CN115873759B - Sarcina barbita strain and application thereof in bone cultural relic protection - Google Patents
Sarcina barbita strain and application thereof in bone cultural relic protection Download PDFInfo
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Abstract
The invention relates to an sarcina barbita strain and application thereof in protecting bone cultural relics, belonging to the field of protecting bone cultural relics. An sarcina barbita strain (Sporosarcina pasteurii) LZG-2107 having a accession number in the chinese microbiological bacterial culture collection center of the committee for common microbiological culture: CGMCC No.24480, the preservation date is 2022, 3 and 8. The invention can produce urease and induce the formation of calcium carbonate, and the formed calcium carbonate can enhance the biomechanical properties of the inside and the surface of fragile bone cultural relics and realize the reinforcement and protection of the bone cultural relics. Meanwhile, the invention also has antibacterial activity on the bone cultural relics in the soil, prevents or slows down the invasion and deterioration of the bone cultural relics by the pathogenic bacteria in the soil, provides a borrowable experience for the development and application of the sarcina baronii, and has good application prospect in the aspect of protecting the bone cultural relics.
Description
Technical Field
The invention belongs to the field of protection of bone cultural relics, and particularly relates to an sarcina baronii strain and application thereof in the protection of bone cultural relics
Background
Bone cultural relics are important basis for researching human development history and natural development history, and are precious and resident in a category containing abundant historical information. After long-time burial, bone cultural relics are affected by various burial environmental factors, such as phenomena that organic matters (mainly bone collagen and the like) are decomposed and the biomechanical performance is obviously reduced due to the fact that the bone cultural relics are easily affected by soil microorganisms. The partial bone relics of the original site exhibition are exposed under different environmental conditions for a long time, so that phenomena such as rapid water loss, various micro attacks and the like are generated, and various degradation manifestations such as cracking, broken, crisp powder, decay and the like are generated. Therefore, the bone relics need to be effectively protected and reinforced to meet the requirements of long-term storage and exhibition.
The organic polymer material is a bone cultural relic protection material with more reports at present and mainly comprises natural resin, nitrovarnish, polyethylene glycol, acrylic resin, polyvinyl acetate, organic silicon and the like, but the organic polymer material has some defects: some organic polymer materials turn yellow and have reduced strength after being used for several years; some organic polymer materials have certain toxicity, and organic solvents used in the operation process of other organic polymer materials can cause toxicity to the health of operators. Modified polymer materials are also gradually applied to the protection of bone cultural relics in recent years, for example, modified water glass materials are used for protecting ancient ivory, and nano TiO is adopted 2 The modified Paraloid B72 (a common acrylic resin type cultural relic protection material) is also applied to the protection of the bone cultural relics, but the modified polymer material has the defect of poor compatibility with the components of the bone cultural relics. The organic-inorganic composite material capable of playing a role in protection is also applied to the protection of bone relics in the present year, for example, a learner mixes the extracted bone collagen with nano apatite to repair the bone relics, but the organic-inorganic composite material has higher manufacturing cost, and organic components in the organic-inorganic composite material are easily decomposed by harmful bacteria in the bone relics and the environment.
Disclosure of Invention
The invention provides an sarcina barbita strain and application thereof in protecting bone cultural relics, and aims to better protect the bone cultural relics.
The technical scheme adopted by the invention is as follows: an sarcina barbita strain (Sporosarcina pasteurii) LZG-2107 having a accession number in the chinese microbiological bacterial culture collection center of the committee for common microbiological culture: CGMCC No.24480, collection date 2022, 3 month 8 days, address: beijing, chaoyang area, north Chen Xili No.1, 3, china academy of sciences, microbiological institute.
The sarcina barbita strain (Sporosarcina pasteurii) LZG-2107 is separated from the surface of bone cultural relics, and has the strain morphological characteristics that: the cell size of the strain is 0.6-0.7μm×1.3μm-2.5 μm, and the strain is rod-shaped and gram-positive. The spore is elliptic, the size is 0.5-0.8 mu m multiplied by 1.0-1.5 mu m, and the spore can move; colony morphological characteristics: the bacterial colony is round, white and opaque, the surface is smooth and moist, and the edge is regular; the physiological and biochemical characteristics are as follows: urease capable of decomposing urea can be produced, and the growth rate is highest at 30 ℃.
