Pseudomonas syringae and application thereof in repairing bone relics

Abstract

The application discloses a Pseudomonas syringae and application of the Pseudomonas syringae in repairing bone relics. (Pseudomonas syringae) The application also provides a biological bacterial agent containing the Pseudomonas syringae strain and application of the biological bacterial agent in inducing calcium carbonate deposition solidification and / or bone relic reinforcement and repair, and a bone relic reinforcement and repair method. The Pseudomonas syringae strain screened in the application has high urease enzyme activity and carbonic anhydrase enzyme activity, the calcium carbonate generation amount is high when the strain or the biological bacterial agent is used to induce calcium carbonate deposition, and the bone relic compression strength can be improved, so that the Pseudomonas syringae strain can be widely applied in many fields such as cultural relic protection and building material reinforcement.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of microbial and material reinforcement engineering, specifically involving a strain of Pseudomonas syringae and its application in the restoration of bone artifacts. Background Technology

[0002] Microbial-induced calcium carbonate deposition (MICP) solidification technology has demonstrated unique application value in many fields due to its excellent ecological compatibility. For example, in the field of building engineering, MIP solidification technology can effectively improve the strength and stability of foundations and soils without disrupting the ecological balance; in the field of water conservancy engineering, MIP solidification technology can assist in seepage prevention treatment, effectively blocking water infiltration paths through the generated calcium carbonate precipitation, thereby improving the seepage prevention performance of water conservancy projects. Because of these significant advantages, MIP solidification technology has received widespread attention and in-depth research, showing broad development prospects.

[0003] The mechanism of MICP (Microbial Inhibition of Carbonate) solidification technology includes photosynthetic biological-induced deposition of calcium carbonate, sulfate-reducing bacteria-induced deposition of calcium carbonate, nitrogen cycle-induced deposition of calcium carbonate, and other biochemical processes-induced deposition of calcium carbonate. Among these, nitrogen cycle-induced deposition is particularly crucial, further encompassing urea degradation and denitrification mechanisms. Urea degradation is the most common deposition mechanism due to its intuitive principle, ease of control, and high efficiency, capable of generating large amounts of carbonate precipitate in a short time. During urea degradation deposition, typical microorganisms such as *Bacillus pasteurellii* produce a key mineralizing enzyme—urease—through metabolic activity. When the urea concentration in the environment is high, the urease secreted by bacteria catalyzes the hydrolysis of urea molecules, producing ammonia and carbamic acid. Subsequently, carbamic acid spontaneously hydrolyzes, rapidly converting into one molecule of carbonic acid and one molecule of ammonia. Ammonia dissolves in water, increasing the pH of the solution and promoting an increase in alkalinity. This increase in alkalinity drives the conversion of dissolved inorganic carbon in the water into carbonate ions, significantly increasing their concentration. If a source of calcium ions is present in the environment, calcium carbonate precipitate can form. Meanwhile, the surface of microbial cells often contains a large number of negatively charged functional groups, which can effectively adsorb Ca²⁺ in the solution. When the adsorbed calcium ions encounter the surrounding enriched carbonate ions, the bacterial cell serves as a nucleation site, gradually forming calcium carbonate crystals. The final precipitated crystals can reinforce and repair materials.

[0004] In the microbial-induced calcium carbonate precipitation and consolidation process, urease and carbonic anhydrase play a central role. Urease catalyzes the decomposition of urea into ammonia and carbon dioxide, while carbonic anhydrase enhances the solubility of carbon dioxide in water, thereby promoting its conversion. Existing research mainly focuses on common species such as *Bacillus pasteurellii* and *Bacillus mucilaginosus*. *Bacillus pasteurellii*, due to its high urease activity but slightly lower carbonic anhydrase activity, is often combined with other strains, such as *Bacillus mucilaginosus* and *Bacillus subtilis*, for calcium carbonate deposition. However, while *Bacillus subtilis* possesses high carbonic anhydrase activity, it lacks urease activity, thus its effectiveness in calcium carbonate deposition or bone artifact consolidation is poor. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a strain of Pseudomonas syringae and its application in the restoration of bone artifacts, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention specifically adopts the following technical solution.

[0007] The first aspect of this invention protects a strain of Pseudomonas syringae, SARIP-03, with accession number CCTCC NO: M 20251626.

[0008] Another aspect of the present invention protects a biological agent comprising the strain SARIP-03 as described above.

[0009] Another aspect of the present invention protects a composition comprising the strain SARIP-03 as described above or the biological agent as described above, and a calcification solution.

[0010] Another aspect of the present invention protects the use of the strain SARIP-03 described above, or the biological agent described above, or the composition described above, in inducing calcium carbonate deposition and solidification, and / or strengthening and / or restoring bone artifacts.

[0011] Another aspect of the present invention protects a method for reinforcing or repairing bone artifacts, comprising the following steps: treating the bone artifacts with the strain SARIP-03 as described above, or the biological agent as described above, or the composition as described above.

