Spore-induced germinator and application thereof in promoting germination and growth of spores of Haerella coagulatum

Abstract

The invention discloses a spore induction germinating agent and application thereof in promoting germination and growth of spores of Haemadella coagulans. The spore induction germinating agent is prepared from the following components: 30 to 100 mmol / L of D-glucose, 30 to 100 mmol / L of trehalose, 50 to 100 mmol / L of L-alanine, 50 to 100 mmol / L of inosine and 30 to 100 mmol / L of Ca < 2 + >-DPA. The method is simple and easy to operate, and the key technology is preparation of the spore germinator, water bath thermal activation and spore germination, so that the spore germination rate of the Haihndersiella coagulum is increased to 95%, and a theoretical basis is provided for germination of the Haihndersiella coagulum and exertion of probiotic efficacy after germination. The germination agent disclosed by the invention can be used for promoting rapid germination of the spores of the Haiderrikia coagulatum in livestock and poultry breeding and aquaculture, and effectively exerts the efficacy of the probiotics.

Description

Technical Field

[0001] The invention belongs to the field of spore germination induction, and particularly relates to a spore germination induction agent and application thereof in promoting the germination and growth of spores of Hyndrickella coagulans. Background Art

[0002] Heyndrickxia coagulans, formerly known as Bacillus coagulans, is a facultative anaerobic Gram-positive bacterium with the dual functions of lactic acid bacteria and Bacillus. It can produce both lactic acid and spores, and compared with other probiotics, it has stronger resistance to stress, high temperature, gastric acid and bile salts. Studies have shown that Heyndrickxia coagulans has multiple physiological activities such as regulating intestinal flora, promoting animal digestion and absorption, and immune regulation. It is called the king of probiotics and an animal growth promoter. In 2013, my country's Ministry of Agriculture officially included it in the "Feed Additive Variety Catalog" and it is widely used as an antibiotic substitute in livestock and poultry feed including aquaculture, laying eggs and ducks, broiler chickens and ducks, and weaned piglets.

[0003] When the external environment is suitable and nutrient-rich, Bacillus hendrixogenes reproduces vegetatively; if it encounters extreme conditions or nutrient deficiency, it induces spore production. Spores can withstand extreme conditions such as feed pelleting process and gastric juice, and are easier to preserve than nutrient cells. They are the main application form of microbial agents, but microbial agents can only grow and reproduce when they germinate into nutrient cells in the intestine, producing metabolites such as L-lactic acid and coagulin to exert probiotic activity. The survival rate of Bacillus hendrixogenes in the gastrointestinal tract is very high, but the germination rate of spores in the intestine is less than 10%. Studies have shown that the germination rate of Bacillus hendrixogenes spores is less than 70% under the action of germination agents (AGFK), D-fructose, L-asparagine and other germination factors commonly used by Bacillus. Therefore, it is of great significance to find an effective germination agent to improve the germination rate of Bacillus hendrixogenes to maximize the efficacy of probiotics in its production and application. Summary of the invention

[0004] Aiming at the problem of low germination rate of spores of Hyndrickella coagulans, the present invention provides a spore germination inducing agent and application thereof in promoting germination and growth of spores of Hyndrickella coagulans.

[0005] The spore germination inducing agent of the present invention comprises the following components: D-glucose 30-100 mmol / L, trehalose 30-100 mmol / L, L-alanine 50-100 mmol / L, inosine 50-100 mmol / L, Ca 2+-DPA30-100 mmol / L (30-100 mmol / L CaCl2, 30-100 mmol / L DPA).

[0006] The spore inducing agent uses 10 mmol / L Tris-HCl and 10 mmol / L NaCl as a compound solvent.

[0007] The invention discloses an application of the spore inducing germination agent in promoting the spore germination and growth of Hyndrickella coagulans.

[0008] The following steps are involved: Step 1: Spore collection and purification 1-1. Streak the coagulans on BC solid medium, culture at 40°C for 24 hours, pick out a single colony and transfer it to BC liquid medium, and culture it at 40°C and 180 r / min for 12 hours under shaking conditions.

[0009] 1-2. Transfer the coagulated Heindrixella liquid to the spore-forming medium at an inoculum of 1-5%, shake culture at 40℃, 180 r / min for 24 hours, and then culture anaerobically for 48 hours. When the spore conversion rate reaches more than 95%, collect the spores by centrifugation at 4℃, 8000 r / min for 10 minutes.

