CN113481142A - Efficient germinant of clostridium sporogenes spores and application thereof - Google Patents

Efficient germinant of clostridium sporogenes spores and application thereof Download PDF

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CN113481142A
CN113481142A CN202110892599.4A CN202110892599A CN113481142A CN 113481142 A CN113481142 A CN 113481142A CN 202110892599 A CN202110892599 A CN 202110892599A CN 113481142 A CN113481142 A CN 113481142A
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germinant
spores
clostridium sporogenes
spore
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黄忠民
潘治利
索标
郭鼐
艾志录
雷萌萌
黄婉婧
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Henan Agricultural University
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Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a high-efficiency germinator for clostridium sporogenes spores and application thereof. The germinant is selected from one or more of the following mixtures: l-alanine, KCl, CaCl2. According to the invention, the germination rate of the spores of the clostridium sporogenes is greatly improved by selecting the germinant.

Description

Efficient germinant of clostridium sporogenes spores and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a high-efficiency germinator for clostridium sporogenes spores and application thereof.
Background
The bacillus can produce spores under the condition of starvation or accumulation of harmful metabolites to a certain degree, the spores can survive and sleep in extreme environments, and once a proper growth environment is detected, the spores can begin to germinate into nutrients. Spores generated by the clostridium sporogenes have strong resistance to severe environment, and the spores can be killed by adopting high-pressure steam sterilization at 121 ℃ for 15min in industry. But the spore can lose the resistance to the external severe environment after being germinated into a nutrient body, and the heat resistance is greatly reduced.
The spores will have an activation process prior to germination. Activation is typically achieved by activating the spores through appropriate conditions of pH, temperature, heating time, and the like. In addition, the spores can also be germinated by adding an inducer, the action mechanism of the germination inducer is to combine with a specific receptor on the inner membrane of the spores to conduct spore germination signals, so that the spores are activated, the cortex of the spores is hydrolyzed, the core becomes loose and enters a germination state, the heat resistance of the activated spores is still strong, most of stains can be resisted, and the spores have refractivity. The spores start to germinate once activated, and if the germinant is removed midway, the germination process does not stop.
By combining a spore germination mechanism, exploring and utilizing the optimal germination conditions of the spores, germinating the spores into a nutrient body and then killing the spores, and providing a new idea for killing the spores. The method explores the efficient germinant, is applied to the sterilization of the low-acidity meat canned food, can obviously reduce the sterilization intensity, reduces the problem of soft tissues of meat raw materials after high-temperature sterilization, and has important practical application value.
Disclosure of Invention
The invention aims to provide a germination agent for clostridium sporogenes spores, and by adopting the germination agent, the clostridium sporogenes spores can obtain higher germination rate.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a germinant for spores of clostridium sporogenes, said germinant being selected from one or a mixture of more than one of the following: l-alanine, KCl, CaCl2。
The germinant is added in the form of aqueous solution, and the mass concentration of the germinant KCl aqueous solution is 0.41-0.63% in the proportion that the volume ratio of the germinant KCl aqueous solution to the liquid of the spores to be germinated is 1: 0.8-1.2; if calcium chloride is used as the germinant, under the same conditions, CaCl2The concentration of the aqueous solution is 0.05-0.88%. If it is adoptedWhen L-alanine is used as the germinant, the concentration of the L-alanine aqueous solution is 14-22mmol/L under the same conditions.
Preferably, the germinant is L-alanine, KCl, CaCl2The mixture aqueous solution of (1) has L-alanine concentration of 14-22mmol/L, KCl concentration of 0.41-0.63%, CaCl2The concentration is 0.05-0.88%;
more preferably, the aqueous mixture has an L-alanine concentration of 16.8mmol/L, KCl of 0.5% and CaCl2The concentration was 0.06%.
Preferably, the pH of the clostridium sporogenes spore suspension added with the germinant is adjusted to 4-6, and the mixture is bathed for 15-25min at 70-90 ℃ of the water bath temperature.