[0012] This invention screened and obtained a strain of *Pseudomonas syringae*, SARIP-03, with high activity of both carbonic anhydrase and urease. A bio-agent formed by combining SARIP-03 with *Bacillus pasteurellii*, which has high urease activity, exhibits both high urease and carbonic anhydrase activity levels. When using this bio-agent to determine calcium carbonate formation, the bio-agent formed by *Pseudomonas syringae* SARIP-03 and *Bacillus pasteurellii* synergistically promoted the efficiency of microbial induced calcification, significantly increasing the amount of calcium carbonate formed. After reinforcing ivory fragments with this bio-agent, compressive strength testing showed that the bio-agent formed by *Pseudomonas syringae* SARIP-03 and *Bacillus pasteurellii* synergistically induced calcification, significantly improving the compressive strength.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1) The novel strain of Pseudomonas syringae SARIP-03 of the present invention is a strain naturally isolated from soil. Its carbonic anhydrase activity is much higher than that reported in the existing literature, reaching 4.02 U / mL, and its urease activity can reach 1.11 U / mL.

[0015] 2) When the biological agent formed by the combination of Pseudomonas syringae SARIP-03 and Bacillus pasteurellii is used to induce calcium carbonate precipitation, the amount of calcium carbonate generated by the two strains is significantly higher than that of the single strain. Under the same conditions, the amount of calcium carbonate generated by the two strains is 26.23% higher than that of Bacillus pasteurellii alone and 5.65 times higher than that of Pseudomonas syringae alone.

[0016] 3) When the biological agent formed by the combination of Pseudomonas syringae SARIP-03 and Bacillus pasteurellii is used to reinforce bone artifact fragments, the compressive strength of the bone artifacts after double-strain reinforcement is significantly higher than that of the single strain; under the same conditions, the compressive strength after double-strain induction is 85.47% higher than that of Bacillus pasteurellii alone. Attached Figure Description

[0017] Figure 1 The figure shown is a sequence comparison result of Pseudomonas syringae SARIP-03 and related species in the GenBank database in Example 1 of the present invention.

[0018] Figure 2 The figure shown is a growth curve of Pseudomonas syringae SARIP-03 in Example 2 of the present invention.

[0019] Figure 3 The graph shows the enzyme activity curves of urease and carbonic anhydrase in *Pseudomonas syringae* SARIP-03, as shown in Example 2 of the present invention.

[0020] Figure 4 The diagram shown is a schematic of the calcium carbonate deposition apparatus used in Embodiment 4 of the present invention.

[0021] Figure 5 The diagram shown is a schematic of the calcification apparatus used in Embodiment 5 of the present invention.

[0022] Figure 6 The graphs shown are from Example 4 of the present invention, which measure the amount of calcium carbonate produced and the enzyme activities of urease and carbonic anhydrase after calcium carbonate precipitation using Pseudomonas syringae SARIP-03, Bacillus pasteurellii, and biological agents.

[0023] Figure 7 The image shows the compressive strength test results of the ivory fragments from the Jinsha Site reinforced with Bacillus pasteurellus and biological agents in Example 5 of the present invention.

[0024] Figure 8 The image shown is a photograph of the reinforcement and restoration of ivory fragments from the Jinsha Site using a biological agent formed by combining *Pseudomonas syringae* SARIP-03 and *Bacillus pasteurellus* in Example 5 of this invention. Detailed Implementation

[0025] The first aspect of this invention protects a strain of *Pseudomonas syringae*, SARIP-03, with accession number CCTCC NO: M 20251626. The strain SARIP-03 protected by this invention was screened and isolated from soil, and identified as *Pseudomonas syringae* by morphological observation and 16S rRNA gene sequencing.

[0026] The colony morphology of *Pseudomonas syringae* is as follows: round, solitary, short rod-shaped, without capsules, does not produce spores, and has polar flagella; when grown on LB solid medium, it forms round colonies with smooth edges, uniform texture, white, glossy, and opaque appearance, and is identified as a Gram-negative bacterium; the culture temperature is 20–40℃, the optimum temperature is 30℃, the lethal temperature is 50℃, the pH range is 6.1–8.8, the optimum pH is 7, it is aerobic, and does not require light.

[0027] In some embodiments, the 16S rDNA sequence of the strain SARIP-03 includes the sequence shown in SEQ ID No. 1.

[0028] In some embodiments, the urease activity of strain SARIP-03 is 1.11 U / mL, and its carbonic anhydrase activity is 4.02 U / mL. In a specific embodiment, the urease activity can be detected by the following method: strain SARIP-03 is inoculated into LB liquid medium containing 4 g / L urea and cultured in a shake flask at 30°C and 200 rpm for 24 h. The OD of the bacterial culture is then measured. 600 The value was 6.82. Urease activity was determined by conductivity, and carbonic anhydrase activity was determined by spectrophotometry. The specific measurement method was as follows: 1 volume of bacterial culture was mixed with 9 volumes of 1.1 mol / L urea solution. The change in conductivity of the solution was measured using a conductivity meter over 5 minutes. The average conductivity change over 5 minutes (unit: mS / cm / min) multiplied by the dilution factor (10-fold) was the initial enzyme activity of the bacterial culture, i.e., the urease activity mentioned above. This value reflects the ability of the bacterial culture to hydrolyze urea. Enzyme activity is defined as the amount of enzyme required to hydrolyze 1 mmol of urea solution per minute at room temperature; one enzyme activity unit (U) is defined as this.

[0029] The commercially available strain *Pseudomonas syringae* ATCC19875 was cultured on LB liquid medium at 30°C under shaking conditions. The OD of the bacterial culture was... 600 The carbonic anhydrase activity was 3.0, with no urease activity detected, and the carbonic anhydrase activity was 2.03 U / mL. Compared with the type strain *Pseudomonas syringae* ATCC19875, the carbonic anhydrase activity of the strain SARIP-03 in this application was increased by 98.0%.

