CN110982763A - Stabilizer of clostridium perfringens and application thereof - Google Patents
Stabilizer of clostridium perfringens and application thereof Download PDFInfo
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Abstract
The invention provides a stabilizing agent of clostridium perfringens, wherein the solvent is water, and the solute comprises sodium citrate, L-carnosine, L-cysteine hydrochloride, skimmed milk powder and sorbitol. The stabilizing agent for the quality control bacteria of the clostridium perfringens can ensure that the freeze-drying survival rate of the clostridium perfringens reaches more than 90 percent, the freeze-drying process hardly loses, the stability of the freeze-drying process is ensured, and the bacteria content of the clostridium perfringens freeze-dried powder is not changed in the long-term storage process.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a clostridium perfringens stabilizer and application thereof.
Background
The clostridium perfringens is a gram-positive crude brevibacterium, is the most common clostridium in pathogenic bacteria of gas gangrene clinically, can decompose sugar in muscle and connective tissue, generate a large amount of gas, cause severe emphysema of the tissue, further influence blood supply and cause large-area necrosis of the tissue. The bacteria are widely distributed in external environments such as human and animal excreta, soil, sewage and the like. Therefore, the inspection of the bacterium is standardized by the current GB8538-2016 national food safety standard 'inspection method for natural mineral water for drinking' and the GB 4789.13-2012 national food safety standard 'inspection for clostridium perfringens'. The quality control strain is used as a positive control in the microbial test, can verify the test method, can perform capability verification on testers, and can check and accept a microbial culture medium, so that the quality control in the microbial experiment process is ensured, and the quality control strain is a standard substance in the microbial test process. The quantitative quality control strain is a development trend of quality control standard substances in the existing microbial detection process due to various advantages of convenient and quick use, improvement of detection efficiency and the like.
The stabilizer is an effective substance for protecting cells of strains in the freeze drying process, different strains have different physiological and metabolic characteristics, different stress resistance in the freeze drying process and respective preference in the selection of the stabilizer, so that different strains need to be screened to obtain the stabilizer with the best effect and protect the activity of the cells to the maximum extent. At present, no preparation method aiming at the quantitative quality control strain of clostridium perfringens exists, and a stabilizing agent capable of quantitatively storing the clostridium perfringens is lacked. Therefore, the preparation method of the stabilizer for quantitatively storing the clostridium perfringens and the quality control bacteria thereof is of great significance.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention aims to provide a clostridium perfringens stabilizer and application thereof.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
a stabilizer for clostridium perfringens, wherein a solvent is water, and solutes of the stabilizer comprise the following raw materials: sodium citrate, L-carnosine, L-cysteine hydrochloride, skimmed milk powder and sorbitol.
In some embodiments, the mass composition of the solute in the above-mentioned stabilizer is: 1-3 parts of sodium citrate, 0.5-2 parts of L-carnosine, 1-2 parts of L-cysteine hydrochloride, 5-10 parts of skimmed milk powder and 1-5 parts of sorbitol.
In some embodiments, the mass composition of the solute in the above-mentioned stabilizer is: 1 to 3 percent of sodium citrate, 0.5 to 2 percent of L-carnosine, 1 to 2 percent of L-cysteine hydrochloride, 5 to 10 percent of skimmed milk powder and 1 to 5 percent of sorbitol.
In a second aspect of the present invention, there is provided:
a quantitative quality control product for clostridium perfringens is prepared by uniformly mixing clostridium perfringens bacterial liquid with the stabilizer, quantifying, subpackaging and freeze-drying.
In some embodiments, the ratio of Clostridium perfringens bacteria to stabilizer (dry weight) is 300-1000CFU/(0.1-0.3) g stabilizer (dry weight). Thus, the bacteria can be fully protected by the stabilizer, and the stabilizer cannot be excessively wasted.