The invention collects spores of clostridium sporogenes, prepares spore suspension and screens germinants. The principle of germinants in promoting spore germination is mainly due to the fact that receptor proteins on the inner membrane of the spore conduct germination signals after receiving the signal molecules, and germination events are started. Germinants for spores generally include amino acids, sugar purine nucleosides, and the like, and there are also some chemicals that promote germination of spores. No report about germination agents of clostridium sporogenes exists at present, and the spore germination agents are generally not suitable for germination of clostridium sporogenes in a screening process, for example, common germination agents such as NaCl, glucose, fructose, vitamin E and the like have poor germination effects on clostridium sporogenes, but L-alanine, KCl, CaCl and the like provided by the invention are used2The germinant has good effect. If the L-alanine is independently adopted as the germinant, the germination rate of the spores of the clostridium sporogenes can reach 85 percent; if KCl is independently adopted as the germinant, the germination rate of the spores of the clostridium sporogenes can reach 89%; if CaCl is used alone2When the bacillus sporogenes is used as a germinant, the germination rate of the spores of the clostridium sporogenes can reach 82 percent; when any two combinations are adopted, the invention discovers that the interaction of the L-alanine and the KCl is very strong, and the influence of the L-alanine on the germination rate of spores is larger; l-alanine and CaCl2In the interaction of (1), CaCl2The influence on the germination rate of spores is larger; when potassium chloride and calcium chloride are used simultaneously, CaCl2The influence on the germination rate of spores is larger. In a specific ratioAfter the three components are combined, the germination rate can reach 91 percent.
Compared with the prior art, the invention has the following advantages:
according to the invention, by selecting the germinant, the germination rate of the spores of the clostridium sporogenes is greatly improved, the spores are germinated into a nutrient body first and then killed, and a new thought and the feasibility of low-intensity sterilization are provided for the problem of spore killing. The high-efficiency germinant is applied to the sterilization of low-acidity meat canned food, the sterilization strength can be obviously reduced, and the problem of soft tissues of meat raw materials due to high-temperature sterilization is solved，Has important practical application value.
Drawings
FIG. 1 is a graph showing the correspondence between different L-alanine concentrations and germination rates in examples 1 to 5;
FIG. 2 is a graph of the correspondence between different potassium chloride concentrations and germination rates for examples 6-10;
FIG. 3 is a graph of the correspondence between different calcium chloride concentrations and germination rates for examples 11-15;
FIG. 4 is a graph showing the correspondence between the different glucose concentrations and germination rates of comparative examples 1 to 5.
Detailed Description
The technical solution of the present invention is illustrated by the following specific examples, but the scope of the present invention is not limited thereto:
the spore test unit used in the following examples was a spore concentration of 108CFU/mL of 1mL thioglycolate medium liquid can be obtained by:
(1) strain activation
Opening a strain freeze-drying tube (clostridium sporogenes (10385), purchased from China industrial microorganism preservation management center, 0 generation strain, and packaged in a freeze-drying tube) in an aseptic operation platform, adding 1mL of thioglycollate fluid culture medium, dissolving the freeze-dried strain, transferring 0.1mL of freeze-dried bacterium liquid into a glass bottle filled with 10mL of thioglycollate fluid culture medium, placing the glass bottle at the opening of the glass bottle by using a sterile cotton plug, and culturing for 24 hours in an anaerobic box at 35 ℃.
(2) Subculturing of strains
0.1mL of the above activated fresh culture was added to 10mL of thioglycollate medium and cultured at 35 ℃ for 24 hours, and this was repeated twice.
(3) And 7 days later, taking a small amount of culture solution for spore staining microscopic examination, and finding that a large amount of spores exist. Taking the culture solution into a sterile centrifuge tube, centrifuging at 2000r/min at 4 ℃ for 10min, and taking the supernatant; centrifuging at 4 deg.C for 10min at 6000r/min to obtain bottom precipitate, adding sterile water, mixing, centrifuging for 3 times, adding sterile water, mixing, and adjusting concentration to 108CFU/mL, obtaining spore suspension, namely a spore test unit, and storing the spore suspension in a refrigerator at 4 ℃ for later use.
The water used is sterile water.
Examples 1 to 5
1ml of L-alanine aqueous solutions with different concentrations are respectively added into a spore test unit, and the treatment is repeated for three times under the conditions that the water bath temperature is 80 ℃, the water bath time is 20min and the pH value is 5. The concentrations of the added L-alanine were: 10mmol/L, 13mmol/L, 16mmol/L, 19mmol/L, 22mmol/L, corresponding germination rates of 71%, 77%, 85%, 81%, 80%, respectively. The correspondence between concentration and germination rate is seen in figure 1.
