl 9.4
CaCl2·2H2O 9.3
MgSO4·7H2O 32.5
The results of the above tests on the microorganism-loaded composite gel beads prepared in comparative example 2 and examples 6 to 8 are shown in table 5:
TABLE 5 respiratory Activity test results
Sample (I) Respiratory activity (%)
Comparative example 2 88.4
Example 6 93.4
Example 7 94.7
Example 8 89.3
As can be seen from the analysis in Table 5, the respiratory activity of the nitrifying bacteria in the composite gel beads loaded with microorganisms prepared in example 6 is significantly higher than that of comparative example 2, which shows that the cytotoxicity of the gel beads can be effectively reduced by performing surface modification on chitosan microspheres by using N- (1, 8-dimethylimidazo [1,2-a ] quinoxalin-4-yl) -1, 2-ethylenediamine and then performing cross-linking with sodium alginate. The porosity of the composite gel sphere prepared in example 7 is equivalent to that of example 6, and the effect of example 8 is equivalent to that of comparative example 2, indicating that the addition of N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidineacetamide, which is cross-linked with other components, does not negatively affect the cytotoxicity of the gel sphere.
2. Restoration of activity of embedded nitrobacteria
Storing the prepared composite gel ball loaded with the microorganisms for 6 months at normal temperature, adding the gel ball into water for swelling, and collecting a strain concentrated solution; an intermittent domestication mode is adopted to restore activity, SBR is adopted during intermittent domestication, carrier particles are added according to the volume filling rate of a reactor, dissolved oxygen is kept at 4mg/L, the temperature is 25-30 ℃, different ammonia nitrogen initial concentrations are adopted during domestication, the formula of a culture solution is shown in table 4, when the concentration is increased, all medicines are increased according to the proportion, and acid or alkali is not added in the domestication process to adjust the pH value of the system. The NH content of the reactor was measured at the same time interval after the start of the reaction4 +-N、NO2 －-N、NO3 －Concentration of-N in NH4 +N is reduced to 10% of the initial concentration, namely an acclimatization period, and after six acclimatization periods, the respiratory activity of the nitrifying bacteria is measured.
The results of the above tests on the microorganism-loaded composite gel beads prepared in comparative example 2 and examples 6 to 8 are shown in table 6:
TABLE 6 respiratory Activity test results
Sample (I) Respiratory activity (%)
Comparative example 2 66.8
Example 6 84.1
Example 7 93.5
Example 8 81.2
As can be seen from the analysis in Table 6, after one month of storage, the respiratory activity of the nitrifying bacteria in the composite gel beads loaded with the microorganisms prepared in example 6 is obviously higher than that of comparative example 2, the respiratory activity reduction rate is 9.96% and is higher than that of comparative example 2 by 24.43%, which shows that the surface modification of the chitosan microspheres by using N- (1, 8-dimethylimidazo [1,2-a ] quinoxalin-4-yl) -1, 2-ethylenediamine, the crosslinking with sodium alginate is performed to prepare the composite gel beads, and the composite gel beads are stored at normal temperature after the microorganisms are loaded, so that the microorganisms can be rapidly reactivated and proliferated, and the storage time is effectively prolonged. The reduction rate of the respiratory activity of nitrobacteria in the composite gel sphere loaded with the microorganisms prepared in the example 8 is 9.07 percent and is obviously lower than that of the comparative example 2, which shows that the storage effect of the gel sphere is enhanced by adding the N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidine acetamide and crosslinking the N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidine acetamide with other components. The reduction rate of the respiratory activity of the nitrobacteria in the composite gel sphere loaded with the microorganism prepared in example 7 is 1.27%, and almost no significant reduction exists, which shows that the storage effect of the gel sphere is better enhanced under the condition that N- (1, 8-dimethylimidazo [1,2-a ] quinoxaline-4-yl) -1, 2-ethylenediamine modified chitosan microspheres and N- (2-amino-2-oxoethyl) -4-hydroxy-2-oxo-1-pyrrolidine acetamide coexist.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims (8)

1. A composite gel pellet comprising:
embedding filler, including N- (1, 8-dimethyl imidazo [1,2-a ] quinoxaline-4-yl) -1, 2-ethylenediamine modified chitosan microspheres;
the framework material, including sodium alginate, forms an interpenetrating or semi-interpenetrating network structure with the embedding filler.
2. The composite gel pellet as claimed in claim 1, wherein: the embedding filler is prepared by preparing an active intermediate through water/oil inverse emulsion crosslinking reaction and then carrying out chemical surface modification by adopting N- (1, 8-dimethylimidazo [1,2-a ] quinoxaline-4-yl) -1, 2-ethylenediamine.
3. The composite gel pellet as claimed in claim 1, wherein: the embedding filler and the framework material form gel spheres through a cross-linking reaction under the action of a catalyst.
4. Use of N- (1, 8-dimethylimidazo [1,2-a ] quinoxalin-4-yl) -1, 2-ethylenediamine according to claim 1 for increasing the porosity and compressive strength of gel beads.
5. Use of the composite gel beads according to claim 1 for preparing a microorganism-immobilized carrier.
6. A hydrogel immobilized microorganism preservation method comprises fixing microorganism in hydrogel immobilized carrier by embedding method, and storing at room temperature; the hydrogel immobilization carrier comprises the composite gel beads according to claim 1.
7. The method for preserving hydrogel-immobilized microorganism according to claim 6, wherein: the microorganism is one or more of an aerobic microorganism, a facultative microorganism, or a microorganism that is dormant for more than 2 hours or survives for more than 2 hours under anaerobic conditions.
8. The method for preserving hydrogel-immobilized microorganism according to claim 6, wherein: the use of said composite gel beads for prolonging the preservation time of microorganisms.
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