Method for inducing spore germination of alicyclobacillus acidoterrestris

Abstract

The invention provides a method for inducing spores of acid soil alicyclobacillus to germinate, and the method for inducing germination comprises the following steps: mixing the spores of acid soil alicyclobacillus with an acidic solution with the pH value of 2-5 to obtain a mixed solution; and carrying out incubation treatment on the mixed solution. By means of the method, the self-germination effect of the spores of the alicyclobacillus acidoterrestris can be achieved, the spores are induced to germinate without the help of external means such as ultrahigh pressure and nutrient germinator addition, the time is short, the efficiency is high, the cost is low, the spores of the alicyclobacillus acidoterrestris in sterilized food can be controlled, the food safety is improved, and the application value is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology. Specifically, the present application relates to a method for inducing the germination of Bacillus acidiproducens spores. BACKGROUND

[0002] Bacterial spores, also known as endospores, are a kind of dormant body formed by Bacillus and Clostridium under extreme conditions such as nutrient deficiency or environmental stress. The dormant spores have a complex and dense multi-layer structure, have no metabolic activity, and show strong resistance to heat, radiation, pressure and most chemicals, so they are difficult to be completely killed. The spores in the dormant state can sense the external environment, and once the environment is suitable and the nutrients are sufficient, the spores will germinate and restore to the vegetative state. The germination of spores in food will cause food spoilage and foodborne diseases and other hazards.

[0003] The "germination-inactivation" strategy aims to induce bacterial spores to germinate using artificial means, reduce their resistance, and then combine other means to make the spores more easily killed. Currently, bacterial spores can be induced to germinate by non-thermal processing technology and adding nutrient germinants, and then killed.

[0004] Bacillus acidiproducens is an acidophilic and thermophilic, non-pathogenic spore-forming bacterium. Its spores can survive and further germinate in low-acid juice after pasteurization, and produce guaiacol and other off-flavor substances in the process, causing spoilage of acidic foods such as juice, causing significant economic losses to the juice processing industry.

[0005] However, the germination of Bacillus acidiproducens spores still needs to be studied. SUMMARY

[0006] The present application aims to at least partially solve the technical problems existing in the prior art. To this end, the present application proposes a method for efficiently inducing the germination of Bacillus acidiproducens spores. Using this method, the "self-germination" effect of Bacillus acidiproducens spores can be achieved without the need for external means such as ultra-high pressure and the addition of nutrient germinants to induce spore germination. The method is time-saving, efficient, and low-cost, and is conducive to controlling Bacillus acidiproducens spores in high-acid foods after pasteurization, improving food safety, and having high application value.

[0007] In one aspect of the present application, a method for inducing the germination of Bacillus acidiproducens spores is provided. According to embodiments of the present application, the method comprises: mixing Bacillus acidiproducens spores with an acidic solution having a pH value of 2-5 to obtain a mixed solution; and incubating the mixed solution.

[0008] The inventors of the present application have creatively found that incubating Bacillus laevolacticus spores in an acidic solution with a pH value of 2-5 can efficiently promote spore germination and make the spores lose resistance without the help of external means such as ultrahigh pressure and the addition of nutrient germinants, and the treatment efficiency is high and the cost is low.

[0009] According to the embodiments of the present application, the method for inducing Bacillus laevolacticus spore germination described above can further have the following additional technical features: According to the embodiments of the present application, the temperature of the incubation treatment is 18-40°C, and the time is 50-70 min. Illustratively, the temperature of the incubation treatment is 18°C, 20°C, 22°C, 25°C, 28°C, 30°C, 32°C, 35°C, 38°C, 40°C, and the time is 50 min, 52 min, 55 min, 58 min, 60 min, 62 min, 65 min, 68 min, 70 min. The inventors have obtained the above-mentioned preferred incubation conditions through a large number of experiments, and if the temperature or time is too high or too low, the spore germination cannot be promoted well. Among them, the preferred temperature is 37°C, and the time is 60 min.