As can be seen from the graph, after the treatment by adding the L-alanine aqueous solution with different concentrations, the spore germination rate of the clostridium sporogenes is gradually increased from 71% to 85% when the addition amount of the L-alanine is increased from 10mmol/L to 16mmol/L, and the spore germination rate of the clostridium sporogenes is in a descending trend when the concentration of the L-alanine is continuously increased from 16 mmol/L.
Examples 6 to 10
1ml of potassium chloride aqueous solution with different concentrations is added into a spore test unit, and the treatment is repeated for three times under the conditions that the water bath temperature is 80 ℃, the water bath time is 20min and the pH value is 5. The mass concentrations of the added potassium chloride aqueous solution are respectively as follows: 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, corresponding germination rates of 69%, 78%, 89%, 82%, 74%, respectively. The correspondence between concentration and germination rate is seen in figure 2.
As can be seen from the graph, after the treatment of KCl aqueous solutions with different concentrations, the spore germination rate of the clostridium sporogenes is gradually increased from 69% to 89% when the spore germination rate of the clostridium sporogenes is increased from 0.3% to 0.5% along with the addition amount of KCl, and the spore germination rate of the clostridium sporogenes is in a descending trend when the KCl concentration is continuously increased from 0.5%.
Examples 11 to 15
1ml of calcium chloride aqueous solution with different concentrations is added into a spore test unit, and the treatment is repeated for three times under the conditions that the water bath temperature is 80 ℃, the water bath time is 20min and the pH value is 5. The concentrations of the added potassium chloride aqueous solution are respectively as follows: 0.01%, 0.04%, 0.07%, 0.10%, 0.13%, corresponding germination rates of 70%, 78%, 82%, 76%, 72%, respectively. The correspondence between concentration and germination rate is seen in figure 3.
As can be seen, the CaCl was concentrated at different concentrations2After the aqueous solution treatment, the germination rate of spores of the clostridium sporogenes follows CaCl2When the addition amount is increased from 0.01% to 0.07%, the spore germination rate of the clostridium sporogenes is gradually increased from 70% to 82% in CaCl2When the concentration is continuously increased from 0.07%, the spore germination rate of the clostridium sporogenes is in a descending trend.
Examples 16 to 29
Adding 1ml of aqueous solution of germinant with different concentrations into a spore test unit, and repeatedly treating for three times under the conditions that the water bath temperature is 80 ℃, the water bath time is 20min and the pH value is 5. The added germinant is the combination of L-alanine, potassium chloride and calcium chloride, and the specific concentration of the aqueous solution and the corresponding germination rate are detailed in the following table.
Figure BDA0003196699660000061
Wherein, the germination rate of the embodiment 29 reaches 91.86%, the germination under the same condition is continued by using the remaining spores which are not germinated after the treatment, and the germination rate of the obtained second-time spores is 76.34%, namely the germination rate of the spores can reach 97.87% after two continuous germination treatments.
Comparative examples 1 to 5
1ml of glucose aqueous solution with different concentrations is added into the spore test unit, and the treatment is repeated for three times under the conditions that the water bath temperature is 80 ℃, the water bath time is 20min and the pH value is 5. The concentrations of the added glucose solution were: 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, the corresponding germination rates are 72%, 75%, 81%, 77%, 76%, respectively. The correspondence between concentration and germination rate is seen in figure 4.
As can be seen from the graph, after the treatment of glucose with different concentrations, the germination rate of the spores of the clostridium sporogenes is gradually increased from 72% to 81% when the addition amount of the glucose is increased from 0.6% to 1.0%, and the germination rate of the spores of the clostridium sporogenes is in a descending trend when the concentration of the glucose is continuously increased from 1.0%. In terms of the using amount, the germination optimization condition of the spores of the clostridium sporogenes is much worse than that of potassium chloride with the same using amount, the germination rate of the spores is not obviously improved, and meanwhile, the maximum germination rate is relatively low. NaCl, fructose, vitamin E are less effective at germination of spores of Clostridium sporogenes than glucose.

