CN112625910A - Low-temperature protective agent capable of automatically setting core - Google Patents
Low-temperature protective agent capable of automatically setting core Download PDFInfo
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Abstract
The invention discloses a low-temperature protective agent capable of automatically setting nucleus, which relates to the technical field of bacteria cryopreservation and comprises water, glycerol and inactivated pseudomonas syringae; wherein the concentration of the glycerol is 5-20% (v/v); the concentration of the inactivated pseudomonas syringae is 107～109CFU/ml. The invention also provides a preparation method of the cryoprotectant capable of automatically setting nucleus, which comprises the following steps of 1: adding glycerol into distilled water, stirring, sterilizing at high temperature under high pressure to obtain sterilized glycerol aqueous solution, and refrigerating; step 2: after the pseudomonas syringae is subjected to subculture and ultrasonic sterilization, obtaining inactivated pseudomonas syringae; and step 3: and uniformly mixing the inactivated pseudomonas syringae with the sterilized glycerol aqueous solution. The invention not only reduces the using concentration of the glycerol and the toxic injury, but also improvesThe low-temperature survival rate of the streptococcus thermophilus can also be realized by simultaneously operating a large batch of samples.
Description
Technical Field
The invention relates to the technical field of bacteria cryopreservation, in particular to a cryoprotectant capable of automatically setting nucleus.
Background
Streptococcus thermophilus (s. thermophilus) is an important probiotic, has fermentation capacity and nutritional value, is commonly used in fermented milk, and plays an important role in food production. Cryopreservation is widely used to preserve its viability over a long period of time. The traditional cryoprotectant to mitigate streptococcus thermophilus damage is glycerol, which is toxic to streptococcus thermophilus. Therefore, the concentration of glycerol should be reduced. However, if the concentration of glycerol is reduced, the damage of Intracellular Ice Formation (IIF) is increased, and then the cell death is caused, the traditional method of manual nucleus setting is adopted to induce intracellular dehydration, and the generation of intracellular ice is avoided, but the method is not beneficial to simultaneously freezing and storing a large number of samples.
Some substances, called Ice Nucleating Agents (INA), have structures or surface properties that tend to nucleate a solution, and the mechanism of action is that their surfaces can interact with water molecules, so that the water molecules are arranged in order, and as the number of "ordered" water molecules on the surface of the INA increases, the probability of nucleation is closer to the equilibrium freezing point, and the degree of supercooling is smaller. Examples of INA used in the field of cryopreservation include Cholesterol crystals (CholC) and encapsulated silver iodide. The biological method for controlling nucleation mainly comprises a biological induction nucleating agent, most of which are inactivated bacteria and fungi, the nucleation key lies in Ice-nucleating proteins (INPs) on the outer membrane of bacterial cells, can induce Ice crystal nucleation at the temperature of-2 ℃, such as inactivated pseudomonas syringae (trade name: SNOMAX), the nucleation key is extra-membrane InaZ protein, the biological method can not only arrange water into a regular structure, but also effectively remove latent heat caused by phase change, and meets two conditions for promoting nucleation. Therefore, the inactivated pseudomonas syringae can be adopted to induce solution to nucleate in advance, so that intracellular free water can be dehydrated, the generation of intracellular ice can be avoided, and a large batch of samples can be operated at the same time.
Therefore, those skilled in the art are devoted to develop a cryoprotectant that can be automatically nucleated, not only to reduce toxic damage and reduce costs, but also to improve the low temperature survival rate of streptococcus thermophilus.
Disclosure of Invention
In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a low-temperature protective agent with low cost, convenient for large-scale nuclear setting operation and good preservation effect, and a preparation method thereof.
In order to achieve the aim, the invention provides a cryoprotectant capable of automatically setting nucleus, which is characterized by comprising water, glycerol and inactivated pseudomonas syringae; wherein the concentration of the glycerol is 5-20% (v/v); the concentration of the inactivated pseudomonas syringae is 107～109CFU/ml。
Further, the concentration of the glycerol is 5-10% (v/v).
Further, the glycerol concentration was 5% (v/v).
