CN118006465A - Heat-resistant protective agent for leptospira - Google Patents
Heat-resistant protective agent for leptospira Download PDFInfo
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- CN118006465A CN118006465A CN202410283225.6A CN202410283225A CN118006465A CN 118006465 A CN118006465 A CN 118006465A CN 202410283225 A CN202410283225 A CN 202410283225A CN 118006465 A CN118006465 A CN 118006465A
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- leptospira
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- resistant protective
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- 241000589902 Leptospira Species 0.000 title claims abstract description 57
- 239000003223 protective agent Substances 0.000 title claims abstract description 32
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a leptospira heat-resistant protective agent, which comprises 5-7% w/v peptone, 2-3% w/v nano-phytosterol, 1-2% w/v sodium pyruvate, 1-2% w/v L-arginine, 1-2% w/v soluble starch, 2-3% w/v trehalose, 1-2% w/v vitamin B12 and 3-5% v/v dimethyl sulfoxide. The heat-resistant protective agent provided by the invention can ensure that the activity of leptospira can be effectively protected in the freeze-drying process and within a certain time after freeze-drying, the toxicity and immunogenicity of strains are maintained, the resuscitating and fermenting effects of the strains are good, and the protective agent is simple in preparation method, low in cost, suitable for mass production and convenient for subsequent research and production and use.
Description
Technical Field
The invention belongs to the field of microbial protectants, and particularly relates to a heat-resistant protectant for leptospira.
Background
Leptospirosis (leptospirosis for short) is an acute zoonotic infectious disease caused by pathogenic leptospirosis (leptospirosis for short). China is one of the most abundant countries of leptospira strain resources, provides an important research basis for the production of leptospira vaccines and the detection of epidemic diseases, and brings about no small obstruction to the preservation of the strain due to the growth characteristics of the strain. At present, the method for preserving the leptospira strain mainly comprises room temperature preservation, low temperature preservation, liquid nitrogen preservation and the like, wherein the room temperature preservation has the defects of reduced strain toxicity, strain pollution, time and labor waste, high cost and the like; the low-temperature and liquid nitrogen preservation cost is high, and the strain dehydration and denaturation can be caused, and the resuscitating of the strain and the survival rate after resuscitating can be influenced. In order to ensure stable and high-quality leptospira strains are obtained for a long time, a method and a strategy for long-term preservation of the leptospira strains need to be deeply studied, and a method for preserving leptospira with stability, time saving and low cost is found.
Disclosure of Invention
The invention aims to provide a heat-resistant protective agent for leptospira.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
A heat-resistant protecting agent for leptospira comprises peptone, nano-phytosterol, sodium pyruvate, L-arginine, soluble starch, trehalose, vitamin B 12 and dimethyl sulfoxide.
Further, the heat-resistant protective agent component comprises 5 to 7 percent w/v peptone and 2 to 3 percent w/v nano-phytosterol,
1 To 2 percent of sodium pyruvate, 1 to 2 percent of v L-arginine, 1 to 2 percent of soluble starch, 2 to 3 percent of trehalose, 1 to 2 percent of vitamin B 12 and 3 to 5 percent of dimethyl sulfoxide.
Further, the preparation method of the heat-resistant protective agent comprises the following steps:
1) Weighing peptone, dissolving in water for injection, continuously stirring until the peptone is completely dissolved, then sequentially adding nano-phytosterol, pyruvic acid and L-arginine, and continuously stirring until the peptone is completely dissolved to obtain a first solution;
2) Weighing soluble starch in water for injection, continuously stirring until the soluble starch is completely dissolved, sequentially adding trehalose and vitamin B12, and continuously stirring until the soluble starch is completely dissolved to obtain a second solution;
3) Mixing the first solution and the second solution in equal volume, then fixing the volume, adjusting the PH value to 7.2-7.4, sterilizing at 116 ℃ under high pressure for 20-30 minutes or filtering and sterilizing by a 0.22 mu m filter, and obtaining a third solution;
4) And measuring dimethyl sulfoxide, adding the dimethyl sulfoxide into the third solution, uniformly mixing to obtain the heat-resistant protective agent, and preserving at 2-8 ℃.
Further, the mixing ratio of the heat-resistant protective agent and the leptospira bacteria liquid is 1:2-1:4.
Further, the concentration of the leptospira bacteria solution is 1X 10 8～1×1010 strips/mL.
Compared with the prior art, the invention has the following advantages:
The heat-resistant protective agent provided by the invention can ensure the effective protection of leptospira activity in the freeze-drying process, maintain the virulence and immunogenicity of strains, and has good resuscitating and fermenting effects.