Claims (7)

1. A high-efficiency germinant of spores of clostridium sporogenes, which is characterized in that the germinant is selected from one or more of the following mixtures: l-alanine, KCl, CaCl2。
2. The efficient germinant of clostridium sporogenes spore as claimed in claim 1, wherein the germinant is added in the form of aqueous solution, and the mass concentration of the germinant KCl aqueous solution is 0.41-0.63% in the ratio of 1:0.8-1.2 of the liquid volume ratio of the germinant spore.
3. The efficient germinant of clostridium sporogenes spore of claim 1, wherein said germinant is added in the form of aqueous solution, and said CaCl is calculated in the ratio of 1:0.8-1.2 of liquid volume ratio of spore to be germinated2The concentration of the aqueous solution is 0.05-0.88%.
4. The efficient germinant of clostridium sporogenes spore of claim 1, wherein said germinant is added in the form of aqueous solution, and the concentration of said L-alanine aqueous solution is 14-22mmol/L in the ratio of 1:0.8-1.2 of the liquid volume of spore to be germinated.
5. The efficient germinant of clostridium sporogenes spore of any one of claims 1-4, wherein said germinant is L-alanine, KCl, CaCl2The mixture aqueous solution of (1) has L-alanine concentration of 14-22mmol/L, KCl concentration of 0.41-0.63%, CaCl2The concentration is 0.05-0.88%.
6. The efficient germinant of clostridium sporogenes spore of claim 5, wherein said germinant is L-alanine, KCl, CaCl2The mixture of (1) and (2) in which the L-alanine concentration is 16.8mmol/L, KCl concentration is 0.5%, and CaCl is added2The concentration was 0.06%.
7. The use of a high germination efficiency germinating agent for clostridium sporogenes spores as claimed in claims 1 to 6, wherein the suspension of clostridium sporogenes spores added with germination agent is adjusted to pH 4 to 6 and is bathed in water at a water bath temperature of 70 to 90 ℃ for 15 to 25 min.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114451516A (en) * 2022-03-08 2022-05-10 合肥工业大学 Method for killing spores in food based on twice induced germination and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170281696A1 (en) * 2016-04-05 2017-10-05 Nch Corporation Nutrient Rich Germinant Composition and Spore Incubation Method
CN111808787A (en) * 2020-08-12 2020-10-23 河南农业大学 Method for improving germination rate of spores of clostridium sporogenes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170281696A1 (en) * 2016-04-05 2017-10-05 Nch Corporation Nutrient Rich Germinant Composition and Spore Incubation Method
CN111808787A (en) * 2020-08-12 2020-10-23 河南农业大学 Method for improving germination rate of spores of clostridium sporogenes

Non-Patent Citations (1)

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Title
项丰娟: "肉毒梭状芽孢杆菌芽孢萌发条件及鸡肉罐头低温杀菌方法的研究", 《中国知网硕士电子期刊》, no. 7, 15 July 2015 (2015-07-15), pages 1 - 63 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114451516A (en) * 2022-03-08 2022-05-10 合肥工业大学 Method for killing spores in food based on twice induced germination and application thereof
CN114451516B (en) * 2022-03-08 2024-01-23 合肥工业大学 Method for killing spores in food based on twice induction germination and application thereof

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