Further, the concentration of the inactivated pseudomonas syringae is 108～109CFU/ml。
Further, the concentration of the inactivated pseudomonas syringae is 109CFU/ml。
Further, the refrigeration temperature is less than 4 ℃.
Further, the method is used for cryopreservation of the streptococcus thermophilus.
The invention also provides a preparation method of the cryoprotectant capable of automatically setting the nucleus, which is characterized by comprising the following steps:
step 1: adding glycerol into distilled water, stirring, sterilizing at high temperature under high pressure to obtain sterilized glycerol aqueous solution, and refrigerating;
step 2: after the pseudomonas syringae is subjected to subculture and ultrasonic sterilization, obtaining inactivated pseudomonas syringae;
and step 3: and uniformly mixing the inactivated pseudomonas syringae with the sterilized glycerol aqueous solution.
Further, in the step 3, the inactivated pseudomonas syringae and the sterilized glycerol aqueous solution are uniformly mixed according to a ratio of 1: 1.
Further, in the step 3, the inactivated pseudomonas syringae, the sterilized glycerol aqueous solution and the streptococcus thermophilus suspension are uniformly mixed, ice-bathed for 30 minutes, and then the mixture is frozen and stored at the temperature of minus 80 ℃.
Compared with the prior art, the invention at least has the following beneficial technical effects:
1. the use concentration of the glycerol is reduced, the toxic damage is reduced, and the cost is reduced.
2. The biological induction nucleating agent is added, so that the generation of intracellular ice is avoided, and the freezing and storing activity of the biological material is improved.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is a graph of the effect of glycerol concentration on the survival of Streptococcus thermophilus;
FIG. 2 is a graph of the effect of nucleator concentration on freezing point;
FIG. 3 is a graph showing the effect of nucleator concentration on the survival rate of Streptococcus thermophilus.
Detailed Description
The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.
If there is an experimental method not specified specific conditions, it is usually carried out according to conventional conditions, such as the relevant instructions or manuals.
Example 1 Effect of Glycerol concentration on the survival of Streptococcus thermophilus
Adding glycerol into distilled water respectively to obtain 10% (v/v), 20% (v/v) and 40% (v/v) glycerol aqueous solution, stirring, sterilizing at high temperature and high pressure to obtain sterilized glycerol aqueous solution, and refrigerating for use.
The streptococcus thermophilus is firstly cultured by liquid standing at 37 ℃, streaked and cultured at 37 DEG CCulturing for 24 hr, selecting single strain, liquid culturing at 37 deg.C, centrifuging, resuspending twice, and determining initial colony number N by plate spotting method0。
Mixing glycerol aqueous solutions with different concentrations with Streptococcus thermophilus suspension at a ratio of 1:1, shaking to obtain glycerol mixed solution containing 5% (v/v), 10% (v/v) and 20% (v/v), ice-cooling for 30 min, and freezing at-80 deg.C for 7 days.
Within 7 days, the glycerol mixed solution groups with different concentrations are rewarming in a water bath at 25 ℃, repeatedly freezing and thawing for 7 times, and the number N of the bacterial colonies after 7 th thawing is determined by a plate spotting methodiSurvival rate ofi/N0The results of calculation are shown in fig. 1, the survival rate of the 5% (v/v) glycerol mixture group is the highest and reaches 0.82 ± 0.08, then 10% (v/v) glycerol mixture group, and the survival rate of the 20% (v/v) glycerol mixture group is the lowest, and has a significant difference compared with the 5% (v/v) glycerol mixture group.
EXAMPLE 2 Effect of nucleator concentration on freezing Point
Adding glycerol into distilled water to obtain 10% (v/v) glycerol aqueous solution, stirring, sterilizing at high temperature under high pressure to obtain sterilized glycerol aqueous solution, and refrigerating.
