Method for delaying formation of bacillus spores and application

Abstract

The invention relates to the field of microorganisms, in particular to a method for delaying spore formation of bacillus and application. Sodium selenite is added into a culture medium in the initial logarithmic growth stage of bacillus fermentation culture, so that the formation of spores is delayed; the concentration of the selenite is 0.01 to 0.5 mM. The method achieves a good effect in the treatment or prevention of ulcerative colitis, colorectal cancer and intestinal stress. The invention not only provides a basis for exploring a molecular mechanism of selenium-regulated spore formation, but also verifies that the survival competitiveness and the treatment efficiency of the probiotics can be improved through simple culture medium optimization, and provides a feasible new strategy for enhancing the curative effect of a probiotic therapy in intestinal diseases such as ulcerative colitis and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to a method for delaying the sporulation of Bacillus and application thereof. BACKGROUND

[0002] With the deepening of the study of intestinal microbiome, probiotic therapy has attracted widespread attention in the application of various diseases. Probiotics (such as some strains of Bacillus) can enhance their survival ability by forming spores in adverse environments, and spores can germinate into vegetative cells under certain conditions. The gastrointestinal microenvironment rich in nutrients is the main factor for spore germination. Due to the lack of nutrients in the colon environment and the increased sensitivity of exponential phase vegetative cells, surviving Bacillus will move down to the large intestine and form spores in the lower part of the colon, which is not conducive to the full play of the function of probiotics. Therefore, delaying the sporulation of probiotics is an important way to improve the efficacy of probiotic therapy. There are many factors that affect sporulation, and current studies have shown that some trace elements have a significant impact on the sporulation of Bacillus. Selenium, as one of the essential trace elements for the human body, it is very important to study its influence on the sporulation of Bacillus.

[0003] Chinese patent with publication number CN116286491A provides a kind of compound selenium-rich microbial inoculant and its preparation method, four kinds of selenium-rich culture fermentation liquor are mixed with rice husk powder to make inoculant by stirring, then organic matter degrading inoculant, selenium-rich inoculant, auxiliary inoculant are mixed and prepared into compound selenium-rich microbial inoculant according to certain proportion.

[0004] The existing method for increasing the amount of effective bacteria is usually more complicated, for example, as described in the aforementioned patent (CN116286491A), it is necessary to prepare multiple different selenium-rich microbial fermentation liquids, and then go through multiple mixing and preparation processes. This complex production process not only increases the time and economic cost, but also puts higher challenges on the quality control of industrial production, reduces the universality and repeatability of the technology, and makes it difficult to be stably promoted in standardized treatment.

[0005] When Bacillus passes through the small intestine with relatively rich nutrients, its spores will germinate into vegetative cells and proliferate. However, when the bacteria continue to move down with the contents of the digestive tract to the large intestine and colon area where nutrients are scarce, they will quickly initiate the spore formation program, i.e. "retreat into spores". Spores are metabolically dormant resting bodies and cannot directly exert probiotic functions (such as secreting antibacterial substances, competing for colonization sites, and regulating immunity), which leads to a significant shortage of vegetative cells that can ultimately exert therapeutic effects in the colon, forming an embarrassing situation of "arrival and dormancy".

[0006] The two shortcomings above together lead to a third fundamental defect, which is insufficient efficacy. Because the preparation process limits the efficient and standardized production of the bacterial agent, and the spore-forming of the probiotic bacteria itself is inactivated at the target site, sufficient and active probiotic bacteria cannot effectively colonize and continuously function at the disease site (such as the colon mucosa of patients with colitis). Therefore, even if a large amount of probiotic agent is taken, the number of bacterial colonies that actually reach the colon and remain active is often far below the treatment threshold, making it difficult to produce significant and stable clinical efficacy. SUMMARY

[0007] In view of the problems in the background art, a method for delaying the formation of spores of Bacillus and an application are provided, which not only provides a basis for exploring the molecular mechanism of selenium regulating spore formation, but also confirms that the survival competitiveness and therapeutic efficacy of probiotics can be improved by simply optimizing the culture medium, thereby providing a new and feasible strategy for enhancing the efficacy of probiotic therapy in intestinal diseases such as ulcerative colitis.

[0008] The present application provides a method for delaying the formation of spores of Bacillus, which delays the formation of spores by adding sodium selenite to the culture medium at the early logarithmic growth stage of Bacillus fermentation culture; the concentration of selenite is 0.01-0.5mM.

[0009] Preferably, the specific method steps are as follows:

[0010] S1, inoculate the original Bacillus strain into the culture medium according to the volume ratio of 1:15-25 for activation culture;

[0011] S2, add selenite to the strain obtained in S1 and continue to culture to obtain a bacterial solution.

[0012] Preferably, the culture conditions in S1 are as follows: the shaking speed is 150-220rpm, the temperature is 35-38℃, and the culture time is 4-12h.

[0013] Preferably, the culture conditions in S2 are as follows: the shaking speed is 150-220rpm, the temperature is 35-38℃, and the culture time is 12-48h.