The nano-phytosterol (NanoActive phytosterol) is a novel water-soluble nano-liposome preparation of the phytosterol, and the phytosterol (phytosterol) is a natural physiologically active substance which is very beneficial to human bodies, is known as a key of life and widely exists in plants. Plant sterols have been found to have a cardiovascular disease inhibiting function in animal studies; it also has anticancer, antitumor, antiinflammatory, immunity enhancing, growth regulating, antiviral, and antioxidant effects. The nano active substance of the plant sterol with the grain diameter smaller than 30nm developed by utilizing a nano transmission system (NDS) completely solves the problem of solubility of the plant sterol, ensures the plant sterol to be stably wrapped in the nano carrier, and can greatly increase the absorption efficiency and bioavailability.
Sodium pyruvate is one of the most common pyruvate salts, and has a molecular formula of C 3H3NaO3, and is an endogenous small molecular substance, and sodium pyruvate and pyruvic acid both naturally exist in human bodies and participate in metabolism of various tissues and organs of the whole body. Sodium pyruvate is widely used as a buffer, excipient and antioxidant in medicine, diagnostic reagents and medical devices.
L-arginine is an organic compound with a molecular formula of C 6H14N4O2 and has a certain detoxification effect. It is present in large amounts in protamine and the like, and is also a basic component of various proteins, and is widely available, and belongs to a nutritional supplement.
Soluble starch (solublestarch) is a starch derivative obtained by treating starch with an oxidizing agent, an acid, glycerol, an enzyme or other means. The soluble starch has no reducing substance, stable chemical property, can improve the stability and solubility of the medicine, can be used as a carrier of the medicine, and protects the medicine.
Trehalose is a non-reducing disaccharide consisting of two glucose molecules with molecular formula C 12H22O11. Trehalose is a typical stress metabolite, and can form a unique protective film on the surface of cells under severe environmental conditions such as high temperature, high cold, high osmotic pressure, drying water loss and the like, so that the biological molecular structure is effectively protected from being damaged, and the life process and biological characteristics of a living body are maintained.
Vitamin B 12, also called cobalamin, is a polycyclic compound containing 3-valent cobalt and is the only vitamin containing metal elements. Vitamin B 12 is red crystal powder, has no smell and no taste, is soluble in water, is insoluble in ethanol, is insoluble in acetone, chloroform and diethyl ether, and vitamin B 12 is the only vitamin which can be absorbed only by the help of gastric parietal cell secretion (endogenous factors), participates in the preparation of bone marrow red blood cells, prevents pernicious anemia and prevents cerebral nerves from being damaged.
Dimethyl sulfoxide (DMSO) is a sulfur-containing organic compound, has a molecular formula of C 2H6 OS, has the characteristics of high polarity, high boiling point, good heat stability, aprotic property and water miscibility, can be dissolved in most organic matters such as ethanol, propanol, benzene, chloroform and the like, is known as a universal solvent, and is also a permeability protective agent, so that the DMSO can lower the freezing point of cells, reduce the formation of ice crystals, lighten the damage of free radicals to cells and change the permeability of a biological film to electrolytes, medicines, poisons and metabolites.
Detailed Description
The following detailed description is exemplary and is intended to provide further explanation of the invention. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the invention, but these changes and substitutions fall within the scope of the present invention.
The test methods used in the following examples are conventional methods, and the reagent and drug are from sigma-aldrich, unless otherwise specified.
Example 1 preparation and use of Heat-resistant protectant
1. The heat-resistant protective agent is prepared according to the component content in table 1, and the specific preparation method is as follows:
1.1 preparation of the first solution peptone is precisely weighed in water for injection according to the preparation components and the content of the protective agent shown in Table 1, continuously stirred until the peptone is completely dissolved, then sequentially weighed and added with nano phytosterol, pyruvic acid and L-arginine, and continuously stirred until the peptone is completely dissolved, thus obtaining the first solution.
1.2 Preparation of the second solution soluble starch is accurately weighed in the water for injection according to the preparation components and the content of the protective agent shown in Table 1, continuously stirred until the soluble starch is completely dissolved, then trehalose and vitamin B12 are sequentially added, and continuously stirred until the soluble starch is completely dissolved, thus obtaining the second solution.
