Method for efficiently separating high-yield amylase endophytic bacteria in sorghum grains
Through the combination of ethanol and chitosan-sodium hypochlorite composite disinfectant combined with targeted enrichment and gradient dilution purification, the pollution and screening efficiency of endogenous bacterial isolation in sorghum grains was solved, and the efficient isolation of high-yield amylase strains was achieved, which improved the economic benefits of the brewing process.
Patent Information
- Application Number
- CN202510560761.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-08-05
AI Technical Summary
In the prior art, the isolation of endogenous bacteria in sorghum grains is not thoroughly disinfected, resulting in pollution interference, low screening efficiency of strain functional activity, poor isolation targeting, and insufficient proportion of high-yield amylase strains, which affects the economic benefits of the brewing process.
The combination of ethanol-sodium hypochlorite composite disinfectant was used to disinfect, and then purified by targeted enrichment culture medium and gradient dilution, combined with starch screening medium and Lugo's iodine solution, and finally the amylase activity was measured and the high-yield amylase strains were isolated.
It improves the isolation targeting of endophyte bacteria and the amylase production activity of strains, reduces pollution interference, improves the separation efficiency and enzyme activity, and has environmentally friendly economic benefits.
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Figure CN120424769A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of fermentation engineering microbial separation, and particularly relates to a method for efficiently separating high-amylase-producing endophytic bacteria in sorghum grains. Background Art
[0002] Sorghum, a dual-purpose grain and feed crop, has a wide range of applications in brewing, particularly in the production of baijiu (white liquor) and aged vinegar. Amylase plays a crucial role in the brewing process. Amylase catalyzes the conversion of sorghum starch into fermentable sugars, providing a key substrate for the fermentation stage. Introducing high-yielding amylase strains into the fermentation process helps achieve efficient starch conversion, thereby improving economic returns.
[0003] Plant grains harbor a large number of endophytic bacteria, establishing mutually beneficial symbiotic relationships with their host plants and playing a key role in plant growth and development, environmental adaptation, and grain quality. The chemical composition of grains coevolves with their endophytic bacterial communities. Sorghum grains are rich in starch, and long-term directional selection pressures may have driven the evolution of efficient amylase synthesis and secretion by bacteria within the grains to maximize substrate utilization. Targeted isolation of high-amylase-producing endophytic bacteria from sorghum grains would not only provide an efficient enzyme source for traditional fermentation industries but also provide high-quality candidate strains for the design of novel industrial enzyme preparations. However, the isolation of endophytic bacteria from sorghum grains currently faces numerous technical bottlenecks. For example, incomplete surface disinfection of grains can lead to contamination: sorghum grains have a prominent wrinkled surface structure and a high microbial colonization density. Conventional single disinfectants have insufficient inactivation rates against deep-seated bacteria, resulting in residual contaminating bacteria as high as 15-30%, severely impacting the activity of the endophytic microbiome. In addition, the screening efficiency of strain functional activity is low and the separation targeting is poor: only 30-40% of the isolated strains have amylase activity, and the proportion of high-yield strains (amylase activity > 200 U / mL) is less than 5%. Summary of the Invention
[0004] In view of this, the present invention aims to overcome the shortcomings of the above-mentioned prior art and provide a method for efficiently isolating high-amylase-producing endophytic bacteria from sorghum grains. The method has the characteristics of strong isolation targeting and high amylase-producing activity of the strain.
