CN103113214A - Method for adjusting and controlling structure and shape of calcium oxalate crystals by using colibacillus secretion - Google Patents
Method for adjusting and controlling structure and shape of calcium oxalate crystals by using colibacillus secretion Download PDFInfo
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- CN103113214A CN103113214A CN2013100524290A CN201310052429A CN103113214A CN 103113214 A CN103113214 A CN 103113214A CN 2013100524290 A CN2013100524290 A CN 2013100524290A CN 201310052429 A CN201310052429 A CN 201310052429A CN 103113214 A CN103113214 A CN 103113214A
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Abstract
The invention relates to a method for adjusting and controlling the structure and shape of calcium oxalate crystals by using colibacillus secretion. The method comprises the following steps of: mixing the colibacillus secretion with a matrix solution of which the ratio of [Ca2<+>] to [C2O4<2->] is (19.5-20.5):1, wherein the volume ratio is 1:(8-80); performing constant temperature incubation, and staying overnight; and washing and drying to obtain pure acalculous type calcium oxalate dehydrate crystals or a mixture of the calcium oxalate dehydrate crystals and calcium oxalate monohydrate crystals, wherein the colibacillus secretion is prepared through the steps of inoculating TOP10 single colonies of colibacillus to an LB liquid culture medium, performing low-speed oscillation on the culture medium at a constant temperature, staying the culture medium overnight, and performing high-speed centrifugation on the original bacterium liquid to obtain the colibacillus secretion. The method has the advantages that the calcium oxalate dehydrate crystals formed through colibacillus secretion adjustment and control have the characteristics of uniform size and consistent shape, the content of the calcium oxalate dehydrate crystals can be adjusted according to different additions, and the preparation condition is simple and non-hazardous, so that the method can be widely used in various fields.
Description
Technical field
The invention belongs to technological field of biochemistry, be specifically related to a kind of novel method of the regulation and control of calcium oxalate crystals structure.
Background technology
Urinary system calculus is worldwide common disease, frequently-occurring disease, and human knowledge's lithiasis has the centuries, and its sickness rate is in rising trend in recent years.Calculus is the product of pathology mineralising in organism, belongs to abnormal mineralization.Abnormal biomineralization process relates to the multiple subjects such as medical science, biology, chemistry, crystallography, materialogy and mineralogy, thereby has also caused these domain experts' extensive concern and great attention.Mainly at present the moiety, detection method, treatment plan of research calculus and with macromolecular effect etc.Most of calculus can be mixed two or more composition.Under physiology urine pH value condition, easily form oxalate calculus, thereby calcinm oxalate calculus is the most common.In urinary stone disease (upper lithangiuria), having more than 80% is calcinm oxalate calculus.Wherein Calcium oxalate monohydrate is easy to gather the formation calculus.Think at present, the formation of urinary stone disease is the process of a multifactor participation, is in cell and the interactional result of extracellular activity.But the formation mechanism of calculus is illustrated not yet fully so far, the prevention of calculus be there is no to very good method.Research is found, different inducements, and pattern and the structure of the calculus of generation also are not quite similar, so the formation influence factor of research calcinm oxalate calculus has realistic meaning.
Intestinal bacteria are that the dust Xi Shi lived in the humans and animals enteron aisle belongs to bacterium.Intestinal bacteria are Gram-negative tyrothricin, 0.5 * 1～3 microns of sizes, and whole body flagellum, can move, without gemma.The various saccharides of fermenting is produced acid, aerogenesis, be the normal perch bacterium in the humans and animals enteron aisle, after baby due, with lactation, enter enteron aisle, accompany all the life with the people, its Metabolic activity can suppress the microorganism growth of decomposing protein in enteron aisle, reduces the harm of protein degradation production to human body.Except some bacterial strain can produce enterotoxin, the people is obtained outside gastroenteritis, generally can not cause a disease.Tens kinds more than of the colibacillary endogenous metabolism products such as amino acid, organic carboxyl acid, carbohydrate, nucleosides and derivative thereof detected in intestinal bacteria secretory product.And amino acid, organic carboxyl acid, carbohydrate, nucleosides and derivative thereof etc. have great impact to biomineralization.So, research intestinal bacteria secretory product just has directive significance to human research and treatment lithiasis, for human treatment's lithiasis has been opened up new approach, furtherly, research intestinal bacteria secretory product has important directive significance on the impact of calculus main component calcium oxalate crystal more to the treatment calculus.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method of intestinal bacteria secretory product regulation and control calcium oxalate crystals structure, utilize intestinal bacteria secretory product to obtain the crystal containing non-calculus type calcium oxalate dihydrate, and its content can, by the consumption regulation and control of intestinal bacteria secretory product, can only not obtain pure calculus type Calcium oxalate monohydrate and do not add intestinal bacteria secretory product.