[0010] According to the embodiments of the present application, the acidic solution comprises at least one of a buffer solution, an aqueous hydrochloric acid solution, an aqueous sulfuric acid solution, and an aqueous phosphoric acid solution. In some embodiments, the buffer solution comprises a K-hepes buffer solution. Thereby, a buffer system is provided to facilitate spore germination.

[0011] According to the embodiments of the present application, the pH value of the acidic solution is 2, 2.5, 3, 3.5, 4, 4.5, 5, and the preferred pH value is 4. Thereby, the spore germination effect is better.

[0012] According to the embodiments of the present application, before the incubation treatment, the acidic solution is subjected to heat shock treatment. In the acidic solution system, heat shock cooperates with incubation to further promote spore germination, shorten the germination time, and improve the germination efficiency and germination amount.

[0013] According to the embodiments of the present application, the temperature of the heat shock is 65-80°C, and the time is 20-40 min. In some embodiments, the temperature of the heat shock is 65°C, 68°C, 70°C, 72°C, 75°C, 78°C, 80°C, and the time is 20 min, 22 min, 25 min, 28 min, 30 min, 32 min, 35 min, 38 min, 40 min. The inventors have obtained the above-mentioned preferred heat shock conditions through a large number of experiments, and if the temperature or time is too high or too low, the spore germination cannot be promoted well. Among them, the preferred temperature is 70°C, and the time is 30 min.

[0014] According to the embodiments of the present application, the method comprises: mixing spores of the acid soil Bacillus laitococcus with an acidic solution with a pH value of 4 to obtain a mixed solution; and subjecting the mixed solution to heat shock at 70 DEG C for 30 min and then incubation at 37 DEG C for 60 min. In this way, the "self-germination" effect of the spores of the acid soil Bacillus laitococcus can be efficiently achieved without the need for external means such as ultrahigh pressure and addition of nutrient germinants to induce spore germination, and the treatment efficiency is high, the germination amount is large, and the cost is low.

[0015] In another aspect of the present application, a method for inactivating spores of the acid soil Bacillus laitococcus is provided. According to the embodiments of the present application, the method comprises: treating spores of the acid soil Bacillus laitococcus by using the method for inducing spore germination of the acid soil Bacillus laitococcus described above. In this way, the method described above can effectively promote the germination of the spores of the acid soil Bacillus laitococcus, and the spores lose resistance, and then inactivation is performed to achieve inactivation of the germinated spores and ensure the safety of samples such as food.

[0016] In some embodiments, the inactivation treatment after spore germination can be performed by using conventional means in the art, such as heating, ultrahigh pressure, etc.

[0017] It should be noted that the features and advantages described above for the method for inducing spore germination of the acid soil Bacillus laitococcus also apply to the inactivation method, and will not be described here again.

[0018] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter in the description of embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0019] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the description of the embodiments, taken in conjunction with the following drawings in which: Figure 1 The figure shows the spore germination situation of L-Ala treatment under the condition of pH 4; Figure 2 The figure shows the spore germination situation of AGFK treatment under the condition of pH 4; Figure 3 The figure shows the spore germination situation after heat shock treatment without adding germinants under different pH conditions; Figure 4 The figure shows the microscope photos of spores before and after heat shock treatment under the condition of pH 4, the left photo is the spores before heat shock treatment, and the right photo is the spores after heat shock treatment and incubation at 37 DEG C under the condition of pH 4; Figure 5 The figure shows the spore germination situation under different pH conditions without heat shock treatment; Figure 6Microscope photos of spores without heat shock treatment (buffer pH 4), left photo: spores before heat shock treatment, right photo: spores after incubation at 37℃; Figure 7 Induction of germination of spores of Acidifiskicola lipolytica at different heat shock temperatures under pH 4 condition; Figure 8 Germination of spores of Acidifiskicola lipolytica under different pH and incubation temperature conditions without heat shock treatment; Figure 9 Germination of spores of Acidifiskicola lipolytica under different pH conditions; Figure 10 Microscope photos of spores of Acidifiskicola lipolytica under different pH conditions. DETAILED DESCRIPTION

[0020] The scheme of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product instruction is used. If no manufacturer of the reagent or instrument is specified, it is a conventional product that can be obtained by purchase in the market.