[0014] The present application further provides an application of the above-mentioned method for delaying the formation of spores of Bacillus: centrifuging the bacterial solution obtained after adding selenite to obtain probiotics, and applying the probiotics in the treatment or prevention of ulcerative colitis, colorectal cancer and intestinal stress.

[0015] Compared with the prior art, the present application has the beneficial technical effects that: by adding different concentrations of sodium selenite (Na2SeO3) in the culture medium of Bacillus coagulans (BC), Bacillus licheniformis (BL) and Bacillus subtilis (BS), the influence of selenium concentration on the spore formation rate of Bacillus is systematically evaluated by plate colony counting method, gram staining and other characterization means. The results show that when the concentration of sodium selenite is 0.06mM, the spore formation process of the three kinds of Bacillus can be significantly inhibited under the premise of effectively maintaining the activity of the three kinds of Bacillus, and the spore yield is reduced to the lowest level (BC, BL, BS is reduced to 2%-80%, 2%-53%, 2%-38%), and the optimal intervention concentration suitable for different strains is successfully selected. The method is simple to operate, and the conversion of probiotics from vegetative cells to spores can be effectively delayed by adding a specific concentration of sodium selenite, thereby prolonging the proliferation time. Further animal experiment verifies the application value of the strategy: in the DSS-induced colitis mouse model, the colonization number of Bacillus subtilis (BSSe group) pretreated with 0.06mM sodium selenite in the colon is significantly higher than that of the untreated group (BS group), and effectively alleviates the pathological symptoms such as weight loss, disease activity index (DAI) increase and colon shortening of mice; histopathological analysis further shows that BSSe intervention can significantly reduce the inflammatory damage of colon tissue and almost maintain the complete colon microstructure.

[0016] In summary, the present application not only provides a basis for exploring the molecular mechanism of selenium regulating spore formation, but also confirms that the survival competitiveness and treatment efficiency of probiotics can be improved by simply optimizing the culture medium, providing a feasible new strategy for enhancing the efficacy of probiotic therapy in intestinal diseases such as ulcerative colitis. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 A schematic diagram of the influence of different concentrations of Na2SeO3 on the spore formation rate of Bacillus subtilis;

[0018] Figure 2 A schematic diagram of the influence of different concentrations of Na2SeO3 on the spore formation rate of Bacillus coagulans;

[0019] Figure 3 A schematic diagram of the influence of different concentrations of Na2SeO3 on the spore formation rate of Bacillus coagulans;

[0020] Figure 4 A schematic diagram of the number of Bacillus subtilis colonies in the colon of mice in different groups;

[0021] Figure 5 A schematic diagram of the DAI score of mice in different groups;

[0022] Figure 6 A schematic diagram of the colon morphology of mice in different groups;

[0023] Figure 7 Figure 4 is a HE staining diagram of colon tissue of mice in the same group. DETAILED DESCRIPTION

[0024] Example 1, the present embodiment proposes a method for delaying the formation of Bacillus spores, mainly aiming at Bacillus subtilis, the steps are as follows:

[0025] (1) Bacillus subtilis in the logarithmic growth phase is inoculated in culture medium with a sodium selenite concentration of 0, 0.015, 0.03, 0.06 and 0.125 mM at an inoculation ratio of 4%, and the culture medium formula is 100 mL distilled water, 0.5 g peptone, 0.3 g beef extract and 0.5 g sodium chloride, and the bacterial liquid is collected after 24 h of culture.

[0026] (2) Each group of bacterial liquid obtained in step (1) is centrifuged at 4000 r for 5 min to obtain Bacillus subtilis with delayed spore shedding.

[0027] The spore yield of Bacillus subtilis obtained by the above method is reduced to 2% to 38%, as shown in Table 1. Figure 1

[0028] Example 2, the present embodiment proposes a method for delaying the formation of Bacillus spores, mainly aiming at Bacillus coagulans, the steps are as follows:

[0029] (1) Bacillus coagulans in the logarithmic growth phase is inoculated in culture medium with a sodium selenite concentration of 0, 0.015, 0.03, 0.06 and 0.125 mM at an inoculation ratio of 4%, and the culture medium formula is 100 mL distilled water, 1.0 g peptone, 0.3 g beef extract and 0.5 g sodium chloride, and the bacterial liquid is collected after 24 h of culture.

[0030] (2) Each group of bacterial liquid obtained in step (1) is centrifuged at 4000 r for 5 min to obtain Bacillus coagulans with delayed spore shedding.

[0031] The spore yield of Bacillus subtilis obtained by the above method is reduced to 2% to 38%, as shown in Table 1. Figure 2

[0032] Example 3, the present embodiment proposes a method for delaying the formation of Bacillus spores, mainly aiming at Bacillus coagulans, the steps are as follows:

[0033] (1) Bacillus coagulans in the logarithmic growth phase is inoculated in culture medium with a sodium selenite concentration of 0, 0.015, 0.03, 0.06 and 0.125 mM at an inoculation ratio of 4%, and the culture medium formula is 100 mL distilled water, 1.0 g peptone, 0.3 g beef extract and 0.5 g sodium chloride, and the bacterial liquid is collected after 24 h of culture.​​

[0034] (2) The obtained bacterial liquid in step (1) is centrifuged at 4000 r for 5 min to obtain B. licheniformis with delayed spore formation.