[0030] The *Pseudomonas syringae* strain SARIP-03 of this invention was deposited on July 16, 2025, at the China Center for Type Culture Collection (CCTCC, No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province), with accession number CCTCC NO: M 20251626. It was isolated from soil through screening.

[0031] The strain SARIP-03 of the present invention comprises the fermentation broth or fermentation metabolites of strain SARIP-03.

[0032] Another aspect of the present invention protects a biological agent comprising the strain SARIP-03 as described above.

[0033] In some embodiments, the biological agent further comprises *Bacillus pasteurellii*. The *Bacillus pasteurellii* is selected from *Bacillus pasteurellii* Sarisp1 (see CN118272270A) with accession number CCTCC NO: M 2024524, *Bacillus pasteurellii* SAR1-02 (see CN120399951A) with accession number CCTCC M 2025059, and *Bacillus pasteurellii* with accession number ATCC11859 from the American Center for Type Culture Collection. In one specific embodiment, the *Bacillus pasteurellii* is *Bacillus pasteurellii* Sarisp1 with accession number CCTCC NO: M 2024524.

[0034] In some embodiments, the ratio of viable bacteria of strain SARIP-03 to Bacillus pasteurellus is (0.5-3):1, or it can be (0.5-1.5):1, or it can be (1-2.5):1, or it can be (2.2-3):1, or it can be 0.5:1, 1:1, 2:1, or 3:1.

[0035] In some embodiments, the biological agent may also include components such as culture medium or additives required for culturing the microorganism. For example, the agent may be obtained by culturing *Bacillus pasteurellii* in a culture medium, or by further processing it into a lyophilized powder using other methods such as freeze drying.

[0036] In some specific embodiments, the strain SARIP-03 is inoculated overnight in a test tube containing LB liquid medium with 4 g / L urea, and then inoculated at a rate of 1 v / v% into a conical flask containing 250 mL of LB liquid medium with 4 g / L urea and cultured aerobically in a constant temperature shaker at 30°C and 180-220 rpm for 24 hours to obtain the SARIP-03 bacterial suspension of Pseudomonas syringae.

[0037] In some specific embodiments, the dissolved *Bacillus pasteurellus* Sarisp1 strain is transferred to beef extract peptone solid medium and spread evenly, then placed in a 30°C constant temperature incubator for 24–48 h. After individual colonies have grown, they are transferred to beef extract peptone liquid medium for subculturing. The resulting bacterial culture is stored at 4°C to obtain *Bacillus pasteurellus* Sarisp1 bacterial culture.

[0038] Then, the bacterial suspensions of *Pseudomonas syringae* SARIP-03 and *Bacillus pasteurellis* Sarisp1 were centrifuged to obtain bacterial precipitates; the precipitates were then diluted to OD values ​​using the corresponding supernatants. 600 =10. Centrifuge the diluted *Pseudomonas syringae* SARIP-03, then mix it with diluted *Bacillus pasteurellii* Sarisp1 to obtain the biological agent.

[0039] In some embodiments, the urease activity of the biological agent formed by the strain SARIP-03 and Bacillus pasteurellus Sarisp1 is 40.01 U / mL, which is 1.14 times that of the urease activity of a single Bacillus pasteurellus Sarisp1 bacterial suspension (35.11 U / mL) and 13.80 times that of a single Pseudomonas syringae SARIP-03 bacterial suspension (2.9 U / mL); the carbonic anhydrase activity of the biological agent is 10.09 U / mL, which is 2.78 times that of the carbonic anhydrase activity of a single Bacillus pasteurellus Sarisp1 bacterial suspension (3.63 U / mL) and 1.64 times that of the carbonic anhydrase activity of a single Pseudomonas syringae SARIP-03 bacterial suspension (6.15 U / mL).

[0040] In some embodiments, the dosage form of the biological agent can be multiple, such as liquid agent, powder agent, granular agent, emulsion, or suspension.

[0041] In some embodiments, the microbial agent further includes a carrier.

[0042] In some specific embodiments, the carrier includes a solid carrier or a liquid carrier. The solid carrier includes mineral materials, plant materials, and / or polymeric compounds; the mineral materials may be at least one selected from clay, talc, maifanite, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant materials may be at least one selected from corn flour, soybean flour, rice husk powder, and starch; the polymeric compounds may be polyvinyl alcohol or / and polyethylene glycol. The liquid carrier may be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane or / and dodecane.

[0043] In some embodiments, the microbial preparation may also contain surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc.

[0044] Another aspect of the present invention protects a composition comprising the strain SARIP-03 as described above or the biological agent as described above, and a calcification solution.

[0045] In some embodiments, the calcification solution comprises calcium salt and urea, wherein the molar ratio of the calcium salt to urea can be 1:1 to 3, 1:1 to 2.2, 1:1.8 to 3, or 1:1, 1:2, or 1:3. The calcification solution refers to a system used to induce or regulate calcium salt deposition, and its core components include a calcium source, a phosphate donor, or other substances capable of promoting calcium salt deposition.

[0046] In some embodiments, the calcium salt is selected from one or more of calcium chloride, calcium nitrate, calcium lactate, calcium formate, and calcium acetate. In one specific embodiment, it is calcium chloride.

