Preparation method of microorganism embedding preparation with core-shell structure
The core-shell structure microbial embedding preparation formed by reaction in calcium salt solution solves the problems of microbial escape and coculture technology, and achieves stable and functional microbial embedding, which is suitable for environmental governance and microbial culture.
Patent Information
- Application Number
- CN202411455581.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-18
- Publication Date
- 2025-01-07
AI Technical Summary
Existing microbial embedding preparations have microbial escape problems, resulting in secondary contamination and weakening of functions. The existing co-culture technology has slow development and disadvantages, making it difficult to simulate symbiosis or parasitic relationships and interactions between cells under laboratory conditions. .
Using the preparation method of microbial embedded preparation with core-shell structure, by preparing embedded carriers and nuclear fluids, the coaxial needle device is used to react them in calcium salt solution to form strain spheres to avoid microbial exposure, ensure the stability of microbial cells and Functional.
It has achieved complete avoidance of secondary pollution caused by microbial exposure, ensured the activity and function of microbial organisms, and could play a variety of functions in environmental governance, reduce interspecies competition, and improve the efficiency of microbial culture.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganism solidification, and in particular to a method for preparing a microorganism embedding preparation with a core-shell structure, which has a stable structure and a simple process and can overcome the problem of microorganism escape in existing embedded microorganism preparations. Background Art
[0002] The vast majority of bacteria in the world are still uncultured, and the number of published bacterial species does not exceed 0.2% of the global total. Uncultured groups play a vital role in the global cycle of carbon, nitrogen, sulfur and other elements, the synthesis of novel natural active products, and the surrounding organisms and environment. Although molecular technology can provide a large amount of functional prediction information of uncultured microorganisms, in order to accurately understand and study their specific metabolic pathways, gene functions and their ecological significance, it is necessary to obtain their pure culture strains. One of the key factors for the current unculture of microorganisms is the inability to simulate the symbiotic or parasitic relationship between cells under laboratory conditions, and the various signal molecules secreted by their interactions, available substrates, metabolites and their intermediates, small molecule compounds and enzymes. The co-culture separation and cultivation methods to alleviate this problem have developed slowly, and there are still many disadvantages in the existing co-culture methods. Therefore, it is urgent to develop new co-culture technologies to alleviate the cultivation barriers of uncultured microorganisms.
[0003] The sandwich plate method can be used to design a pure culture method for two symbiotic strains that are not in contact with each other, but this method cannot be applied to the enrichment culture process of marine microorganisms.
[0004] At present, microbial immobilization technology has been applied to the field of environmental governance. For example, physical and chemical methods are used to fix microorganisms on carriers to prepare biofilms for treating organic matter in sewage. Encapsulation technology is a method of using carriers to encapsulate solids, liquids or gases to form microcapsules. It is a commonly used cell immobilization technology. The encapsulation method is to restrict microbial cells to a non-insoluble constraint structure (polymer grid structure), while also allowing the matrix to diffuse and penetrate. The method is simple to operate and has almost no damage to the activity of microbial cells. The entire cell can grow and die in the polymer grid. At the same time, the loose structure can ensure substrate penetration and product diffusion. Encapsulating microorganisms in microcapsules for environmental governance can not only realize the environmental governance capabilities of various functional microorganisms, but also achieve division of labor and cooperation, and reduce the impact of competition.
[0005] Currently, the commonly used embedding carriers are mainly organic carriers such as agar, sodium alginate, polyvinyl alcohol, polyacrylamide, etc. The ideal embedding carrier should be non-toxic to microbial cells, have good mass transfer performance, not easily decomposed by microorganisms, have high strength, can be used for a long time and have low cost. The currently used embedding technology usually directly mixes the microorganisms with the embedding material, and the prepared microcapsules have problems such as microorganism escape. Summary of the invention
[0006] In view of the shortcomings and deficiencies in the prior art, the present invention proposes a method for preparing a microbial embedding preparation with a core-shell structure and its application, which has a novel structure and controllable preparation technology, can completely avoid secondary contamination caused by microbial exposure, and can give full play to the role of functional microorganisms.