The nucleotide sequence of the 16S rDNA of the strain is shown in SEQ ID No. 1.
Application of Sarcina barbita strain (Sporosarcina pasteurii) LZG-2107 in protecting bone cultural relics.
The sarcina barbita (Sporosarcina pasteurii) LZG-2107 has good antibacterial effect on bone cultural relics in soil.
The sarcina barbita (Sporosarcina pasteurii) LZG-2107 has the effect of inducing to produce calcium carbonate to play a role in reinforcing bone cultural relics.
The invention has the beneficial effects that based on the determination of the biomechanical index of the bone cultural relics, the sarcina baronii (Sporosarcina pasteurii) LZG-2107 is separated from the surface of the backbone of a certain mineworker tomb Chen Lieguan DF343 for the first time, and is a new variety. The sarcina baronii LZG-2107 can generate urease and induce to form calcium carbonate, and the formed calcium carbonate can enhance the biomechanical properties of the inside and the surface of fragile bone cultural relics and realize the reinforcement and protection of the bone cultural relics. Meanwhile, the sarcina baronii LZG-2107 also has antibacterial activity on bone cultural relics in soil (the bone cultural relics can be invaded and the biomechanical property of the bone cultural relics is reduced when the sarcina baronii LZG-2107 exists in the soil environment), and prevents or slows down the invasion and the deterioration of the bone cultural relics by the bone cultural relics. Because related researches on the sarcina baronii LZG-2107 are blank, the results provide a borrowable experience for the development and application of the sarcina baronii LZG-2107, and the sarcina baronii LZG-2107 has the effects of inducing calcium carbonate to play a role in reinforcing bone cultural relics and inhibiting bone cultural relics in soil, so that the sarcina baronii LZG-2107 has a good application prospect in the aspect of protecting bone cultural relics.
Drawings
FIG. 1 is a diagram of the energy spectrum analysis of the reinforcement product with calcium carbonate;
FIG. 2 is an XRD analysis of the consolidated product with calcium carbonate;
FIG. 3 is a chart of FTIR analysis of the consolidated product with calcium carbonate;
FIG. 4 is a graph of the microtopography (40000 times) of the consolidated product.
Detailed Description
EXAMPLE 1 isolation, screening and purification of the eight-fold coccus Pasteurensis (Sporosarcina pasteurii) LZG-2107 Strain
After the target spinal surface is gently scraped by a sterile absorbent cotton swab, the absorbent cotton swab is rapidly stored in a sterile container in a sealed manner, and the absorbent cotton swab is placed into a sterile water with a certain volume in a sterile super clean bench to sufficiently shake and elute microorganisms possibly existing in a sample, so as to obtain an eluent (bacterial liquid A).
1ml of bacterial liquid A is added into 100ml of culture medium A, and shake culture is carried out for 24 hours in a constant temperature shaking table at 30 ℃ and 200 revolutions per minute to obtain a bacterial enrichment liquid.
100. Mu.L of the bacteria-enriched liquid was dropped into a culture plate (culture plate B) prepared from the medium B and applied, and after the application, the culture plate was cultured in a constant temperature incubator at 30℃for 48 hours, and whether or not a characteristic colony of purple color was present was observed.
The characteristic bacterial colony which is purple on the culture plate B is picked by a sterile toothpick and placed in a culture medium C, and shake culture is carried out for 24 hours in a constant temperature shaking table at 30 ℃ and 200 revolutions per minute, thus obtaining bacterial liquid (bacterial liquid C).
100 mu L of bacterial liquid C is coated on a culture plate B again, the plate is cultured in a constant temperature incubator at 30 ℃ for 24 hours after coating, and then purple characteristic bacterial colonies are picked and cultured in the culture medium C to obtain bacterial suspension. The step is repeated for 3 times to obtain the target strain.
Medium A (g/L): 10g of yeast extract powder, 20g of ammonium chloride, 8.2g of anhydrous sodium acetate and 60.6g of urea, and the natural pH value.
Medium B composition (g/L): 10g of peptone, 3g of beef extract, 5g of sodium chloride, 15g of bacterial agar powder, 2mL of 1.6% bromocresol purple and 15g of agar powder, and the natural pH value.
Medium C composition (g/L): 10g of yeast extract powder, 20g of ammonium chloride, 5g of urea and pH9.0.