[0047] In some embodiments, the concentration of the calcium salt, based on the total volume of the calcification solution, can be 0.5–10 mol / L, 0.5–2.2 mol / L, 1.8–4.8 mol / L, 3.6–8.6 mol / L, or 6.5–10 mol / L. In one specific embodiment, it is 1 mol / L.

[0048] In some embodiments, the concentration of urea, based on the total volume of the calcification solution, can be 0.5–10 mol / L, 0.5–2.2 mol / L, 1.8–4.8 mol / L, 3.6–8.6 mol / L, or 6.5–10 mol / L. In one specific embodiment, it is 2.5 mol / L.

[0049] In some embodiments, the concentration ratio of the strain SARIP-03 to the concentration of the calcification solution is (1 × 10⁻⁶). 9 ~10×10 9 cfu / mL: 1 mol / L, or 1×10 9 CFU / mL: 1 mol / L, 1.5 × 10⁻⁶ 9 cfu / mL: 1 mol / L, 2×10 9 CFU / mL: 1 mol / L, 2.5 × 10⁻⁶ 9 cfu / mL: 1 mol / L, 3×10 9 CFU / mL: 1 mol / L, 3.5 × 10⁻⁶ 9 cfu / mL: 1 mol / L, 4×10 9 CFU / mL: 1 mol / L, 4.5 × 10⁻⁶ 9 cfu / mL: 1 mol / L, 5×10 9 CFU / mL: 1 mol / L, 5.5 × 10⁻⁶ 9 cfu / mL: 1 mol / L, 6×10 9 CFU / mL: 1 mol / L, 6.5 × 10⁻⁶ 9 cfu / mL: 1 mol / L, 7×10 9 CFU / mL: 1 mol / L, 7.5 × 10⁻⁶ 9 cfu / mL: 1 mol / L, 8×10 9 cfu / mL: 1 mol / L.

[0050] Another aspect of the present invention protects the use of the strain SARIP-03 described above, or the biological agent described above, or the composition described above, in inducing calcium carbonate deposition and solidification, and / or strengthening and / or restoring bone artifacts.

[0051] In some implementations, the object to be processed is selected from bone artifacts. Bone artifacts, in accordance with the "Regulations on the Management of Cultural Relics Protection Projects," refer to various cultural relics made of animal bones, horns, teeth, and other bone materials.

[0052] The bone artifact reinforcement described in this invention refers to treating bone artifacts through physical or chemical methods to improve their mechanical strength, durability, and stability, and to prevent further damage caused by environmental factors (such as humidity, temperature changes, microbial erosion, etc.).

[0053] The bone artifact restoration described in this invention refers to the process of repairing artifacts damaged by natural degradation (hydrolysis, oxidation), mechanical damage, or human destruction in order to restore their integrity, stability, and historical value.

[0054] In some embodiments, the bone artifact is unearthed ivory. Unearthed ivory refers to ivory artifacts excavated from archaeological sites, such as ancient tombs, ruins, or other archaeological locations. Due to prolonged burial, unearthed ivory typically suffers varying degrees of damage, such as dehydration, cracking, deformation, and corrosion. After reinforcement and restoration, unearthed ivory is ultimately provided to museums, becoming important exhibits for the public to understand the ancient Shu civilization through ancient ivory. Therefore, the protection of unearthed ivory has significant historical and archaeological value for understanding ancient culture, art, and craftsmanship.

[0055] Another aspect of the present invention protects a method for reinforcing or repairing bone artifacts, comprising the following steps: treating the bone artifacts with the strain SARIP-03 as described above, or the biological agent as described above, or the composition as described above.

[0056] In some embodiments, the method is as follows: using strain SARIP-03 or the biological agent and calcification solution as described above, or using the composition described above to treat bone artifacts, specifically by first treating the bone artifacts with the biological agent described above, and then treating the bone artifacts with the calcification solution.

[0057] In some embodiments, the treatment of unearthed ivory with the above-mentioned biological agent and calcification solution is repeated more than 6 times, for example, 6, 7, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times.

[0058] In some embodiments, after the treatment is completed, the treated bone artifacts are dried for 24 to 30 hours, for example, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, or 48 hours.

[0059] The method and optimized conditions developed in this application are used to reinforce and repair unearthed ivory with special pores and structures. The compressive strength of the reinforced and repaired unearthed ivory reaches 16.08 MPa, and the micropores in the unearthed ivory are completely filled, meeting the requirements for the structural stability of bone cultural relics. This provides a solution for the long-term reinforcement or repair of highly fragile bone cultural relics.

[0060] The biological agent formulated using the strain SARIP-03 of this invention and *Bacillus pasteurellus* (especially *Bacillus pasteurellus Sarisp1*) increased calcium carbonate production from 5.68 g to 7.17 g, a 26.2% increase, compared to *Bacillus pasteurellus* alone; and increased calcium carbonate production by 5.65 times compared to *Pseudomonas syringae* SARIP-03 alone. Furthermore, when the biological agent formulated using the strain SARIP-03 and *Bacillus pasteurellus* of this invention was used for bone artifact reinforcement, the compressive strength increased from 8.67 MPa to 16.08 MPa, an 85.47% increase, compared to *Bacillus pasteurellus* alone. Compared to a commercially available biological agent formulated using the strain *Pseudomonas syringae* ATCC19875 and *Bacillus pasteurellus*, the calcium carbonate production increased from 5.972 g to 7.17 g, a 20.1% increase. In summary, the strain SARIP-03 and the biological agent containing strain SARIP-03 of the present invention can be widely used in many fields such as cultural relic protection and building material reinforcement, and have broad application prospects.