[0010] The spore transformation rate was evaluated by the malachite green staining method. The specific method was to add a drop of sterile water on a clean glass slide, spread the bacteria, dry, heat fix by flame back and forth 20 times, cover and stain with saturated malachite green at room temperature for more than 15 minutes, wash with water, cover with carbolic acid fuchsin stain for 1-2 minutes, wash with water, dry and observe under a microscope.

[0011] 1-3. In order to obtain 100% pure spores, immerse the spore suspension in an 80°C water bath for 15 minutes to remove the vegetative bodies and germinating cells. In order to effectively remove the culture medium residues and cell debris, wash the spores three times with pre-cooled sterilized ultrapure water to purify the spores. Adjust the concentration of the purified spores to 10 8 CFU / mL, stored at 4℃ for future use.

[0012] Step 2: Spore germination The absorbance of the spore suspension was adjusted to OD 600nm≈1.0, transfer to a 60-75℃ water bath for 10-20min, then cool to room temperature, centrifuge at 8000 r / min for 2min, add the spore inducing agent (volume 1mL:1mL), transfer to an incubator, and culture at 40-45℃, 150-200 r / min for 1-3h. Detect the absorbance of the spore suspension at 600 nm. Shake the test tube for at least 30s before detection to evenly distribute the spores.

[0013] The invention is simple and easy to operate, and the key technology lies in the preparation of spore germination agent, water bath heat activation and spore germination, so that the spore germination rate of coagulans hyendricksiella is increased to 95%, providing a theoretical basis for the germination of coagulans hyendricksiella and the probiotic effect after germination. The germination agent of the invention can be used in livestock and poultry breeding and aquaculture to promote the rapid germination of coagulans hyendricksiella spores and effectively exert the probiotic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Result graph of the germination rate of the spores of Hyndrickella coagulans in Example 1.

[0015] FIG. 2 is a graph showing the germination rate of spores of Bacillus heindrix in Example 2. DETAILED DESCRIPTION

[0016] Various specific embodiments of the present invention are described in detail below. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0017] BC medium (g / L): 5g / L yeast powder, 10g / L peptone, 10g / L sodium chloride, 5g / L glucose, pH 7.0, 1.5% agar powder added to the solids. Sterilize at 121℃ for 20min.

[0018] Sporulation medium (g / L): 2g / L glucose, 10g / L tryptone, 3g / L yeast extract, 10g / L sodium chloride, use bran water instead of distilled water to make up the volume. The bran water is made by weighing 150g bran, soaking it in 1000mL water, boiling it for 10-20min, then filtering it with three layers of gauze, and adding distilled water to the filtrate to 1000mL. Sterilize at 121℃ for 20min. Embodiment 1:

[0019] Step 1: Prepare the germination agent Preparation of germination medium for B. coagulans: 50 mmol / L D-glucose, 50 mmol / L trehalose, 100 mmol / L L-alanine, 100 mmol / L inosine and 50 mmol / L Ca2+ -DPA (50 mmol / L CaCl2, 50 mmol / L L DPA). Nutrient germination agent was prepared using germination buffer (10 mmol / L Tris-HCl, 10 mmol / L NaCl).

[0020] Step 2: Spore collection and purification Streak the coagulant Hyendricksiella on BC solid medium, culture at 40℃ for 24 hours, pick a single colony and transfer it to BC liquid medium, and culture it at 40℃ and 180 r / min for 12 hours. Transfer the coagulant Hyendricksiella liquid to the spore-forming medium with an inoculum of 3%, culture it at 40℃ and 180 r / min for 24 hours, and then culture it anaerobically for 48 hours. The spore transformation rate was evaluated by malachite green staining. When the spore transformation rate reached more than 95%, the spores were collected by centrifugation at 4℃ and 8000 r / min for 10 min. In order to obtain 100% pure spores, the spore suspension was immersed in an 80℃ water bath for 15 minutes to remove the vegetative bodies and germinating cells. In order to effectively remove the culture medium residues and cell debris, the spores were purified by washing them repeatedly three times with pre-cooled sterilized ultrapure water. The purified spores were adjusted to a concentration of 10 8 CFU / mL, stored at 4℃ for future use.