1.3 Preparation of heat-resistant protective agent the first solution and the second solution are mixed in equal volume, then the volume is fixed, the PH value is regulated to 7.2-7.4, and the high-pressure sterilization is carried out at 116 ℃ for 20-30 minutes or the filtration sterilization is carried out by a 0.22 mu m filter, thus obtaining the third solution. And accurately measuring dimethyl sulfoxide according to the preparation components and the content of the protective agent shown in the table 1, adding the dimethyl sulfoxide into the third solution, and uniformly mixing to obtain the heat-resistant protective agent.
TABLE 1 formulation ingredients and content of leptospira heat resistant protectant
2 Leptospira strain freeze-drying
2.1 Strain recovery: inoculating the liquid nitrogen preserved leptospira strain into EMJH, culturing at 30+/-1 ℃ for 7-10 days, sampling, counting the bacterial cells under a dark field microscope, and harvesting bacterial liquid with the quantity of 10 9/mL, wherein the bacterial liquid is used as a resuscitated strain after being purely inspected to be qualified.
2.2 First-order seed preparation: inoculating resuscitated strain into EMJH culture medium according to 10% proportion, culturing at 30+ -1deg.C for 7-10 days, sampling, counting thallus under dark field microscope, and collecting bacterial liquid with the number of 10 9/mL, and pure checking to obtain first-stage seed.
2.3 Preparation of secondary seeds: inoculating the first-stage seeds into EMJH culture medium according to 10% proportion, culturing at 30+/-1 ℃ for 7-10 days, sampling, counting thalli under a dark field microscope, harvesting bacterial liquid with the quantity of 10 9/mL, and taking the bacterial liquid as the second-stage seeds after pure inspection.
2.4 Culturing with Leptospira icterus bacterial liquid as an example
Inoculating the second-level seeds of leptospira icterus into triangular flasks containing EMJH culture medium according to 10%, culturing for 5-7 days in a constant-temperature shaking incubator at 30+/-1 ℃, sampling, counting under a dark-field microscope, taking a heat-resistant protective agent and bacterial suspension to mix according to a ratio of 1:2 when the bacterial count result reaches more than 10 9.0/mL, subpackaging into penicillin bottles with 3mL specifications, subpackaging 1mL each bottle, and freeze-drying in a freeze dryer, wherein the freeze-drying procedure is shown in table 2.
Table 2 lyophilization procedure
3 Leptospira species survival rate
The original volume of the freeze-dried leptospira strain was reconstituted, counted by a bacterial counting plate under a dark field microscope, and the freeze-dried survival rate was calculated, and the results are shown in table 3. The result shows that the survival rate of leptospira of the protective agent 3-5 is high and can reach more than 88 percent.
The survival rate of the protective agent 6 is the lowest when no nano plant sterol and sodium pyruvate are added.
TABLE 3 results of Leptospira freeze-drying survival rate
Taking the formulation of the heat-resistant protective agent 3-5 as an example, mixing with bacterial suspension according to the ratio of 1:3 to 1:4, subpackaging into penicillin bottles with 3mL specifications, subpackaging 1mL each bottle, freeze-drying in a freeze dryer, freeze-drying according to the freeze-drying procedure shown in Table 2, re-dissolving the original volume of the freeze-dried leptospira strain, counting by a bacterial counting plate under a dark field microscope, and calculating the freeze-drying survival rate, wherein the result is shown in Table 4.
TABLE 4 Leptospira freeze-drying survival results
Example 2 detection of freeze-dried bacteria of leptospira
1. Routine examination of leptospira freeze-dried strains
The specific detection method comprises the following steps: the appearance, residual moisture, vacuum, solubility and purity of the lyophilized strain were measured according to the "chinese veterinary pharmacopoeia" of 2020 edition and the results are shown in table 5.
TABLE 5 routine examination of leptospira species after lyophilization
It can be seen from tables 3 to 5 that the leptospira lyophilized from the heat-resistant protectants 3, 4, 5 has a higher survival rate after lyophilization, and the appearance, vacuum, residual moisture, and purity meet the requirements.
2. Accelerated stability test
The freeze-dried leptospira strain is placed at 37+/-1 ℃ for 7 days, 14 days, 21 days and 28 days, and after the original volume is redissolved, the bacterial count plate is used for counting under a dark field microscope, the survival rate of the leptospira strain stored at 37+/-1 ℃ for different times is calculated, and the result is shown in table 6.
TABLE 6 accelerated stability test leptospira survival
As can be seen from the results in Table 6, the survival rate of the leptospira strain after freeze-drying is not less than 80% after the leptospira strain is placed at 37+/-1 ℃ for 7 days, 14 days, 21 days and 28 days, which shows that the heat-resistant protective agent can ensure the activity and recovery effect of the leptospira within a certain period of time.