[0021] Example 1 In this example, the germination of spores of Acidifiskicola lipolytica was induced according to the following method: 1. Spores of Acidifiskicola lipolytica were added into sterile K-hepes buffer with pH 4 to obtain a mixture.

[0022] 2. The mixture was subjected to heat shock treatment at 75℃ for 30 min, and then incubated at 37℃ for 60 min, while the release of DPA was determined.

[0023] Example 2 The germination of spores of Acidifiskicola lipolytica was induced according to the method of Example 1, except that in step 1, 0 mM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM of L-alanine or 0x, 0.5x, lx, 4x, 8x, 12x, 15x AGFK was added into the buffer, respectively.

[0024] Example 3 The germination of spores of Acidifiskicola lipolytica was induced according to the method of Example 1, except that the pH of the buffer was 2, 3, 5, 6, 7, 9, respectively.

[0025] Example 4 The germination of spores of Acidifiskicola lipolytica was induced according to the method of Example 3, except that no heat shock treatment was performed.

[0026] Example 5 The method of Example 3 was used to induce the germination of Bacillus acidiproducens spores, except that Bacillus subtilis spores (Example 5-1) Bacillus subtilis ) and Bacillus cereus spores (Example 5-2) Bacillus cereus ) were used instead of Bacillus acidiproducens spores.

[0027] Example 6 The method of Example 1 was used to induce the germination of Bacillus acidiproducens spores, except that the heat shock treatment was performed at 60°C, 65°C, and 75°C.

[0028] Example 7 The method of Example 4 was used to induce the germination of Bacillus acidiproducens spores, except that Experiment 1: no heat shock treatment, incubation temperature was 4°C, time was 12 days, and the pH of the buffer was 4.

[0029] Experiment 2: no heat shock treatment, incubation temperature was 4°C, time was 12 days, and the pH of the buffer was 7.

[0030] Experiment 3: no heat shock treatment, incubation temperature was 20°C, time was 12 days, and the pH of the buffer was 4.

[0031] Experiment 4: no heat shock treatment, incubation temperature was 20°C, time was 12 days, and the pH of the buffer was 7.

[0032] Experiment 5: no heat shock treatment, incubation temperature was 37°C, time was 12 days, and the pH of the buffer was 7.

[0033] Test Example The amount of pyridinedicarboxylic acid (DPA) released in the mixture during the incubation of Examples 1-7 was measured, and the results are shown below. As shown in Figures 1 and 2, when the heat shock treatment was performed at 75°C for 30 min and the incubation was performed at 37°C for 60 min, the DPA release amount of Bacillus acidiproducens spores did not increase with increasing concentration of the nutrient germinant L-alanine, AGFK, or L-alanine+AGFK, indicating that the Bacillus acidiproducens spores did not germinate better after the addition of the nutrient germinants. Figure 1 Figure 2 As shown in Figures 3 and 4, when the heat shock treatment was performed at 75°C for 30 min and the incubation was performed at 37°C for 60 min, the DPA release amount of Bacillus acidiproducens spores did not increase with increasing concentration of the nutrient germinant L-alanine, AGFK, or L-alanine+AGFK, indicating that the Bacillus acidiproducens spores did not germinate better after the addition of the nutrient germinants.

[0034] As shown in Figures 5 and 6, when the heat shock treatment was performed at 75°C for 30 min and the incubation was performed at 37°C for 60 min, the DPA release amount of Bacillus acidiproducens spores did not increase with increasing concentration of the nutrient germinant L-alanine, AGFK, or L-alanine+AGFK, indicating that the Bacillus acidiproducens spores did not germinate better after the addition of the nutrient germinants. Figure 3 Figure 4 As shown in Figures 7 and 8, when the heat shock treatment was performed at 75°C for 30 min and the incubation was performed at 37°C for 60 min, the DPA release amount of Bacillus acidiproducens spores did not increase with increasing concentration of the nutrient germinant L-alanine, AGFK, or L-alanine+AGFK, indicating that the Bacillus acidiproducens spores did not germinate better after the addition of the nutrient germinants.​​

[0035] By Figure 5 and Figure 6 As shown, without heat shock treatment at 75℃ / 30min, without adding germinant, adjusting the pH value of buffer, incubating at 37℃ for 30min, the spores still do not germinate, and the spores still appear bright under microscope after treatment.