In some embodiments, the clostridium perfringens content of the quantitative quality control product is 300-. Of course, other contents can be selected according to actual needs. The variation of the bacteria content is not more than 20%.
In a third aspect of the present invention, there is provided:
a preparation method of a clostridium perfringens quantitative quality control product comprises the following steps:
(1) recovering the strain: inoculating clostridium perfringens to a recovery culture medium, and performing recovery passage;
(2) and (3) enrichment: inoculating the recovered clostridium perfringens into a proliferation culture medium, and culturing and proliferating to obtain clostridium perfringens bacterial liquid;
(3) counting: uniformly mixing the clostridium perfringens bacterial liquid with the stabilizer, counting and subpackaging to obtain quantitative clostridium perfringens bacterial liquid;
(4) freeze-drying: and (4) freeze-drying the quantified clostridium perfringens bacterial liquid, and sealing to obtain the quantified quality control product of clostridium perfringens.
In some embodiments, the proliferation medium in step (2) comprises, in mass fractions, 0.5% to 2% peptone, 0.5% to 2% soytone, 0.3% to 1.5% beef powder, 0.2% to 1% yeast powder, 0.01% to 1.2% soluble starch, 0.1% to 0.2% L-cysteine hydrochloride, 0.01% to 0.05% sodium glycerophosphate.
In some embodiments, the proliferation medium in step (2) comprises, in mass fractions, 1% peptone, 1% soytone, 1% beef powder, 0.5% yeast powder, 0.1% soluble starch, 0.2% L-cysteine hydrochloride, 0.02% sodium glycerophosphate.
In some embodiments, the resuscitation medium in step (1) is columbia agar medium.
In some embodiments, the freeze drying in step (4) is performed by fast freezing with liquid nitrogen, then placing the frozen.
In some embodiments, the vacuum drying conditions are: the vacuum degree is 10Pa-100Pa, the drying temperature is-35 ℃ to-15 ℃, and the drying time is 20h-48 h; the analysis drying conditions are as follows: the drying temperature is 0-25 ℃.
In some embodiments, the counting method described in step (3) is an absorbance standard curve method.
The invention has the beneficial effects that:
the stabilizing agent used in the invention can ensure that the freeze-drying survival rate of the clostridium perfringens reaches more than 90%, the freeze-drying process hardly loses, the stability of the freeze-drying process is ensured, and the bacterium content of the clostridium perfringens freeze-dried powder is not changed in the long-term storage process.
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Fig. 1 is a graph of the clostridium perfringens content over time for each of the compositions of the examples and comparative examples.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
For convenience of comparison, the operations of strain recovery, enrichment, counting and lyophilization were as follows:
strain resuscitation
Clostridium perfringens (ATCC 13124: purchased from American type culture Collection) is streaked and inoculated into Columbia agar medium, and is subjected to anaerobic recovery passage at a culture temperature of 36 +/-1 ℃ and identified.
Enrichment of bacteria
Inoculating the recovered and identified clostridium perfringens into a special culture medium for the clostridium perfringens, wherein the culture medium comprises the following components: 1% of peptone, 1% of soytone, 1% of beef powder, 0.5% of yeast powder, 0.1% of soluble starch, 0.2% of L-cysteine hydrochloride and 0.02% of sodium glycerophosphate, wherein the pH value is 6.8 +/-0.2, and the mixture is placed in a constant-temperature oscillator at 36 ℃ for culture and proliferation, the rotating speed is 150rpm/min, and the culture is carried out for 18 hours.
Counting
And establishing a standard curve of the absorbance value and the plate count bacterium content by using the enriched clostridium perfringens suspension. The equation is: y ═ 6E +08) X- (1E +07), R20.997. And (3) diluting the clostridium perfringens suspension liquid in the step (2) to a proper concentration by using a stabilizer with the volume ratio of clostridium perfringens to the stabilizer being 1:10, and measuring an absorbance value by using a spectrophotometer, namely calculating the required clostridium perfringens suspension liquid amount according to the measurement result of the absorbance value.