[0021] Step 3: Spore germination The absorbance of the spore suspension was adjusted to OD 600nm ≈1.0, transferred to a 75℃ water bath for 15 min and cooled to room temperature. Take 7 10mL sterilized centrifuge tubes and add 1mL of spore suspension to each tube. Centrifuge at 8000 r / min for 2 min and then add 1mL of the spore inducing agent, 50 mmol / L D-glucose, 50 mmol / L trehalose, 100 mmol / L L-alanine, 100 mmol / L inosine and 50 mmol / L Ca 2+-DPA (50 mmol / L CaCl2, 50 mmol / L DPA), with germination buffer (10 mmol / L Tris-HCl, 10 mmol / L NaCl) as blank control. Immediately measure the absorbance of the spore suspension at 600 nm, and then transfer the centrifuge tube to an incubator at 40-45℃, 150-200 r / min shaking culture for 2 h. Detect the absorbance of the spore suspension at 600 nm, and shake the test tube for at least 30 seconds before detection to make the spores evenly distributed. The optical density of the coagulation of the spore system of Hendrixella at 600nm decreases with the increase of the germination rate (the spores have high refractive index, and the absorbance increases after germination). When the germination rate reaches 100%, the optical density decreases to 50% to 60% of the initial value, and within this range, the decrease in optical density is inversely proportional to the increase in the spore germination rate, that is, the spore germination rate is twice the decrease in optical density.

[0022] The results are as follows Figure 1 As shown, when the germination agent was added, the germination rate of Bacillus hyendricus was 95.4%, which was higher than the germination rates of 50 mmol / L D-glucose, 50 mmol / L trehalose, 100 mmol / L L-alanine, 100 mmol / L inosine and 50 mmol / L Ca2+-DPA groups. The results indicate that the germination agent for Bacillus hyendricus in the present invention can well promote the germination of Bacillus hyendricus spores. Embodiment 2:

[0023] Step 1: Spore collection and purification Streak the coagulant Hendrixella on LB solid medium, culture at 40℃ for 24 hours, pick a single colony and transfer it to LB liquid medium, and culture it at 40℃ and 180 r / min for 12 hours. Transfer the coagulant Hendrixella liquid to the spore-forming medium with an inoculum of 3%, culture it at 40℃ and 180 r / min for 24 hours, and then culture it anaerobically for 48 hours. The spore transformation rate was evaluated by malachite green staining. When the spore transformation rate reached more than 95%, the spores were collected by centrifugation at 4℃ and 8000 r / min for 10 min. In order to obtain 100% pure spores, the spore suspension was immersed in an 80℃ water bath for 15 minutes to remove the vegetative bodies and germinating cells. In order to effectively remove the culture medium residues and cell debris, the spores were purified by washing them repeatedly three times with pre-cooled sterilized ultrapure water. The purified spores were adjusted to a concentration of 10 8 CFU / mL, stored at 4℃ for future use.

[0024] Step 2: Prepare the germination agent Preparation of Germination Agent A for Bacillus coagulans: 50 mmol / L D-glucose, 50 mmol / L trehalose, 100 mmol / L L-alanine, 100 mmol / L inosine, and 50 mmol / L Ca 2+ -DPA (50 mmol / L CaCl2, 50mmol / L DPA), and the nutrient germination agent was prepared using germination buffer (10 mmol / L Tris-HCl, 10 mmol / L NaCl).

[0025] Preparation of Germination Agent B for Bacillus coagulans: 30 mmol / L D-glucose, 30 mmol / L trehalose, 100 mmol / L L-alanine, 100 mmol / L inosine, and 50 mmol / L Ca 2+ -DPA (50 mmol / L CaCl2, 50mmol / L DPA), and the nutrient germination agent was prepared using germination buffer (10 mmol / L Tris-HCl, 10 mmol / L NaCl).

[0026] Preparation of Germination Agent C for Bacillus coagulans: 50 mmol / L D-glucose, 50 mmol / L trehalose, 50 mmol / L L-alanine, 50 mmol / L inosine and 50 mmol / L Ca 2+ -DPA (50 mmol / L CaCl2, 50 mmol / L L DPA), and the nutrient germination agent was prepared using germination buffer (10 mmol / L Tris-HCl, 10 mmol / L NaCl).