The present invention solves the problems of the technologies described above taked technical scheme: a kind of method of intestinal bacteria secretory product regulation and control calcium oxalate crystals structure, intestinal bacteria secretory product and [Ca
2+]: [C
2o
4 2-]=(19.5～20.5): 1 Matrix Solution mixes, the volume ratio of intestinal bacteria secretory product and Matrix Solution is 1:(8～80), 37～38 ℃ of constant temperature culture, spend the night, through washing, drying obtains pure non-calculus type calcium oxalate dihydrate crystal or the mixture of non-calculus type calcium oxalate dihydrate crystal and Calcium oxalate monohydrate crystal, wherein, being prepared as of described intestinal bacteria secretory product: by the mono-colony inoculation of intestinal bacteria TOP10 in LB liquid nutrient medium (Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L), under 37～38 ℃ of constant temperature, 100～120rpm shaking culture is spent the night, obtain original bacterium liquid, original bacterium liquid 4000rpm is centrifugal, obtain intestinal bacteria secretory product.
Concrete, the mol ratio of calcium ion and oxalate denominationby can be [Ca
2+]: [C
2o
4 2-]=19.5:1,19.6:1,19.7:1,19.8:1,19.9:1,20:1,20.1:1,20.2:1,20.3:1,20.4:1 or 20.5:1.
The volume ratio of intestinal bacteria secretory product and Matrix Solution can be 1:8,1:8.1,1:8.2,1:8.3,1:8.4,1:8.5,1:9,1:10,1:12,1:14,1:16,1:18,1:20,1:25,1:30,1:35,1:40,1:45,1:50,1:55,1:60,1:65,1:70,1:75 or 1:80.
On the basis of such scheme, in the non-calculus type calcium oxalate dihydrate crystal of gained and the mixture of Calcium oxalate monohydrate crystal, the content of non-calculus type calcium oxalate dihydrate crystal is not less than 15%.
On the basis of such scheme, the volume ratio of described intestinal bacteria secretory product and Matrix Solution is 1:8, and the gained crystal is pure non-calculus type calcium oxalate dihydrate crystal.It is pointed out that the consumption that further suitably increases intestinal bacteria secretory product is also on obtaining the not impact of pure calcium oxalate dihydrate crystal.
On the basis of such scheme, described Matrix Solution is included as the aqueous solution of calcium chloride and sodium oxalate.
On the basis of such scheme, the constant temperature culture time of described intestinal bacteria secretory product in Matrix Solution is 22～26 hours, and the constant temperature culture time in the mono-bacterium colony liquid medium within of described intestinal bacteria TOP10 is 22～26 hours.
On the basis of such scheme, described washing, dry for after discarding supernatant liquor, bottom crystal priority water and ethanol are respectively washed sample twice.
On the basis of such scheme, the preservation of described intestinal bacteria and secretory product thereof is all at 4 ℃.
The invention has the beneficial effects as follows:
The calcium oxalate dihydrate formed with intestinal bacteria secretory product regulation and control has big or small homogeneous, consistent appearance, characteristics that transformation efficiency is high, and can regulate the content of calcium oxalate dihydrate crystal according to the difference of add-on, for research treatment lithiasis provides thinking and method; And step is simple, preparation condition is simple, nothing is dangerous, can be widely used in the aspects such as bio-pharmaceuticals, health care, scientific research, industrial production.
The accompanying drawing explanation
The scanning electron microscope (SEM) photograph that Fig. 1 is comparative sample.