Freeze-drying
Diluting clostridium perfringens suspension with a stabilizer to a proper concentration, subpackaging into penicillin bottles, performing quick freezing by adopting liquid nitrogen after half plugging, placing in a freeze drying box which is pre-cooled to-40 ℃, balancing for 30min, starting a vacuum pump, and performing vacuum drying under the following conditions: the vacuum degree is 20Pa, the drying temperature is-28 ℃, the drying time is 30h, and the analysis drying is carried out after the vacuum drying, wherein the analysis drying conditions are as follows: drying at 18 deg.C until the water content of the sample is less than 3%, and vacuum-pressing.
Of course, the above operations can also be performed using methods well known in the art.
TABLE 1 stabilizers of different compositions (mass%,%)
And (3) testing the uniformity and stability of the clostridium perfringens quantitative quality control bacteria:
and (3) storing the freeze-dried quantitative quality control clostridium perfringens in a refrigerator at the temperature of 2-8 ℃.3 of them were randomly picked each month, and two plates were spread per bottle for counting, the average of which was the number of viable cells per month.
The uniformity and stability of the quantitative quality control bacteria of the clostridium perfringens prepared in the examples are tested according to the general principle and statistical method of standard sample fixed value of GB/T15000.3-2008 standard sample work guide (3), and SPSS single-factor variance analysis is adopted to verify whether the data have significant difference. Wherein, 10 bottles of samples are randomly selected for counting in the uniformity test, 12 months of test data are selected for testing the stability, and the P values are all greater than 0.05 in a 95% confidence interval, which shows that the data have no statistical difference, namely the uniformity and the stability of the prepared quality control bacteria meet the requirements.
Effect of different composition stabilizers on the lyophilization and storage of Clostridium perfringens
The clostridium perfringens is subjected to stable protection by adopting stabilizers with different compositions, and the compositions of the stabilizers are shown in table 1.
Respectively calculating the content of clostridium perfringens in penicillin bottles before and after freeze-drying by adopting a flat plate counting method, and calculating the freeze-drying survival rate, wherein the method for calculating the content of clostridium perfringens before freeze-drying comprises the following steps: sucking 100uL of clostridium perfringens cell suspension in a penicillin bottle, coating the clostridium perfringens cell suspension on a Columbia culture medium, performing anaerobic culture at 37 ℃ for 24 hours, and counting; the method for calculating the content of clostridium perfringens after freeze-drying comprises the following steps: adding sterile normal saline with the same volume before freeze-drying for dissolving and uniformly oscillating, sucking 100uL of bacterial suspension, coating the bacterial suspension on a corresponding agar culture medium for culture, and counting.
The calculation formula of the freeze-drying survival rate is as follows:
the results are shown in table 2:
TABLE 2 Freeze-drying survival rates of Clostridium perfringens in stabilizers of different compositions
As can be seen from table 2, with the stabilizers of the examples, the freeze-drying survival rate was increased to 90%, whereas with the stabilizer of composition B or composition C, the freeze-drying survival rate was also significantly higher than with the stabilizer of composition D.
Effect of different composition stabilizers on Freeze-dried storage of Clostridium perfringens
The clostridium perfringens is subjected to stable protection by using stabilizers with different compositions, and the composition of each stabilizer is shown in table 1. In order to ensure that the number of bacteria at the initial stage of the comparison test is close and the comparison basic state is consistent, the freeze-drying survival rate of the clostridium perfringens in the stabilizers with different compositions is measured according to the table 2, the number of live bacteria before freeze-drying is measured in real time by using an OD standard curve method, the using amount of the clostridium perfringens before freeze-drying is calculated according to the freeze-drying survival rate, and a clostridium perfringens product containing the number of live bacteria of about 1000 CFU/bottle after freeze-drying is prepared.