[0061] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0062] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0063] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0064] In the following examples, the compressive strength was tested using a texture analyzer.

[0065] OD in this application 600 When the value is 1, the corresponding concentration of *Pseudomonas syringae* SARIP-03 or *Bacillus pasteurellis* is approximately 0.9 × 10⁻⁶. 8 cfu / mL.

[0066] Example 1: Isolation and screening of Pseudomonas syringae SARIP-03

[0067] 1.1 Initial screening

[0068] Fresh soil samples were collected from highly alkaline soil in the suburbs of Shanghai. Approximately 1 g of soil sample was weighed and placed in an Erlenmeyer flask containing 9 mL of sterile water. The flask was shaken at 30°C and 150 rpm for 15 minutes, then allowed to settle naturally for 30 minutes. The supernatant was diluted with sterile water at a ratio of 1:1. -3 10 -6 10 -9 Three gradient dilutions were performed to create three experimental groups. The supernatants from each of the three dilution groups were evenly spread onto LB solid medium. After spreading, the cultures were incubated at 30°C for 24–48 hours, resulting in single colonies forming on the LB solid medium. Five different colonies from each group were picked and activated in LB liquid medium tubes. These were then inoculated into 100 mL of LB liquid medium at a 1 v / v% inoculation rate and incubated at 30°C with shaking at 150 rpm for 18–24 hours.

[0069] Take 1 mL of the cultured bacterial solution and add 9 mL of 1.1 mol / L urea solution. Use a conductivity meter to select colonies with significant changes in conductivity after adding the bacterial solution for secondary screening.

[0070] LB solid medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, agar 20 g / L, autoclaved at 115℃ for 30 min.

[0071] LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, autoclaved at 115℃ for 30 min.

[0072] 1.2 Secondary screening

[0073] The colonies with significant changes in conductivity obtained from the initial screening were inoculated into 100 mL of LB liquid medium and cultured in shake flasks at 30°C and 200 rpm for 18-24 h. Their urease activity was then measured using the same method as in step 1.1, resulting in a strain with high urease activity.

[0074] 1.3 Identification of strains

[0075] The strains with high urease activity after secondary screening were placed on LB solid medium and cultured at 30°C for 30 h.

[0076] The colony morphology is as follows: round, solitary, short rod-shaped, without capsules, does not produce spores, and has polar flagella; when grown on LB solid medium, it forms round colonies with smooth edges, uniform texture, white, glossy, and opaque appearance, and has been identified as a Gram-negative bacterium; the culture temperature is 20–40℃, with an optimum temperature of 30℃ and a lethal temperature of 50℃; the pH range is 6.1–8.8, with an optimum pH of 7; it is aerobic and does not require light.

[0077] Genomic DNA was extracted from the bacterial culture of the strain to be identified after shaking flask culture using the Ezup column-based bacterial DNA extraction kit.

[0078] Amplification was performed using universal primers 27F and 1492R. The resulting 16S rDNA sequences were compared with sequences of related species in the GenBank database. The comparison results are shown below. Figure 1 .

[0079] from Figure 1 It can be seen that the strain obtained from the secondary screening is similar to Pseudomonas syringae, with a similarity of 99.93% to strain Pseudomonas syringae LDST1. Therefore, the strain was finally named Pseudomonas syringae SARIP-03.

[0080]

[0081] 27F primer: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 2)

[0082] 1492R primer: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No. 3)

[0083] The *Pseudomonas syringae* strain SARIP-03 of this invention was deposited on July 16, 2025, at the China Center for Type Culture Collection (CCTCC, No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province), with accession number CCTCC NO: M 20251626. It was isolated from soil through screening.

[0084] Example 2 Enzyme activity assay of Pseudomonas syringae SARIP-03

[0085] In Example 2, the urease and carbonic anhydrase activities of *Pseudomonas syringae* SARIP-03 were determined. The following were included:

[0086] 2.1 Obtaining the bacterial suspension of *Pseudomonas syringae* SARIP-03

[0087] The frozen strain SARIP-03 was taken out of the -80℃ freezer and a small amount of bacterial culture was inoculated into a test tube containing LB liquid medium with 4 g / L urea for overnight activation. Then, it was inoculated into 250 mL Erlenmeyer flasks containing 4 g / L urea LB liquid medium at a volume of 1 v / v and cultured aerobically at 30℃ and 180-220 rpm for 75 hours for expansion.

[0088] Bacterial culture was collected at 9h, 12, 21, 24, 30, 36, 48, 60, and 72h, and the OD of the bacterial culture was measured using a spectrophotometer. 600 The results are shown Figure 2 .

[0089] from Figure 2 It can be seen that when the OD of the expanded bacterial culture solution... 600 When the optical density value reaches 4.5, the growth curve is in a plateau phase, indicating that strain SARIP-03 is in a vigorous logarithmic growth phase and has the strongest activity.

[0090] 2.2 Detection of urease and carbonic anhydrase activities in bacterial culture

[0091] The culture was expanded using the same aerobic shaking incubation method as in step 2.1 for 24 hours. Bacterial culture samples were collected at 6, 9, 12, 15, 18, 21, and 24 hours for urease and carbonic anhydrase activity assays. Details are as follows:

[0092] 2.2.1 Detection of urease activity

[0093] When a certain concentration of urea solution is added to the bacterial culture, the concentration of conductive ions in the solution increases per unit time due to the hydrolysis of urea by urease-producing microorganisms, thereby increasing the conductivity of the solution. Furthermore, the amount of urea hydrolyzed is directly proportional to the increase in solution conductivity. Therefore, the change in solution conductivity can be used to characterize the ability of the bacterial culture to hydrolyze urea per unit time (urease activity of the bacterial culture).