EXAMPLE 3 toxicity test of lyophilized bacteria
1. Strains were virulent to hamsters before and after lyophilization
The method comprises the steps of respectively taking the leptospira strains before and after freeze-drying, re-dissolving the freeze-dried strains by water for injection of an original volume, injecting 5 hamsters of 40-50 g into the abdominal cavity, feeding each 1mL in an isolator, continuously observing for 10 days, and recording the morbidity and mortality of the hamsters. Hamsters were purchased from beijing velutinin limited. The results are shown in Table 7.
TABLE 7 toxicity of lyophilized strains on hamster
As can be seen from the results in Table 7, the leptospira species had no change in hamster virulence before and after lyophilization, and all died 5/5. The heat-resistant protective agent disclosed by the invention can ensure that the toxicity of leptospira after freeze-drying is equivalent to that before freeze-drying.
2. Accelerating the virulence of the end-of-stability test period leptospira species to hamsters
Respectively taking acceleration stabilization experiment 37+ -1deg.C, preserving to 28 days leptospira strain, re-dissolving with water for injection of original volume, injecting 5 hamsters 40-50 g into abdominal cavity, feeding each 1mL in isolator, continuously observing for 10 days, and recording hamster morbidity and mortality. Hamsters were purchased from beijing velutinin limited. The results are shown in Table 8.
TABLE 8 toxicity of Leptospira species to hamsters during the accelerated stability test period
As can be seen from the results in Table 8, the leptospira species were preserved at 37.+ -. 1 ℃ for 28 days, and all 5/5 died without change in hamster virulence. The heat-resistant protective agent disclosed by the invention can ensure that the leptospira strain keeps stable against hamster virulence for a certain period of time.
8 Long term stability
The freeze-dried strains of leptospira were stored at 2-8 ℃ and tested for appearance, residual moisture, vacuum, purity, survival rate, and hamster virulence according to the methods described above for 3 months, 6 months, 12 months, 18 months, and 24 months after freeze-drying, respectively, and the experimental results are shown in table 9.
TABLE 9 Leptospira survival within shelf life
From the experimental results in Table 9, the appearance, the residual moisture and the vacuum degree of the leptospira strain are all purely satisfactory when the leptospira strain is stored for 30 months at the temperature of 2-8 ℃, the survival rate of the strain is still more than 80%, and the toxicity of hamsters is unchanged, which shows that the heat-resistant protective agent prepared by the invention can ensure the activity and the toxicity stability of the leptospira for 24 months.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (5)
1. The heat-resistant protective agent for leptospira is characterized by comprising peptone, nano-phytosterol, sodium pyruvate, L-arginine, soluble starch, trehalose, vitamin B 12 and dimethyl sulfoxide.
2. A leptospira heat resistant protectant according to claim 1, wherein the heat resistant protectant composition comprises 5% -7% w/v peptone, 2% -3% w/v nano phytosterol, 1% -2% w/v sodium pyruvate, 1% -2% w/v L-arginine, 1% -2% w/v soluble starch, 2% -3% w/v trehalose, 1% -2% w/v vitamin B 12, 3% -5% v/v dimethyl sulfoxide.
3. The leptospira heat resistant protectant of claim 1, wherein the heat resistant protectant is prepared by the steps of:
1) Weighing peptone, dissolving in water for injection, continuously stirring until the peptone is completely dissolved, then sequentially adding nano-phytosterol, pyruvic acid and L-arginine, and continuously stirring until the peptone is completely dissolved to obtain a first solution;
2) Weighing soluble starch in water for injection, continuously stirring until the soluble starch is completely dissolved, sequentially adding trehalose and vitamin B12, and continuously stirring until the soluble starch is completely dissolved to obtain a second solution;
3) Mixing the first solution and the second solution in equal volume, then fixing the volume, adjusting the PH value to 7.2-7.4, sterilizing at 116 ℃ under high pressure for 20-30 minutes or filtering and sterilizing by a 0.22 mu m filter, and obtaining a third solution;
4) And measuring dimethyl sulfoxide, adding the dimethyl sulfoxide into the third solution, uniformly mixing to obtain the heat-resistant protective agent, and preserving at 2-8 ℃.
4. The heat-resistant protective agent for leptospira according to claim 1, wherein the heat-resistant protective agent is mixed with leptospira bacterial liquid in a ratio of 1:2-1:4.
5. The heat-resistant protective agent for leptospira according to claim 4, wherein the concentration of the leptospira bacterial liquid is 1 x 10 8～1×1010 strips/mL.
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