[0007] The present invention is achieved by the following measures: A method for preparing a microorganism embedding preparation with a core-shell structure, characterized in that an embedding carrier and a core liquid are prepared separately, the prepared core liquid and the embedding carrier are pumped into a coaxial needle device through a peristaltic pump respectively, and then dripped into a sterilized calcium salt solution through the coaxial needle device, the sterilized calcium salt solution is magnetically stirred, and reacted for 0.5 to 24 hours to obtain a strain ball; wherein the preparation of the embedding carrier is specifically as follows: alginate is dissolved in deionized water, heated in a water bath or an oil bath and magnetically stirred to obtain a sol system, the mass fraction of the alginate hydrosol is 1.5% to 5%; the water bath or oil bath temperature is 30°C to 100°C, the time is 0.5 to 10 hours, sterilized, and alginate sol is obtained; The preparation of the nuclear liquid is specifically as follows: in an ultra-clean bench, a sterilized alginate sol is taken, a set strain and specific nutrients necessary for strain growth are added, carbon particles with a particle size of less than 100 nm are added, and the mixture is stirred evenly, wherein the mass ratio of the carbon particles to the sodium alginate is 1 / 100 to 1, and the carbon particles are non-porous carbon nanoparticles or carbon nanofibers or graphite powder or graphene or carbon nanotubes or porous carbon nanoparticles.
[0008] The calcium salt solution in the present invention is prepared by dissolving a calcium salt in deionized water, with a mass fraction of 0.01-0.5 mol / L, and the calcium salt is calcium chloride, calcium nitrate or calcium sulfate. Furthermore, the concentration of the calcium salt aqueous solution is preferably 0.1-0.4 mol / L.
[0009] The alginate described in the present invention is sodium alginate, potassium alginate or other alginates and mixtures thereof. The mass fraction of the alginate sol is preferably 2% to 4%, the water or oil bath temperature is preferably 50° C. to 70° C., and the time is preferably 2 to 6 hours. This is because the concentration of the alginate is too low and the viscosity is too low to be easily formed, the viscosity is too high to be difficult to prepare and form, and the temperature is too high to cause the alginate to deteriorate.
[0010] The reaction time for preparing the strain spheres using the prepared nuclear liquid and the embedding carrier in the present invention is preferably 1-10 hours, and the speed of dripping the calcium salt solution through the coaxial needle device is preferably 0.5-1 drop / s. Too fast will result in poor spherical shape, and too slow will affect the activity of the microorganisms.
[0011] The present invention will contain strain D737 T A mixture of the bacterial suspension, the nucleic acid fragments extracted from Escherichia coli DH5α and 1.5% sodium alginate sol containing 0.1% VGCF (carbon nanofiber) was used as the core liquid of the strain ball, and 3% sodium alginate sol was used as the shell liquid. The shell liquid and the core liquid were pumped into the coaxial needle at the same time by a peristaltic pump, merged at the needle and dripped into 0.1 mol / L calcium chloride solution. After reacting at 4°C for 4 hours, the strain ball was obtained.
[0012] Compared with the prior art, the present invention adopts a one-step molding method to prepare a microbial embedded bacterial agent with a core-shell structure. The structure is novel and the preparation technology is controllable. The core-shell structure can completely avoid secondary pollution caused by microbial exposure and play the role of functional microorganisms. Specifically, the present invention can mix microbial embedded bacterial agents with different functions, which can play multiple functions and avoid the problem of functional microbial death caused by interspecies competition due to the lack of contact between microorganisms. In addition, according to the characteristics and needs of functional microorganisms, the concentration of the nuclear liquid and the content of the bacterial agent are adjusted to prepare specific nuclear sols, and necessary nutrients and supporting materials are added to improve the mechanical strength of the structure, so as to provide support for the subsequent preparation of specific shell sols according to the use environment of the microecological preparations, and to prepare them into microecological preparations through a one-step molding method, so as to play an effective role in environmental governance. BRIEF DESCRIPTION OF THE DRAWINGS Attached Figure 1 The strain D737 was constructed based on the 16S rDNA sequence in the present invention. T Phylogenetic tree of .