Example 2 identification of an sarcina Pacifica strain (Sporosarcina pasteurii) LZG-2107
(1) Morphological and physiological Biochemical characteristics of Strain LZG-2107
Morphological characteristics of the strain: the cell size of the strain is 0.6-0.7μm×1.3μm-2.5 μm, the strain is in the shape of rod, gram positive bacteria and spore ellipse, the size is 0.5-0.8μm×1.0μm-1.5 μm, and the strain can move;
colony morphology characterization: the bacterial colony is round, white and opaque, the surface is smooth and moist, and the edge is regular;
physiological and biochemical characteristics: urease capable of decomposing urea can be produced, and the growth rate is highest at 30 ℃.
(2) Identification of LZG-2107 Strain 16S rDNA
Inoculating the target bacterial liquid into a culture medium D, placing the culture medium D into a constant-temperature shaking table at 30 ℃ for shake culture for 24 hours at 200 revolutions per minute, and performing expansion culture of bacterial to obtain bacterial suspension. The bacterial suspension was sent to Jilin vincristoceme, inc. for sequencing, and the 16S rDNA nucleotide sequence of this strain was as set forth in SEQ ID No. 1.
Medium D composition (g/L): 15g of casein peptone, 5g of soybean peptone, 5g of sodium chloride, 20g of urea and natural pH value.
The species of the strain is determined by Blast similarity comparison with the existing 16S rDNA sequence in NCBI, and the strain is finally determined to be the genus Sporosarcina by combining with morphological characteristics and comparison results of the strain, and is named as Sporosarcina bardans LZG-2107.
EXAMPLE 3 antibacterial Activity test of Sporoboccus barbituric LZG-2107
Experiments on the antibacterial activity of the sarcina bardana LZG-2107 are carried out by referring to an evaluation method of the antibacterial activity of QB/T2738-2012 daily chemical products.
Inoculating the sarcina barbita LZG-2107 bacterial liquid into a culture medium D, placing the sarcina barbita LZG-2107 bacterial liquid into a constant temperature shaking table at 30 ℃ for shake culture for 24 hours at 200 rpm, and performing expansion culture of strains to obtain LZG-2107 bacterial suspension.
The common disease bacteria of bone cultural relics are selected from Aspergillus versicolor (CGMCC 3.3885), acremonium gracilis (CGMCC 3.5919) and Mortierella sparganii (CGMCC 3.14306). Inoculating the sclerotin cultural relic disease bacteria into a PDA liquid culture medium, and placing the culture medium into a constant-temperature shaking table at 30 ℃ for shake culture for 24 hours at 200 revolutions/min to obtain a disease bacteria suspension. Diluting LZG-2107 bacterial suspension and disease bacterial suspension to 1x10 respectively with sterile PBS solution 4 -9x10 4 cfu/mL。
Mixing a certain amount of LZG-2107 bacterial suspension and a certain amount of disease bacterial suspension to form a test sample, placing the test sample into a sterilized test tube, placing for a period of time, sucking 1mL of the test sample, coating the test sample into a PDA solid culture medium plate, placing the PDA solid culture medium plate into a constant temperature incubator at 30 ℃ for culturing for 48 hours, and counting viable bacteria colonies.
The preparation method comprises the steps of replacing LZG-2107 bacterial suspension with sterile PBS, mixing a certain amount of sterile PBS with a certain amount of disease bacterial suspension to form a control sample, placing the control sample into a sterilized test tube, placing for a period of time, sucking 1mL of the control sample, coating the control sample into a PDA solid culture medium plate, placing the PDA solid culture medium plate into a constant temperature incubator at 30 ℃ for culturing for 48 hours, and counting viable bacterial colonies.
The above experiment was repeated 3 times to determine the average colony count of the test sample and the average colony count of the control sample.
PDA medium composition g/L:200g of potato, 20g of glucose, 15g of agar and pH5.4-5.8.
PBS solution: 8.0g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4 、0.24g KH 2 PO 4 Dissolving in 800mL distilled water, adjusting the solution to 7.4 with HCl, and finally adding distilled water to fix the volume to 1L.
The formula of the bacteriostasis rate:
wherein A- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -, a control sample was an average colony count).
B- -average colony count of test samples.