[0027] Preparation of Germination Agent D for Bacillus coagulans: 100 mmol / L D-glucose, 100 mmol / L trehalose, 50 mmol / L L-alanine, 50 mmol / L inosine, and 50 mmol / L Ca 2+ -DPA (50 mmol / L CaCl2, 50 mmol / L DPA), and the nutrient germination agent was prepared using germination buffer (10 mmol / L Tris-HCl, 10 mmol / L NaCl).

[0028] Step 3: Spore germination The absorbance of the spore suspension was adjusted to OD 600nm≈1.0, transfer to a 75℃ water bath for 15 min and cool to room temperature. Take 5 10mL sterilized centrifuge tubes and add 1mL of spore suspension to each tube. Centrifuge at 8000 r / min for 2 min and add 1mL of germination agents A, B, C, and D prepared in step (2) respectively. Use germination buffer (10 mmol / L Tris-HCl, 10 mmol / LNaCl) as blank control. Immediately measure the absorbance of the spore suspension at 600 nm, then transfer the test tube to an incubator at 40-45℃ and shake at 150-200 r / min for 2 h. Detect the absorbance of the spore suspension at 600 nm. Shake the test tube for at least 30 seconds before detection to ensure uniform distribution of the spores. The optical density of the coagulant Heindrix spore system at 600nm decreases with the increase of germination rate (spores have high refractive index, and the absorbance increases after germination). When the germination rate reaches 100%, the optical density decreases to 50% to 60% of the initial value, and within this range, the decrease in optical density is inversely proportional to the increase in spore germination rate, that is, the germination rate of spores is twice the rate of decrease in optical density.

[0029] The results are as follows Figure 2 As shown in the figure, when the germination agent A was added, the germination rate of Bacillus hyendricus was 95.4%, which was higher than the germination rates of the germination agents B, C and D. The results showed that the optimal formula of the germination agent for Bacillus hyendricus in the present invention was 50 mmol / L D-glucose, 50 mmol / L trehalose, 100 mmol / L L-alanine, 100 mmol / L inosine and 50 mmol / L Ca 2+ -DPA (50 mmol / L CaCl2, 50 mmol / L DPA).

Claims

1. A spore inducing agent, characterized in that The components include: D-glucose 30-100mmol / L, trehalose 30-100 mmol / L, L-alanine 50-100 mmol / L, inosine 50-100mmol / L, Ca 2+ -DPA 30-100 mmol / L.

2. The spore germination inducing agent according to claim 1, characterized in that: The spore inducing agent is prepared by using 10 mmol / L Tris-HCl and 10 mmol / L NaCl as a compound solvent.

3. The spore germination inducing agent according to claim 1 or 2, characterized in that The components include: D-glucose 50 mmol / L, trehalose 50 mmol / L, L-alanine 100 mmol / L, inosine 100 mmol / L, Ca 2+ -DPA 50 mmol / L.

4. Use of the spore inducing agent according to claim 1 or 2 in promoting the spore germination and growth of Hyndrickella coagulans.

5. The use according to claim 4, characterized in that The following steps are involved: Step 1: Spore collection and purification 1-1. Streak the coagulant Bacillus on BC solid medium, culture at 40°C for 24 hours, pick a single colony and transfer it to BC liquid medium, and culture it at 40°C and 180 r / min for 12 hours; 1-2. Transfer the coagulated Hendrixella liquid to the spore-forming medium, and culture it anaerobically at 40°C and 180 rpm for 24 hours; 1-3. Soak the spore suspension obtained in 1-2 in an 80°C water bath for 15 minutes to remove the vegetative bodies and germinating cells, wash and purify the spores with sterilized ultrapure water, and adjust the concentration of the purified spores to 10 8 CFU / mL, stored at 4℃ for future use; Step 2: Spore germination The absorbance of the spore suspension was adjusted to OD 600nm ≈1.0, take 1 mL and transfer to 60-75℃ water bath for 10-20min, then cool to room temperature, centrifuge at 8000 r / min for 2min, add the spore inducing agent, and transfer to incubator for culture.

6. The use according to claim 5, characterized in that: In step 1-2, when the spore conversion rate reaches more than 95%, the spores are collected by centrifugation.

7. The use according to claim 5, characterized in that: In step 2, after adding the spore inducing agent, the mixture is transferred to an incubator and cultured at 40-45° C. and 150-200 r / min for 1-3 hours.

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