[0094] The relationship between the amount of urea hydrolyzed and the change in conductivity is as follows:

[0095] Urea hydrolysis capacity (mM / min) = Change in conductivity (mS / cm / min) × 11.11

[0096] That is, using the method reported in the literature (Whiffin V S. Microbial CaCO3 precipitation for the production of bbiocemenet. Pertj, Austalia: Murdoch University, 2004), the standard curve R... 2 =0.9988.

[0097] The specific measurement method is as follows: Mix 1 volume of bacterial culture with 9 volumes of 1.1 mol / L urea solution, and measure the change in conductivity of the solution over 5 minutes using a conductivity meter. Multiply the average conductivity change over 5 minutes (unit: mS / cm / min) by the dilution factor (10-fold) to obtain the initial enzyme activity of the bacterial culture. This value reflects the ability of the bacterial culture to hydrolyze urea. Enzyme activity is defined as the amount of enzyme required to hydrolyze 1 mmol of urea solution per minute at room temperature, which is one enzyme activity unit (U).

[0098] 2.2.2 Detection of carbonic anhydrase activity

[0099] Carbonic anhydrase not only catalyzes the hydration reaction of CO2, but also has esterase activity, which can be used to catalyze the hydrolysis of sulfonates, carboxylic esters, phosphate esters, etc. Therefore, the enzyme activity of carbonic anhydrase can be indirectly represented by esterase activity.

[0100] The specific method is as follows:

[0101] 1) Prepare a 3 mmol / L p-nitrobenzene acetate solution and a 15 mmol / L Tris-H2SO4 buffer solution (pH 7.6);

[0102] 2) Constructing a standard curve for p-nitrophenol: Weigh 0.0417 g of p-nitrophenol, dissolve it, and dilute to 100 mL to obtain a 3 mmol / L p-nitrophenol stock solution. Take 10 test tubes and add the solution according to the proportions in Table 1, mixing well. Simultaneously, use the same volume of distilled water as a blank control. Construct a standard curve, with R0... 2 =0.9906.

[0103] Table 1

[0104] Mother liquor / mL 0.15 0.3 0.45 0.6 0.75 0.9 1.05 1.2 1.35 1.5 Distilled water / mL 9.85 9.7 9.55 9.4 9.25 9.1 8.95 8.8 8.65 8.5 <![CDATA[OD 400 ]]> 0.026 0.064 0.099 0.128 0.167 0.18 0.224 0.242 0.279 0.287

[0105] 3) Determination of carbonic anhydrase activity in bacterial culture:

[0106] 1.9 mL of Tris-H2SO4 buffer (pH 7.6), 0.1 mL of the test bacterial solution, and 1.0 mL of p-nitrophenyl acetate solution were added sequentially to the cuvette. After reacting for 5 min at room temperature, the absorbance was measured at 400 nm using a UV spectrophotometer. The cuvette without the test bacterial solution was used as a blank control.

[0107] Enzyme activity is defined as the amount of enzyme required to hydrolyze 1 μmol of p-nitrophenyl acetate solution per minute at room temperature, which is one enzyme activity unit.

[0108] The urease and carbonic anhydrase activity curves of *Pseudomonas syringae* SARIP-03 were determined, and the results are as follows: Figure 3 As shown.

[0109] from Figure 3 It can be seen that the OD of the bacterial solution 600 The activity of urease and carbonic anhydrase in *Pseudomonas syringae* SARIP-03 was 6.82, with 1.11 U / mL and 4.02 U / mL, respectively.

[0110] Example 3 Preparation of biological inoculant

[0111] In this embodiment, *Pseudomonas syringae* SARIP-03 and *Bacillus pasteurellii* Sarisp1 were combined to form a biological agent, and the urease and carbonic anhydrase activities of the biological agent were investigated. These included the following:

[0112] 3.1 Obtaining *Pseudomonas syringae* SARIP-03 and *Bacillus pasteurellis* Sarisp1 bacterial suspensions

[0113] 3.1.1 Obtaining the bacterial suspension of *Pseudomonas syringae* SARIP-03

[0114] Take the frozen strain SARIP-03 from the -80℃ freezer, use an inoculation loop to take a small amount of bacterial solution and inoculate it into a test tube containing LB liquid medium with 4 g / L urea for overnight activation. Then, inoculate it into a 250 mL Erlenmeyer flask containing 4 g / L urea LB liquid medium at a 1 v / v inoculation rate and incubate it in an aerobic shaking incubator at 30℃ and 180-220 rpm for 24 hours.

[0115] 3.1.2 Obtaining the bacterial culture of Bacillus pasteurellii Sarisp1

[0116] The dissolved Pasteurella Sarisp1 strain was transferred to beef extract peptone solid medium and spread evenly. It was then incubated in a 30°C incubator for 24–48 h. After individual colonies grew, the colonies were transferred to beef extract peptone liquid medium for subculture. The resulting bacterial culture was stored at 4°C.