[0014] Attached Figure 2 Schematic diagram of the structure of the coaxial needle device in an embodiment of the present invention.
[0015] Attached Figure 3 It is a strain ball obtained in the example of the present invention.
[0016] Attached Figure 4 Schematic diagram of the growth curve of strains in different culture media in the embodiments of the present invention.
[0017] Attached Figure 5 In the present invention, DNA enzyme culture medium was used to treat D737 T Detection results of extracellular nucleic acids.
[0018] Attached Figure 6 1 is the dilution curve of each sample at different enrichment periods in the embodiment of the present invention.
[0019] Attached Figure 7 Schematic diagram of T test of diversity index in an embodiment of the present invention.
[0020] Attached Figure 8 1 is a Venn diagram of OUT overlap levels between samples at different enrichment periods in an embodiment of the present invention.
[0021] Attached Fig. 9 4 is a bar graph showing the relative abundance distribution of microorganisms at the family level in the embodiments of the present invention.
[0022] Attached Fig.10 This is a diagram showing the differences of high-abundance groups in different treatments in an embodiment of the present invention. DETAILED DESCRIPTION The present invention will be further described below in conjunction with embodiments.
[0024] Example: This example provides a method for preparing a microbial embedding preparation with a core-shell structure, specifically, a method for preparing a strain ball preparation in which a strain having the function of degrading extracellular nucleic acids is embedded in a sodium alginate embedding carrier.
[0025] The strain used in this example is: Marine Slippery Bacteria New Species D737 T , strain D737 was obtained by genome sequencing T The full-length 16S rDNA sequence of D737 was 1489 bp. The existing 16S sequence in GenBank (accession number OP162633) was updated. The 16S rDNA sequence was compared again using the EzTaxon database and found to be similar to that of D737. T The 16SrRNA gene sequence with the highest similarity is M. albidiflavum FB208 T (98.1%), followed by M.flexuosum DSM21950 T (98%), compared with other M. fragile JCM 15579 T (97.1%), a new species of the genus Marine Nematodes. The phylogenetic tree was constructed using NJ, ML and MP. The results showed that D737 T It formed an independent subcluster with the same genus. Marinilabiliaceae The strains of other genera clustered together to form a monophyletic group ( Figure 1 ); In this example, a Figure 2 The coaxial needle device shown in the figure will be 100ml OD 600 D737 of 0.6 TA mixture of strain suspension, 5 ml (1 g / L) DNA extract and 1.5% sodium alginate sol containing 0.1% VGCF (VGCF is a carbon nanofiber, which can improve the mechanical properties of the strain ball after addition) was used as the core liquid of the strain ball, and 3% sodium alginate sol was used as the shell liquid. The shell liquid and the core liquid were pumped into the coaxial needle at the same time by a peristaltic pump, merged at the needle and dripped into 0.1 mol / L calcium chloride solution. After reacting at 4°C for 4 hours, the strain ball was taken out and placed in an enrichment bottle containing marine sediments for enrichment culture, and samples were taken to test the microbial groups of the enrichment liquid and the outer shell of the strain ball.
[0026] Through the above means, the experimental parameters were adjusted to prepare a sodium alginate sphere with a stable core-shell structure. 0.1% carbon material was added to the sphere to build a composite interface and increase the mechanical strength of the material. Figure 3 It can be seen that the prepared rubber balls are complete in shape and uniform in size.