The pseudomonas syringae is firstly subjected to liquid culture for 20 hours under the conditions of 26 ℃ and 200r/min, then single bacteria are picked after solid subculture for 24 hours under the condition of 26 ℃, and the liquid culture is switched to 18 ℃ for standing culture for 4 hours after 20 hours under the same condition so as to induce the expression of InaZ protein. After the pseudomonas syringae is subjected to subculture (third generation), the concentration reaches 109CFU/ml (group C), ultrasonic sterilizing, sequentially diluting 10 times and 100 times to concentration of 108CFU/ml (group B) and 107CFU/ml (group A).
Respectively mixing inactivated Pseudomonas syringae with different concentrations with sterilized 10% (v/v) glycerol aqueous solution at a ratio of 1:1, oscillating uniformly to obtain 5% (v/v) glycerol mixed solution groups containing different concentrations of nucleating agents, i.e. 3 groups of cryoprotectants capable of automatically nucleating, and measuring the freezing point of each group of cryoprotectants by a differential scanning calorimeter, as shown in figure 2, and mixing with 5% (v/v) glycerol aqueous solution groupsCompared with the 5% (v/v) glycerol mixed solution with different concentrations of the nucleating agent, the freezing point of the mixed solution is improved, and particularly, the mixed solution contains 10 percent of the nucleating agent9The freezing point of the 5% (v/v) glycerol mixed liquor group of CFU/ml inactivated Pseudomonas syringae is obviously improved.
Example 3 Effect of nucleator concentration on the survival Rate of Streptococcus thermophilus
Adding glycerol into distilled water to obtain 20% (v/v) glycerol aqueous solution, stirring, sterilizing at high temperature under high pressure to obtain sterilized glycerol aqueous solution, and refrigerating.
The pseudomonas syringae is firstly subjected to liquid culture for 20 hours under the conditions of 26 ℃ and 200r/min, then single bacteria are picked after solid subculture for 24 hours under the condition of 26 ℃, then the liquid culture is carried out for 20 hours under the same condition, and then the temperature is changed to 18 ℃ for standing culture for 4 hours so as to induce the expression of InaZ protein. After the pseudomonas syringae is subjected to subculture (third generation), the concentration reaches 109CFU/ml (group C), ultrasonic sterilizing, sequentially diluting 10 times and 100 times to concentration of 108CFU/ml (group B) and 107CFU/ml (group A).
Respectively and uniformly mixing inactivated pseudomonas syringae with different concentrations with a sterilized 20% (v/v) glycerol aqueous solution and a streptococcus thermophilus suspension according to a ratio of 1:1:2 to prepare 5% (v/v) glycerol low-temperature protective agent containing nucleating agents with different concentrations for protecting streptococcus thermophilus, carrying out ice bath for 30 minutes, and carrying out cryopreservation at-80 ℃ for 7 days.
After 7 days, repeated freeze thawing is carried out for 7 times under the same conditions, the colony number after 7 times of thawing is measured, the survival rate is calculated, and the result is shown in figure 3, compared with a 5% (v/v) glycerol aqueous solution group, the survival rate of a 5% (v/v) glycerol protective agent group added with different concentrations of the nucleating agent is improved, particularly 10% (v/v) glycerol protective agent is added9The 5% (v/v) glycerol protectant group of CFU/ml inactivated Pseudomonas syringae promoted survival to 0.93 + -0.04, both FIG. 2 and FIG. 3 show 10%9The 5% (v/v) glycerol protective agent for CFU/ml inactivated pseudomonas syringae has stronger nuclear retention capacity.
Cryoprotectants are important factors that affect the quality and viability of the preserved biological material. In order to reduce the toxic damage and intracellular ice damage of the conventional protective agent, namely 20% (v/v) glycerol to the streptococcus thermophilus, so as to ensure the viable count and the bacterial activity to the maximum extent, the invention reduces the using concentration of the glycerol and compares the protective effects of each group, and the result shows that the 5% (v/v) glycerol has the minimum toxicity and the best protective effect, but the survival rate is still lower than 90%. The reason is probably that the glycerol concentration is reduced, the generation probability of the intracellular ice is increased, therefore, the addition of the inactivated pseudomonas syringae induces the extracellular solution to nucleate in advance, so that the intracellular free water can be dehydrated, the generation of the intracellular ice is avoided, and a large batch of samples can be operated simultaneously.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (10)
1. A cryoprotectant capable of automatically setting nucleus is characterized by comprising water, glycerol and inactivated pseudomonas syringae; wherein the concentration of the glycerol is 5-20% (v/v); the concentration of the inactivated pseudomonas syringae is 107～109CFU/ml。