The freeze-dried products of the clostridium perfringens prepared by different stabilizers are stored for a long time (the storage temperature is 2-8 ℃, the storage time is 12 months), the bacterium content in each freeze-dried product is measured every month, and the change of the clostridium perfringens content in each composition along with the time is shown in figure 1.
As can be seen from fig. 1, the clostridium perfringens products prepared by using the compositions a1 (example 1) and a2 (example 2) have the bacterium content swinging up and down at 1000 CFU/bottle within the storage period of 12 months, and the variation range is small; while the bacteria content of compositions B-C can be kept at a high level during the initial storage period (months 1-3), the content of Clostridium perfringens begins to decrease significantly after month 4, while the content of Clostridium perfringens is decreased during the initial storage period in group D.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A stabilizer of clostridium perfringens, the solvent is water, and the stabilizer is characterized in that: the solute comprises the following raw materials: sodium citrate, L-carnosine, L-cysteine hydrochloride, skimmed milk powder and sorbitol.
2. The stabilizer according to claim 1, characterized in that: the mass composition of the solute is as follows: 1-3 parts of sodium citrate, 0.5-2 parts of L-carnosine, 1-2 parts of L-cysteine hydrochloride, 5-10 parts of skimmed milk powder and 1-5 parts of sorbitol.
3. The stabilizer according to claim 2, characterized in that: the mass composition of the solute is as follows: 1 to 3 percent of sodium citrate, 0.5 to 2 percent of L-carnosine, 1 to 2 percent of L-cysteine hydrochloride, 5 to 10 percent of skimmed milk powder and 1 to 5 percent of sorbitol.
4. A quantitative quality control product of clostridium perfringens is characterized in that: is prepared by uniformly mixing clostridium perfringens bacterial liquid and the stabilizer of any one of claims 1 to 3, quantifying, subpackaging and freeze-drying.
5. The quantitative quality control product of claim 4, wherein: the content of the clostridium perfringens is 300-1000 CFU/bottle.
6. A preparation method of a clostridium perfringens quantitative quality control product comprises the following steps:
(1) recovering the strain: inoculating clostridium perfringens to a recovery culture medium, and performing recovery passage;
(2) and (3) enrichment: inoculating the recovered clostridium perfringens into a proliferation culture medium, and culturing and proliferating to obtain clostridium perfringens bacterial liquid;
(3) counting: uniformly mixing clostridium perfringens bacterial liquid with the stabilizer of any one of claims 1-3, counting and subpackaging to obtain quantitative clostridium perfringens bacterial liquid;
(4) freeze-drying: and (4) freeze-drying the quantified clostridium perfringens bacterial liquid, and sealing to obtain the quantified quality control product of clostridium perfringens.
7. The method of claim 6, wherein: the proliferation culture medium in the step (2) comprises the following raw materials in percentage by mass: 0.5 to 2 percent of peptone, 0.5 to 2 percent of soytone, 0.3 to 1.5 percent of beef powder, 0.2 to 1 percent of yeast powder, 0.01 to 1.2 percent of soluble starch, 0.1 to 0.2 percent of L-cysteine hydrochloride and 0.01 to 0.05 percent of sodium glycerophosphate.
8. The method of claim 6, wherein: and (4) performing freeze drying by adopting liquid nitrogen for quick freezing, then placing the frozen.
9. The method of manufacturing according to claim 10, wherein: the vacuum drying conditions are as follows: the vacuum degree is 10Pa-100Pa, the drying temperature is-35 ℃ to-15 ℃, and the drying time is 20h-48 h; the analysis drying conditions are as follows: the drying temperature is 0-25 ℃.
10. The method of claim 6, wherein: the counting method in the step (3) is an absorbance standard curve method.
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| CN112300960A (en) * | 2020-10-21 | 2021-02-02 | 北京市理化分析测试中心 | Clostridium perfringens microbial inoculum and preparation method and application thereof |
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