[0117] Beef extract peptone solid medium: 3 g / L beef extract, 5 g / L peptone, 20 g / L urea, 20 g / L agar, autoclaved at 115℃ for 30 min.

[0118] Beef extract peptone liquid culture medium: 3 g / L beef extract, 5 g / L peptone, 20 g / L urea, autoclaved at 115℃ for 30 min.

[0119] 3.2 Preparation of biological inoculants

[0120] The bacterial suspensions of *Pseudomonas syringae* SARIP-03 obtained in step 3.1.1 and *Bacillus pasteurellii* Sarisp1 obtained in step 3.1.2 were measured and their bacterial concentrations (OD) were obtained. 600 The cells were then centrifuged at 8000 rpm for 10 min to remove the supernatant, yielding two different bacterial pellets. The corresponding bacterial pellets were then diluted with the supernatant for each strain until the OD value of the bacterial culture was reached. 600 Given 10, OD was obtained respectively. 600 =10 OD *Pseudomonas syringae* SARIP-03 bacterial suspension (also known as 10 OD *Pseudomonas syringae* SARIP-03 bacterial suspension), OD 600 =10% Pasteurella Sarisp1 bacterial suspension (also known as 10OD Pasteurella Sarisp1 bacterial suspension).

[0121] Then, the 10OD *Pseudomonas syringae* SARIP-03 bacterial suspension was centrifuged at 8000 rpm for 10 min to obtain bacterial precipitate A. The bacterial precipitate A was then mixed thoroughly with a 10OD *Bacillus pasteurellis* Sarisp1 bacterial suspension. Finally, the OD was measured. 600 =20 biological agent (also known as OD20 biological agent).

[0122] 3.3 Urease and Carbonic Anhydrase Activities of Biological Agents

[0123] The urease and carbonic anhydrase activities of the 10OD *Pseudomonas syringae* SARIP-03 bacterial suspension, the 10OD *Bacillus pasteurellii* Sarisp1 bacterial suspension, and the 20OD biological agent obtained in step 3.2 were determined.

[0124] The measurement method is the same as in Example 2.

[0125] See results Figure 6 and Figure 7 .

[0126] from Figure 6 and Figure 7 It can be seen that the urease activity of the biological agent is 40.01 U / mL, which is 1.14 times that of the urease activity of a single Bacillus pasteurellus Sarisp1 bacterial suspension (35.11 U / mL) and 13.80 times that of the urease activity of a single Pseudomonas syringae SARIP-03 bacterial suspension (2.9 U / mL).

[0127] from Figure 6 and Figure 7 It can be seen that the carbonic anhydrase activity of the biological agent is 10.09 U / mL, which is 2.78 times that of the carbonic anhydrase activity of a single Bacillus pasteurellus Sarisp1 bacterial suspension (3.63 U / mL) and 1.64 times that of the carbonic anhydrase activity of a single Pseudomonas syringae SARIP-03 bacterial suspension (6.15 U / mL).

[0128] In summary, the urease and carbonic anhydrase activities of the biological agent are significantly superior to those of single Bacillus pasteurellus Sarisp1 and single Pseudomonas syringae SARIP-03.

[0129] Example 4: Efficiency of biological agents inducing calcium carbonate deposition

[0130] The calcification process used 10OD *Pseudomonas syringae* SARIP-03 bacterial suspension, 10OD *Bacillus pasteurellii* Sarisp1 bacterial suspension, and 20OD biological agent obtained in Example 3. The specific efficiency of calcium carbonate deposition is as follows:

[0131] Use such as Figure 4The centrifuge tubes shown are prepared by weighing empty centrifuge tubes in advance and setting up a 30 mL system. Add 5 mL of each bacterial culture and 25 mL of 1 mol / L calcification solution (the molar mass ratio of urea to calcium chloride is 1:1) to the system. After adding the solution, let it stand at room temperature for 24 h. Separate the supernatant from the generated calcium carbonate precipitate, discard the supernatant, and then put the centrifuge tubes into an oven to dry. After drying, weigh the tubes and subtract the weight of the pre-weighed centrifuge tubes to obtain the weight of the generated calcium carbonate. Thus, the amount of induced calcium carbonate precipitate can be obtained.

[0132] from Figure 6 It can be seen that, due to the high activity of urease and carbonic anhydrase in the biological agent, the calcification rate is fast. The calcium carbonate production obtained by the synergistic induction of the two bacteria was 7.17g, which is 26.23% higher than that of Bacillus pasteurellis alone (5.68g) and 5.65 times higher than that of Pseudomonas syringae alone (SARIP-03) alone (1.27g).

[0133] Example 5: Compressive strength induced by biological agents

[0134] The 10OD *Pasteurella sativa* Sarisp1 bacterial suspension obtained in Example 3 and the 20OD biological agent were used. The reinforcement test is detailed below:

[0135] Use such as Figure 5 The calcification device shown consists of a wire mesh with a support frame on top, supporting the ivory, and a container for waste liquid at the bottom. Above the wire mesh is a syringe device that drips various bacterial solutions and calcification solutions onto the ivory fragments.

[0136] The specific procedure for calcification is as follows:

[0137] 1) Select several ivory fragments from the Jinsha Site that need to be reinforced and weigh them. Select those with similar weights for subsequent experiments.

[0138] 2) Place the selected ivory fragments on the reinforcement device and use an injection device to drop 50 μL of each bacterial solution onto the ivory fragments.