[0027] Using RAST database to analyze D737 T The genome of strain D737 was annotated and related genes were found. T The genome of the bacteria has a large number of enzymes related to nucleic acid degradation and synthesis and genes related to nucleic acid degradation. Extracellular nucleases are the most critical enzymes for bacteria to utilize DNA in the environment. After the extracellular nucleases degrade DNA macromolecules into small molecules, they further enter the cell through phosphate transporters.
[0028] In this case, the addition of DNA extract to the modified 2216E medium significantly increased the growth rate of the strain (see attached Figure 4 ), D737 T The degradation zone on the culture medium shows that it has the ability to degrade extracellular DNA, such as Figure 5 As shown, it can be seen that the experimental results are consistent with the gene prediction results. Therefore, during the culture process, the additional addition of nucleic acid fragments extracted from Escherichia coli DH5α can promote its growth. The strain is filtered through a 0.22µm filter and added to the extracted nucleic acid solution, which can quickly degrade the nucleic acid fragments.
[0029] The performance of the microbial embedding preparation obtained in this example is analyzed below: First, check out D737 T In this case, a total of 84 16S rRNA amplicon data were obtained, and the dilution curves of each sample at different enrichment periods are shown in Figure 6 ,As can be seen from the figure, as the sample sequencing depth increases, the curve no longer increases significantly, ,which indicates that the sequencing amount is sufficient and ,can be used for subsequent analysis.
[0030] Based on the number of sequences obtained by sequencing and the number of OTUs obtained by clustering, the α and β diversity indices of the samples in the control group and the experimental group were calculated to see whether the differences were statistically significant (see Appendix Figure 7 and Table 1).
[0031] Table 1 β diversity difference table: The Alpha diversity index reflects the number of species in the community and their relative abundance. Comparing the alpha diversity index of different samples can show the diversity differences of different samples. It can relatively objectively reflect the species diversity of the community. At the same time point, there are statistical differences between the samples of the control group and the experimental group, that is, there are differences in the diversity and uniformity of the communities of the experimental group and the control group. The Beta diversity index reflects the difference in species composition between communities in different habitats or the replacement rate of species along the environmental gradient, which is also called between-habitat diversity. At the same time point, there are statistical differences between the samples of the control group and the experimental group, that is, there are differences in the species of the communities of the experimental group and the control group.
[0032] The Venn diagram can be used to count the number of common and unique OTUs in multiple samples, and can intuitively show the similarity and overlap of the number of OTUs in environmental samples. Among them, A: embedded live bacteria-surface, B: embedded dead bacteria-liquid, C: embedded dead bacteria-surface, D: embedded live bacteria-liquid, K: sampling on day 0. From the Venn diagram, it can be seen that there are 1683 species that are common to each component. In addition to K0, there are 295 groups that are common groups in the samples of embedded strains, and 569 groups belong to common groups except K0 and samples of embedded inactivated-sphere surface. Most groups belong to common groups.
[0033] In the ball surface samples, the embedded live bacteria had more microbial groups than the embedded inactivated bacteria, that is, the substances produced by the degradation of extracellular nucleic acids contributed to the changes in the surrounding microbial groups. In the liquid samples, the changes in the number of microbial groups between the embedded live bacteria and the embedded inactivated bacteria were not large, so the contribution to the changes in the microbial groups was not as good as that of the ball surface.
[0034] The abundance ratios of the top 20 groups in each treatment and at different times were further plotted. Consistent with the results of the Venn diagram, the microbial species of the embedded live bacteria in the ball surface samples were more diverse than those of the embedded inactivated bacteria. Compared with the microbial groups on the ball surface, the differences in the groups of the embedded live bacteria and the embedded inactivated bacteria in the liquid were not obvious (see Appendix Fig. 9 ).