TABLE 1 inhibition ratio of sarcina Babesia Babesii LZG-2107 to bone cultural relics disease bacteria
The results in Table 1 show that the sarcina bararum LZG-2107 has good growth inhibition effect on bone cultural relics disease bacteria Aspergillus versicolor, acremonium gracilis and Sparganium sacculus.
The following specifically describes the configuration and the effects of the implementation of the bone relic reinforcing liquid on the compressive strength and the porosity of the bone relic by examples 4 to 6.
Example 4
In a 100mL system, the feed contains 2.0mol/L calcium chloride, 2.0 percent of urea, 2.0 percent of sarcina bardana LZG-2107, and the balance of water. Under the condition of room temperature, soaking and reinforcing the bone relic (No. I) for 24 hours, taking out and airing to obtain the reinforced bone relic (No. I-1), and measuring the compressive strength and the porosity of the bone relic (No. I) and the bone relic (No. I-1).
Example 5
In a 100mL system, the feed contains 2.0mol/L calcium chloride, 2.0 percent of urea, 2.0 percent of sarcina bardana LZG-2107, and the balance of water. And under the condition of room temperature, soaking and reinforcing the bone relic (No. I) for 48 hours, taking out and airing to obtain the reinforced bone relic (No. I-2), and measuring the compressive strength and the porosity of the bone relic (No. I-2). .
Example 6
In a 100mL system, the feed contains 2.0mol/L calcium chloride, 2.0 percent of urea, 2.0 percent of sarcina bardana LZG-2107, and the balance of water. And under the condition of room temperature, soaking and reinforcing the bone relic (No. I) for 72 hours, taking out and airing to obtain the reinforced bone relic (No. I-3), and measuring the compressive strength and the porosity of the bone relic (No. I-3).
TABLE 2 compressive Strength and porosity of bone relics
The results in Table 2 show that the compressive strength and the porosity of the bone cultural relics are remarkably improved after the eight-fold coccus pasteuris LZG-2107 is soaked and reinforced.
In conclusion, the sarcina baronii LZG-2107 provided by the invention is natural bacteria, is safe and harmless, greatly protects the safety of operators, remarkably improves the mechanical properties of bone cultural relics reinforced by the sarcina baronii LZG-2107, and can inhibit the growth of bone cultural relics by identifying that the reinforced product is calcium carbonate and the biocompatibility of the calcium carbonate and the bone cultural relics is extremely high. Therefore, the strain has definite application effect and application prospect for strengthening and inhibiting bacteria of bone relics.
Claims (4)
1. An sarcina barbita strain (Sporosarcina pasteurii) LZG-2107, characterized in that: the preservation number of the microbial strain in the common microorganism center of the China Committee for culture Collection of microorganisms is as follows: CGMCC No.24480, collection date 2022, 3 month 8 days, address: beijing, chaoyang area, north Chen Xili No.1, 3, china academy of sciences, microbiological institute.
2. An sarcina barbita strain (Sporosarcina pasteurii) LZG-2107 as claimed in claim 1, wherein: the sarcina barbita strain (Sporosarcina pasteurii) LZG-2107 is separated from the surface of bone cultural relics, and the strain morphological characteristics are as follows: the cell size of the strain is 0.6-0.7μm×1.3-2.5 μm, the strain is in the shape of a rod, gram-positive bacteria and spore ellipse, and the cell size is 0.5-0.8μm×1.0-1.5 μm and can move; colony morphological characteristics: the bacterial colony is round, white and opaque, the surface is smooth and moist, and the edge is regular; the physiological and biochemical characteristics are as follows: urease capable of decomposing urea can be produced, and the growth rate is highest at 30 ℃.
3. An sarcina barbita strain (Sporosarcina pasteurii) LZG-2107 as claimed in claim 1, wherein: the nucleotide sequence of the 16S rDNA of the strain is shown in SEQ ID No. 1.
4. The application of sarcina barbita strain (Sporosarcina pasteurii) LZG-2107 in protecting bone cultural relics, which is characterized in that the protection has an inhibiting effect on aspergillus versicolor CGMCC3.3885, acremonium graminis CGMCC3.5919 and Sparganium barbituric bacteria CMGCC3.14306, and has the effect of inducing calcium carbonate to play a role in reinforcing bone cultural relics.
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