[0139] 3) Add 50 μL of 1 mol / L calcification solution (the molar mass ratio of urea to calcium chloride is 1:1) to the ivory fragment using an injection device.

[0140] 4) Repeat steps 2) and 3) more than 15 times.

[0141] 5) Place the reinforced ivory sample in a 50℃ oven and dry for 24 hours.

[0142] 6) Place the dried ivory sample at room temperature to allow it to re-moisten for 48 hours.

[0143] 7) Place the ivory sample that has become damp again into a 50℃ oven to dry.

[0144] 8) Take photos of the reinforced ivory sample.

[0145] See results Figure 7 See the actual photos after reinforcement. Figure 8 .

[0146] from Figure 7 It can be seen that, due to the high activity of urease and carbonic anhydrase in the biological agent, the calcification speed is fast. The compressive strength of the dual-bacterial synergistic induction and reinforcement was 16.08 MPa, which is 85.47% higher than that of the calcified sample using a single Bacillus pasteurellus Sarisp1 (8.67 MPa).

[0147] Comparative Example 1

[0148] The difference between Comparative Example 1 and Example 4 is that *Pseudomonas syringae* ATCC19875, purchased from Baosai Biotechnology, was used instead of *Pseudomonas syringae* SARIP-03. All other steps were the same as in step 3.2, and OD was obtained. 600 =20 biological agent.

[0149] The culture medium for *Pseudomonas syringae* ATCC19875 was LB liquid medium.

[0150] *Pseudomonas syringae* ATCC19875 was inoculated into LB broth and cultured at 30°C on a shaker at 200 rpm until the OD of the bacterial culture reached [missing value]. 600 At a concentration of 3.0, no urease activity was detected, and the carbonic anhydrase activity was 2.03 U / mL.

[0151] The bio-initiative, formulated with *Bacillus pasteurellii* to form a 20 OD, was used for calcium carbonate deposition. The measured calcium carbonate production was 5.972 g, representing only a 5.14% increase compared to the production using only *Bacillus pasteurellii* (5.68 g). Compared to the calcium carbonate production of Comparative Example 1 (5.972 g), the calcium carbonate production of the bio-initiative in Example 4 (7.17 g) was increased by 20.01%.

[0152] The *Pseudomonas syringae* SARIP-03 strain of this invention is a naturally isolated strain with urease activity reaching 1.11 U / mL and carbonic anhydrase activity reaching 4.02 U / mL, significantly higher than that of *Pseudomonas syringae* strains in the prior art. When combined with *Bacillus pasteurellii* to form a bio-agent that synergistically induces calcification and is used for calcium carbonate deposition, the calcium carbonate production is 7.17 g, which is 26.23% higher than that using *Bacillus pasteurellii* alone (5.68 g); 5.65 times higher than that using *Pseudomonas syringae* SARIP-03 alone (1.27 g); and 20.01% higher than that using a bio-agent formed by combining *Pseudomonas syringae* ATCC19875 and *Bacillus pasteurellii* (7.17 g). Furthermore, when the biological agent of the present invention is used for the reinforcement and restoration of bone artifacts, it improves the calcification rate by 85.47% compared to calcified samples using a single Bacillus pasteurellus (8.67 MPa).

[0153] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A strain of *Pseudomonas syringae* ( Pseudomonas syringae The strain SARIP-03, with accession number CCTCCNO: M 20251626, is used.

2. The strain SARIP-03 as described in claim 1, characterized in that, The 16S rDNA sequence of the strain SARIP-03 is shown in SEQ ID No.

1.

3. A biological agent, characterized in that, It contains the strain SARIP-03 as described in any one of claims 1-2.

4. The biological agent as described in claim 3, characterized in that, The biological agent also includes Bacillus pasteurellus, which is selected from one or more strains with accession numbers CCTCC NO: M 2024524, CCTCC M 2025059 and ATCC11859.

5. The biological agent as described in claim 4, characterized in that, The ratio of viable bacteria of strain SARIP-03 to Bacillus pasteurellus was (0.5–3):

1.

6. A composition, characterized in that, Includes the strain SARIP-03 as described in any one of claims 1-2 or the biological agent as described in any one of claims 3-5, and calcification solution.

7. The composition according to claim 6, characterized in that, The calcification solution comprises calcium salt and urea, wherein the molar ratio of the calcium salt to urea is 1:(1-3); And / or, the ratio of the concentration of the strain SARIP-03 to the concentration of the calcification solution is (1×10⁻⁶). 9 ~10×10 9 cfu / mL: 1mol / L.

8. The composition according to claim 7, characterized in that, The calcium salt is selected from one or more of calcium chloride, calcium nitrate, calcium lactate, calcium formate, and calcium acetate; And / or, based on the total volume of the calcification solution, the concentration of the calcium salt is 0.5 to 10 mol / L.

9. Use of the strain SARIP-03 as described in any one of claims 1-2, or the biological agent as described in any one of claims 3-5, or the composition as described in any one of claims 6-7 in inducing calcium carbonate deposition and solidification, and / or strengthening and / or restoring bone artifacts.

10. A method for reinforcing or repairing bone artifacts, characterized in that, The procedure includes the following steps: treating bone artifacts with the strain SARIP-03 as described in any one of claims 1-2, or the biological agent as described in any one of claims 3-5, or the composition as described in any one of claims 6-7.

11. The method as described in claim 10, characterized in that, The bone artifacts were selected from unearthed ivory.

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