The scanning electron microscope (SEM) photograph that Fig. 2 is embodiment 1 sample.
Fig. 3 be comparative sample (curve a) with the X-ray diffractogram of embodiment 1 sample (curve b).
The scanning electron microscope (SEM) photograph that Fig. 4 is embodiment 2 samples.
The scanning electron microscope (SEM) photograph that Fig. 5 is embodiment 3 samples.
Embodiment
Further illustrate the present invention below by embodiment.
Comparative example
Prepare respectively the calcium chloride of 100mM and the sodium oxalate solution of 5mM.The calcium chloride solution that to get 40ml concentration be 100mM, in the 100ml Erlenmeyer flask, adds the sodium oxalate solution that 40ml concentration is 5mM, is placed in 37.5 ℃ of constant incubators and places 24 hours.Discard supernatant liquor, bottom crystal successively water, ethanol is respectively washed sample twice, and the gained sample is placed in the furnace pot Air drying and is placed on sample hose and preserves.Scanning electron microscope (SEM) photograph is obtained by field emission scanning electron microscope (Philips company, XL-30E, SEM), sets sweep voltage 3kV, and as can be seen from Figure 1, product is the Calcium oxalate monohydrate of sheet.Further by Fig. 3, (the curve a) content of known calcium oxalate dihydrate is 0%.
Embodiment 1
The preparation of intestinal bacteria secretory product:
The mono-bacterium colony of intestinal bacteria TOP10 of preserving under 4 ℃ of pickings, be inoculated in the LB liquid nutrient medium, the shaking bath device that is placed in 37.5 ℃ of constant temperature obtains original bacterium liquid in 24 hours with the 110rpm shaking culture, and the centrifugal intestinal bacteria secretory product that obtains of original bacterium liquid 4000rpm, be placed under 4 ℃ and save backup.
The preparation of non-calculus type calcium oxalate dihydrate crystal:
Prepare respectively the calcium chloride of 100mM and the sodium oxalate solution of 5mM.The calcium chloride solution that to get 40ml concentration be 100mM, in the 100ml Erlenmeyer flask, adds intestinal bacteria secretory product 10ml, then add the sodium oxalate that 40ml concentration is 5mM in Erlenmeyer flask, and concussion is placed in 37.5 ℃ of constant incubators and places 24 hours.Discard supernatant liquor, bottom crystal successively water and ethanol is respectively washed sample twice, and the gained sample is placed in the furnace pot Air drying and is placed on sample hose and preserves.Scanning electron microscope (SEM) photograph is obtained by field emission scanning electron microscope (Philips company, XL-30E, SEM), sets sweep voltage 3kV, and as can be seen from Figure 2, product is the calcium oxalate dihydrate of sheet.Further the content by the known calcium oxalate dihydrate of Fig. 3 (curve b) is 100%.
Embodiment 2
The preparation of intestinal bacteria secretory product is identical with embodiment 1.
Prepare respectively the calcium chloride of 100mM and the sodium oxalate solution of 5mM.The calcium chloride solution that to get 40ml concentration be 100mM, in the 100ml Erlenmeyer flask, adds intestinal bacteria secretory product 1ml, then add the sodium oxalate that 40ml concentration is 5mM in Erlenmeyer flask.Concussion is placed in 37.5 ℃ of constant incubators and places 24 hours.Discard supernatant liquor, bottom crystal successively water, ethanol is respectively washed sample twice.Gained sample (Fig. 4) is placed in the furnace pot Air drying and is placed on sample hose and preserves.Recording the shared percentage composition of calcium oxalate dihydrate is 15%, and all the other are the Calcium oxalate monohydrate crystal, illustrates that the intestinal bacteria secretory product of minute quantity just has pair control action kou of caoxalate crystal formation.
Embodiment 3
The preparation of intestinal bacteria secretory product is identical with embodiment 1.