[0035] We further analyzed whether there were statistical differences in the abundance of high-abundance taxa between the experimental group and the control group at each time point (see Appendix Fig.10), the groups with significant differences include AM997541_f, Bacteroidaceae , Caminicella_ f, Clostridiaceae , Lachnospiraceae , Marinifilaceae , Marinilabiliaceae , Oscillospiraceae , Paludibacteraceae , Prolixibacteraceae , Sphaerochaetaceae , Vibrionaceae In liquid samples, the microbial abundance of embedded live bacteria is higher than that of embedded inactivated bacteria. Marinifilaceae , Marinilabiliaceae , Oscillospiraceae , Prolixibacteraceae , Vibrionaceae , AM997541_f, etc.
[0036] In summary, this case used the embedding method to investigate the situation in which strains with extracellular nucleases provide nutrients to microorganisms in sediments and affect the composition of microbial communities during enrichment culture. The results show that the embedding culture strategy can affect the composition of microbial communities without direct contact with microorganisms, thereby providing support for the subsequent embedding of certain key groups in the ocean into strain balls to improve the culture efficiency of microorganisms that interact with them.
Claims
1. A method for preparing a microorganism embedding preparation having a core-shell structure, characterized in that: Prepare embedding carrier and nuclear liquid respectively, pump the prepared nuclear liquid and embedding carrier into coaxial needle device respectively through peristaltic pump, drip into sterilized calcium salt solution through coaxial needle device, keep magnetic stirring of sterilized calcium salt solution, and react for 0.5~24h to obtain strain ball; wherein, the preparation of embedding carrier is specifically as follows: dissolve alginate in deionized water, heat in water bath or oil bath and magnetically stir to obtain sol system, the mass fraction of alginate hydrosol is 1.5%~5%; the water bath or oil bath temperature is 30℃~100℃, the time is 0.5~10h, sterilize, and obtain alginate sol; The preparation of the nuclear liquid is specifically as follows: in an ultra-clean bench, a sterilized alginate sol is taken, a set strain and specific nutrients necessary for strain growth are added, carbon particles with a particle size of less than 100 nm are added, and the mixture is stirred evenly, wherein the mass ratio of the carbon particles to the sodium alginate is 1 / 100 to 1, and the carbon particles are non-porous carbon nanoparticles or carbon nanofibers or graphite powder or graphene or carbon nanotubes or porous carbon nanoparticles.
2. The method for preparing a microorganism embedding preparation with a core-shell structure according to claim 1, characterized in that: The calcium salt solution is prepared by dissolving a calcium salt in deionized water, with a mass fraction of 0.01-0.5 mol / L, and the calcium salt is calcium chloride, calcium nitrate, or calcium sulfate.
3. The method for preparing a microorganism embedding preparation with a core-shell structure according to claim 2, characterized in that: The concentration of the calcium salt aqueous solution is 0.1~0.4 mol / L.
4. The method for preparing a microorganism embedding preparation with a core-shell structure according to claim 1, characterized in that: The alginate is sodium alginate, potassium alginate or other alginate and a mixture thereof. The mass fraction of the alginate sol is 2% to 4%. The water or oil bath temperature is 50° C. to 70° C., and the bath time is 2 to 6 hours.
5. The method for preparing a microorganism embedding preparation with a core-shell structure according to claim 1, characterized in that: The reaction time for preparing the strain ball using the prepared nuclear solution and the embedding carrier is 1 to 10 hours, and the speed of dripping the calcium salt solution through the coaxial needle device is 0.5 to 1 drop / s.
6. The method for preparing a microorganism embedding preparation with a core-shell structure according to claim 1, characterized in that: The strain D737 T A mixture of the bacterial suspension and DNA extracted from large intestine DH5α and 1.5% sodium alginate sol containing 0.1% carbon nanofiber VGCF was used as the core liquid of the strain ball, and 3% sodium alginate sol was used as the shell liquid. The shell liquid and the core liquid were pumped into the coaxial needle at the same time by a peristaltic pump, merged at the needle and dripped into 0.1 mol / L calcium chloride solution. After reacting at 4°C for 4 hours, the strain ball was obtained.