[0036] By Figure 7 As shown, the buffer is at pH 4 under heat shock induction at 70℃, and the germination effect is better.

[0037] By Figure 8 As shown, without heat shock treatment and without adding germinant, under acidic conditions, incubate at 4℃, 20℃, and 37℃, respectively, after 12 days, the spores incubated at 37℃ have obvious DPA release, indicating that heat shock treatment is not a necessary condition for the germination of acid soil Alcanivorax spores, and acid soil Alcanivorax spores without heat shock treatment can also germinate.

[0038] In order to verify that this germination stimulation is only effective for acid soil Alcanivorax spores, Bacillus subtilis and Bacillus cereus spores were subjected to 70℃ / 30min heat shock in buffers at pH 3, 4, 5, 6, and 7, respectively, and then incubated at 37℃ for germination determination. As shown in Figure 9 As shown, the Bacillus subtilis and Bacillus cereus spores do not release DPA, and the acid soil Alcanivorax spores release a large amount of DPA under acidic conditions, and almost no release under neutral conditions at pH 7. Phase contrast microscopy also verifies this conclusion, and 75℃ / 30min heat shock combined with 37℃ treatment under acidic conditions has species specificity for acid soil Alcanivorax spores, and can significantly improve the germination effect of acid soil Alcanivorax spores, but has no effect on Bacillus subtilis and Bacillus cereus spores Figure 10 ).

[0039] In summary, the acidic conditions of 75℃ / 30min heat shock combined with 37℃ incubation are a means to specifically induce the germination of acid soil Alcanivorax spores.

[0040] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The ranges and values should be construed and interpreted as covering values adjacent and approximately around them. For numerical ranges the endpoints are included and the endpoints are independently combinable with each other, with the single points and with other points, to form new numerical ranges. The numerical ranges are specifically disclosed.

[0041] Although the embodiments of the present application have been shown and described above, it is understood that the above embodiments are exemplary, and are not to be interpreted as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A method of inducing the germination of spores of Paenibacillus albus in an acid soil, characterized in that, The application relates to a method for inducing the germination of acid soil Bacillus cyclohexanicus spores. The acid soil Bacillus cyclohexanicus spores are mixed with an acid solution with a pH value of 2-5 to obtain a mixed solution. The mixed solution is subjected to incubation treatment.

2. The method of claim 1, wherein, The temperature of the incubation treatment is 18-40 DEG C, and the time is 50-70 min.

3. The method of claim 1, wherein, The acid solution comprises at least one of a buffer solution, an aqueous hydrochloric acid solution, an aqueous sulfuric acid solution and an aqueous phosphoric acid solution.

4. The method of claim 3, wherein, The buffer solution comprises a K-hepes buffer solution.

5. The method of claim 1, wherein, The pH value of the acid solution is 4. The temperature of the incubation treatment is 37 DEG C, and the time is 60 min.

6. The method of claim 1, wherein, Before the incubation treatment, the acid solution is subjected to heat shock treatment.

7. The method of claim 6, wherein, The temperature of the heat shock treatment is 65-80 DEG C, and the time is 20-40 min.

8. The method according to claim 6 or 7, characterized in that, The temperature of the heat shock treatment is 70 DEG C, and the time is 30 min.

9. The method of claim 1, wherein, The application relates to a method for inducing the germination of acid soil Bacillus cyclohexanicus spores. The acid soil Bacillus cyclohexanicus spores are mixed with an acid solution with a pH value of 4 to obtain a mixed solution. The mixed solution is subjected to heat shock treatment at 70 DEG C for 30 min and then incubation treatment at 37 DEG C for 60 min.

10. A method of inactivating spores of Acidamyloliquefaciens Paenibacillus comprising, The application relates to a method for inducing the germination of acid soil Bacillus cyclohexanicus spores. The acid soil Bacillus cyclohexanicus spores are treated by the method for inducing the germination of acid soil Bacillus cyclohexanicus spores according to any one of claims 1-9.

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