[0005] To achieve the above object, the technical solution of the present invention is as follows:
[0006] A method for efficiently isolating high-amylase-producing endophytic bacteria from sorghum grains comprises the following steps:
[0007] Step 1, surface disinfection: Soak the sorghum kernels in ethanol, transfer the kernels to a chitosan-sodium hypochlorite compound disinfectant solution, gently stir during soaking, and finally rinse the kernels with sterile distilled water; take the last rinse solution and evenly spread it on LB solid medium for culture. If no colonies appear on the LB solid medium, the sorghum kernels are considered to have passed the surface disinfection;
[0008] Step 2, targeted enrichment: Surface-sterilized sorghum grains are fully ground into fine powder, sieved, mixed with enrichment culture medium, and cultured with shaking to prepare a bacterial suspension;
[0009] Step 3, separation and purification: dilute the bacterial suspension with sterile water in a gradient manner, diluting 10 3 times, 10 4 times, 10 5 times, 10 6 times, and evenly spread the bacterial suspension at each dilution concentration on LB solid medium, invert and culture, pick single colonies with different morphological characteristics, streak inoculate on LB solid medium for purification, and obtain purified strains;
[0010] Step 4, initial screening of strains: The purified strains were inoculated onto starch screening medium and cultured in an inverted manner. After the culture was completed, Lugol's iodine solution was added to the starch screening medium to cover the surface of the medium. After standing, the iodine solution was poured out and the colonies were observed for the presence of a transparent zone. The diameter D of the transparent zone and the diameter d of the colony were measured. Strains with a D / d ratio greater than 1.5 were considered to be amylase-producing strains.
[0011] Step 5, strain rescreening: inoculate the amylase-producing strain obtained in the initial screening into a seed culture medium for shaking culture. When the OD600 of the culture medium is 1, transfer it to the basic fermentation medium and continue shaking culture; take the fermentation liquid and centrifuge it to collect the supernatant, i.e., the crude enzyme liquid; use 3,5-dinitrosalicylic acid colorimetry to determine the amylase activity in the crude enzyme liquid; strains with amylase activity >200 U / mL are considered high-amylase-producing strains.
[0012] In an optional embodiment, in step 1, 75% ethanol is used to soak the sorghum kernels for 5 minutes to remove fat-soluble pollutants and some surface microorganisms; after soaking, the sorghum kernels are transferred to a chitosan-sodium hypochlorite composite disinfectant and soaked for 10 minutes, stirring gently 2-3 times during the soaking to achieve enhanced sterilization and protect the activity of endophytic bacteria; finally, the sorghum kernels are washed 5 times with sterile distilled water to remove residual disinfectant and prevent endophytic bacteria from being killed.
[0013] In an optional embodiment, the preparation method of the chitosan-sodium hypochlorite composite disinfectant in step 1 is as follows: acetic acid solution is slowly added to the chitosan powder at a ratio of 100 mL of 1% acetic acid solution per 0.5 g of chitosan powder and stirred until it is completely dissolved to prepare a chitosan solution with a concentration of 0.5%; then, sodium hypochlorite stock solution is slowly added to the chitosan solution until the effective chlorine concentration is 0.5-1% (because the sorghum grain epidermis is firm, the surface is wrinkled, and the microbial colonization density is high, if the effective chlorine concentration is lower than 0.5%, it is not possible to quickly and effectively kill microorganisms on the surface of the sorghum grain; if the effective chlorine concentration is higher than 1%, the strong oxidizing property of the high concentration of effective chlorine may penetrate the interior of the grain and non-specifically kill the target endophytes), stirring while adding to avoid violent reactions; finally, NaHCO3 is used to adjust the pH value of the mixed solution to 5.9±0.1 to maintain the stability of sodium hypochlorite. In the above technical solution, the chitosan solution forms a film on the surface of the grain, effectively destroying the cell membranes of pathogenic microorganisms on the grain surface and exerting an antibacterial effect. Furthermore, the chitosan film can activate the grain's defense mechanism, increasing its resistance to pathogens and preventing endophytic bacteria from being inhibited by pathogens. Sodium hypochlorite can be used to rapidly kill surface-attached microorganisms. Combining the film-forming and antibacterial properties of chitosan with the bactericidal properties of sodium hypochlorite can improve the disinfectant's efficiency in inactivating microorganisms on the grain surface. Furthermore, compared to traditional single disinfectants, the composite disinfectant can significantly increase the survival rate of endophytic bacteria in the grain.
[0014] In an optional embodiment, the sieving in step 2 is through a 100-200 mesh standard sieve; the rotation speed of the shaking culture is 200 r / min, the temperature is 37° C., and the time is 24 h.