Prepare respectively the calcium chloride of 100mM and the sodium oxalate solution of 5mM.The calcium chloride solution that to get 40ml concentration be 100mM, in the 100ml Erlenmeyer flask, adds intestinal bacteria secretory product 5ml, then add the sodium oxalate that 40ml concentration is 5mM in Erlenmeyer flask.Concussion is placed in 37.5 ℃ of constant incubators and places 24 hours.Discard supernatant liquor, bottom crystal successively water, ethanol is respectively washed sample twice.Gained sample (Fig. 5) is placed in the furnace pot Air drying and is placed on sample hose and preserves.The percentage composition that records calcium oxalate dihydrate is 43.4%, and all the other are the Calcium oxalate monohydrate crystal, and the crystallisate ratio increase with the increase calcium oxalate dihydrate of intestinal bacteria secretory product consumption is described.
Claims (7)
1. the method for an intestinal bacteria secretory product regulation and control calcium oxalate crystals structure, is characterized in that: intestinal bacteria secretory product and [Ca
2+]: [C
2o
4 2-]=(19.5～20.5): 1 Matrix Solution mixes, the volume ratio of intestinal bacteria secretory product and Matrix Solution is 1:(8～80), 37～38 ℃ of constant temperature culture, spend the night, through washing, drying obtains pure non-calculus type calcium oxalate dihydrate crystal or the mixture of non-calculus type calcium oxalate dihydrate crystal and Calcium oxalate monohydrate crystal, wherein, being prepared as of described intestinal bacteria secretory product: by the mono-colony inoculation of intestinal bacteria TOP10 in the LB liquid nutrient medium, under 37～38 ℃ of constant temperature, 100～120rpm shaking culture is spent the night, obtain original bacterium liquid, original bacterium liquid 4000rpm is centrifugal, obtain intestinal bacteria secretory product.
2. intestinal bacteria secretory product according to claim 1 regulates and controls the method for calcium oxalate crystals structure, it is characterized in that: in the non-calculus type calcium oxalate dihydrate crystal of gained and the mixture of Calcium oxalate monohydrate crystal, the content of non-calculus type calcium oxalate dihydrate crystal is not less than 15%.
3. intestinal bacteria secretory product according to claim 1 and 2 regulates and controls the method for calcium oxalate crystals structure, it is characterized in that: the volume ratio of described intestinal bacteria secretory product and Matrix Solution is 1:8, and the gained crystal is pure non-calculus type calcium oxalate dihydrate crystal.
4. the method for intestinal bacteria secretory product regulation and control calcium oxalate crystals structure according to claim 1, is characterized in that: the aqueous solution that described Matrix Solution is calcium chloride and sodium oxalate.
5. intestinal bacteria secretory product according to claim 1 regulates and controls the method for calcium oxalate crystals structure, it is characterized in that: the constant temperature culture time of described intestinal bacteria secretory product in Matrix Solution is 22～26 hours, and the constant temperature culture time in the mono-bacterium colony liquid medium within of described intestinal bacteria TOP10 is 22～26 hours.
6. intestinal bacteria secretory product according to claim 1 regulates and controls the method for calcium oxalate crystals structure, it is characterized in that: described washing, dry for after discarding supernatant liquor, bottom crystal priority water and ethanol are respectively washed sample twice.
7. intestinal bacteria secretory product according to claim 1 regulates and controls the method for calcium oxalate crystals structure, and it is characterized in that: the preservation of described intestinal bacteria and secretory product thereof is all at 4 ℃.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103884746A (en) * | 2014-03-06 | 2014-06-25 | 同济大学 | Regulation and control method for thermodynamics and dynamics of melamine on lithogenesis |
| CN106093364A (en) * | 2016-05-27 | 2016-11-09 | 长春迪瑞医疗科技股份有限公司 | A kind of physiology crystallizes in-vitro simulated and verification method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102115440A (en) * | 2010-12-31 | 2011-07-06 | 马艳荣 | Method for producing calcium oxalate by using carbide slag |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102115440A (en) * | 2010-12-31 | 2011-07-06 | 马艳荣 | Method for producing calcium oxalate by using carbide slag |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103884746A (en) * | 2014-03-06 | 2014-06-25 | 同济大学 | Regulation and control method for thermodynamics and dynamics of melamine on lithogenesis |
| CN106093364A (en) * | 2016-05-27 | 2016-11-09 | 长春迪瑞医疗科技股份有限公司 | A kind of physiology crystallizes in-vitro simulated and verification method |
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