[0035] The yield of the spores of the B. licheniformis obtained by the above method is reduced to 2% to 53%, as shown in Table 1. Figure 3

[0036] In Examples 1, 2 and 3, different concentrations of Na2SeO3 are added to the culture medium of B. coagulans (BC), B. licheniformis (BL) and B. subtilis (BS), respectively. The effects of different concentrations of selenium on the spore formation rate of the bacillus spores are studied by plate colony counting method and Gram staining. The optimal concentration of Na2SeO3 for the culture medium of BC, BL and BS is 0.06 mM. Under the premise that the activity is not inhibited, the three kinds of bacillus spores show the weakest sporulation capacity. This provides a basis and evidence for prolonging the proliferation time of probiotics, improving the therapeutic effect of probiotic therapy, and further studying the key genes and signal pathways of selenium regulating spore formation.

[0037] In Example 4, the application of the method for delaying the formation of bacillus spores is proposed. The method achieves good results in the treatment or prevention of ulcerative colitis, colorectal cancer and intestinal stress. Taking Example 1 as an example, the concentration of Na2SeO3 is 0.06 mM. The application includes the following steps:

[0038] In this experiment, healthy C57BL / 6 male mice (Beijing Spero Biological Co., Ltd.) were used as animal models and randomly divided into three groups: blank control group (Control), modeling group (DSS), B. subtilis group (DSS+BS), and sodium selenite-treated B. subtilis group (DSS+BSSe). Each group had 7 mice. The treatment methods for the B. subtilis group (DSS+BS) and the sodium selenite-treated B. subtilis group (DSS+BSSe) were as follows: from the 1st to the 7th day of the experiment, the mice were given 100 μL of B. subtilis solution (DSS+BS) and sodium selenite-treated B. subtilis solution (DSS+BSSe) for free drinking of distilled water. From the 8th to the 14th day of the experiment, the mice were given 3% DSS solution. The treatment method for the modeling group (DSS) was as follows: from the 1st to the 7th day of the experiment, the mice were given 100 μL of normal saline by gavage and free drinking of distilled water. From the 8th to the 14th day of the experiment, the mice were given 3% DSS solution. The treatment method for the blank control group (Control) was as follows: during the experiment, the mice in the control group were given 100 μL of normal saline by gavage and free drinking of distilled water. The body weight and disease activity index (DAI) scores of the mice were recorded every day. After the treatment ended, the colon tissues were collected, plated to detect the concentration of B. subtilis, and subjected to H&E staining for further analysis.​

[0039] As shown in Figure 4 , it can be seen that at 48h, the number of probiotics in the colon of the BSSe group is significantly increased compared with the BS group. From the experimental results ( Figure 5 ), it can be seen that the body weight of the healthy control group of mice increases slightly, and the DAI remains at a low level, indicating that the mice are in good health, and the DAI value of the mice treated by BSSe is lower. As shown in Figure 6 , the colon length of the DSS model group of mice is only about 4.8cm, while the colon length of the BSSe intervention group is significantly increased to about 6cm. In addition, by analyzing the pathological sections of the colon tissue, as shown in Figure 7 , from the cell morphology and arrangement, the colon tissue of the healthy control group shows no signs of damage or inflammation. After DSS induction, the colon tissue shows obvious epithelial damage and colonic inflammation. The BSSe group treatment significantly reduces the inflammatory condition and almost retains the complete colon microscopic structure morphology, indicating that BSSe can alleviate the symptoms of acute colonic inflammation and colon tissue damage, and significantly relieve colitis.

[0040] The embodiments of the application are described in detail above with reference to the drawings, but the application is not limited thereto, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the application.

Claims

1. A method for delaying the formation of Bacillus spores, characterized in that, Adding sodium selenite to the culture medium during the early logarithmic growth phase of Bacillus fermentation culture delayed spore formation. The concentration of selenite is 0.01-0.5 mM.

2. The method for delaying Bacillus spore formation according to claim 1, characterized in that, The specific steps are as follows: S1. Inoculate the original Bacillus strain into the culture medium at a volume ratio of 1:15-25 for activation culture; S2. Add selenite to the bacterial culture obtained in S1 and continue culturing to obtain bacterial solution.

3. The method for delaying Bacillus spore formation according to claim 2, characterized in that, The culture conditions in S1 are: shaking speed 150-220 rpm, temperature 35-38℃, culture time 4-12 h.

4. The method for delaying Bacillus spore formation according to claim 2, characterized in that, The cultivation conditions in S2 are: shaking speed 150-220 rpm, temperature 35-38℃, and cultivation time 12-48 h.

5. The application of the method for delaying Bacillus spore formation as described in claim 1, characterized in that, Probiotics were obtained by centrifuging the bacterial solution after adding selenite. The probiotics were then applied to the treatment or prevention of ulcerative colitis, colorectal cancer, and intestinal irritability.

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