2. The autonucleatable cryoprotectant of claim 1, wherein the glycerol concentration is 5-10% (v/v).
3. The autonucleatable cryoprotectant of claim 2, wherein the glycerol concentration is 5% (v/v).
4. The autonuclearly cryoprotectant of claim 3, wherein said inactivated Pseudomonas syringae is present at a concentration of 108～109CFU/ml。
5. The autonucleable of claim 4The cryoprotectant of (a), wherein the inactivated pseudomonas syringae has a concentration of 109CFU/ml。
6. The cryoprotectant capable of self-nucleation according to any one of claims 1 to 5, wherein the refrigeration temperature is less than 4 ℃.
7. The cryoprotectant capable of self-nucleation according to any one of claims 1 to 5, for the cryopreservation of Streptococcus thermophilus.
8. A preparation method of the cryoprotectant capable of automatically nucleating as claimed in any one of claims 1 to 5, characterized by comprising the following steps:
step 1: adding glycerol into distilled water, stirring, sterilizing at high temperature under high pressure to obtain sterilized glycerol aqueous solution, and refrigerating;
step 2: after the pseudomonas syringae is subjected to subculture and ultrasonic sterilization, obtaining inactivated pseudomonas syringae;
and step 3: and uniformly mixing the inactivated pseudomonas syringae with the sterilized glycerol aqueous solution.
9. The method for preparing the cryoprotectant capable of automatically nucleating as claimed in claim 8, wherein in the step 3, the inactivated pseudomonas syringae and the sterilized glycerol aqueous solution are uniformly mixed according to a ratio of 1: 1.
10. The method for preparing the cryoprotectant capable of automatically nucleating according to claim 8, wherein in the step 3, the inactivated pseudomonas syringae, the sterilized glycerol aqueous solution and the streptococcus thermophilus suspension are uniformly mixed, ice-bathed for 30 minutes, and then placed at-80 ℃ for cryopreservation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114766541A (en) * | 2022-05-24 | 2022-07-22 | 梧州市桂粤供应链有限公司 | Low-temperature quick-freezing sleep method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87104158A (en) * | 1986-05-15 | 1988-02-17 | 赛尔系统有限公司 | Biological cryoprotection |
| CN102978150A (en) * | 2012-11-19 | 2013-03-20 | 陕西科技大学 | Streptococcus thermophilus composite antifreezing factor |
| CN107635402A (en) * | 2015-04-16 | 2018-01-26 | 阿西姆普托特有限公司 | For controlling the device of the ice nucleus formation in biological specimen to be freezed |
| CN108027203A (en) * | 2015-07-22 | 2018-05-11 | 北卡罗来纳-查佩尔山大学 | Fluid means and relevant operation and analysis method with the freeze thaw valve using ice nucleator |
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2020
- 2020-12-23 CN CN202011541770.9A patent/CN112625910A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87104158A (en) * | 1986-05-15 | 1988-02-17 | 赛尔系统有限公司 | Biological cryoprotection |
| CN102978150A (en) * | 2012-11-19 | 2013-03-20 | 陕西科技大学 | Streptococcus thermophilus composite antifreezing factor |
| CN107635402A (en) * | 2015-04-16 | 2018-01-26 | 阿西姆普托特有限公司 | For controlling the device of the ice nucleus formation in biological specimen to be freezed |
| CN108027203A (en) * | 2015-07-22 | 2018-05-11 | 北卡罗来纳-查佩尔山大学 | Fluid means and relevant operation and analysis method with the freeze thaw valve using ice nucleator |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114766541A (en) * | 2022-05-24 | 2022-07-22 | 梧州市桂粤供应链有限公司 | Low-temperature quick-freezing sleep method |
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