[0015] In an optional embodiment, the ratio of mixing the fine powder and the enrichment culture medium in step 2 is 1 g / 100 mL.
[0016] In an optional embodiment, the formula of the enrichment culture medium in step 2 is: 5g / L soluble starch, 5g / L sorghum grain starch, 10g / L peptone, 1g / L K2HPO4, 1g / L NaCl, 0.5g / L MgSO4, 0.1-0.5% (w / v) activated carbon, 50mg / L Cycloheximide, and the pH is adjusted to 6.8. In the above technical solution, the activated carbon content in the enrichment culture medium is 0.1-0.5% (w / v), and its function is to adsorb growth and metabolic inhibitors produced by endophytic bacteria during the culture process. If the content is lower than 0.1%, the activated carbon cannot effectively adsorb the inhibitors, and the growth and enzyme production of endophytic bacteria will be inhibited; if the content is higher than 0.5%, excessive activated carbon will adsorb nutrients in the culture medium, resulting in a decrease in the growth rate of the bacteria. The sorghum grain starch in the enrichment medium should be extracted from mature sorghum grains of the same variety. This starch serves to construct a host biomimetic system and induce the endophytic bacteria to synthesize and secrete amylase. The concentration should be maintained at 5g / L. A concentration that is too low will have a negligible effect, while a concentration that is too high will inhibit bacterial growth.
[0017] In an optional embodiment, the temperature of the inverted culture in step 3 is 37° C. and the time is 2-5 days.
[0018] In an optional embodiment, the temperature of the inverted culture in step 4 is 37° C. and the time is 24 hours.
[0019] In an optional embodiment, the rotation speed of the shaking culture in step 5 is 200 r / min.
[0020] In an optional embodiment, the formula of the basic fermentation medium in step 5 is: glucose 20 g / L, peptone 20 g / L, K2HPO4 1 g / L, NaCl 1 g / L, pH 7.0.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] The present invention provides a method for efficiently isolating high-amylase-producing endophytic bacteria from sorghum grains. The method has a simple separation process and strong separation targeting. The separated endophytic bacteria have high amylase-producing activity, are environmentally friendly and pollution-free, and have obvious environmental and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is a graph showing the amylase activity of the nine high-yielding endophytic amylase strains in Example 1. DETAILED DESCRIPTION
[0024] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.
[0025] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.
[0026] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.
[0027] Example 1
[0028] The sorghum variety Jiuxiangnuo No. 10 was used for the experiment.
[0029] Step 1: Surface disinfection. Soak the sorghum kernels in 75% ethanol for 5 minutes. Transfer the kernels to a chitosan-sodium hypochlorite disinfectant solution and soak for 10 minutes, gently stirring three times. Finally, rinse the kernels five times with sterile distilled water. Take 50 μL of the final rinse solution and evenly spread it on LB solid medium. Incubate at 37°C for 24 hours. If no bacterial colonies appear on the LB solid medium after 24 hours, the sorghum kernels are considered to have passed surface disinfection. The chitosan-sodium hypochlorite composite disinfectant is prepared as follows: 100 mL of 1% acetic acid solution is added to 0.5 g of chitosan powder. The acetic acid solution is slowly added to the chitosan powder and stirred until completely dissolved, resulting in a 0.5% chitosan solution. A sodium hypochlorite solution is then slowly added to the chitosan solution to achieve an effective chlorine concentration of 0.5%, stirring to avoid a violent reaction. Finally, the pH of the mixture is adjusted to 5.9 ± 0.1 with NaHCO₃ to maintain the stability of the sodium hypochlorite. Store in dark and use within 2 hours of preparation.
[0030] Step 2, targeted enrichment, aims to selectively amplify amylase-producing bacterial strains. Surface-sterilized sorghum grains were thoroughly ground into fine powder in a clean bench, and the ground powder was sieved through a 200-mesh standard sieve. 100 mL of enrichment culture medium was mixed for every 1 g of fine powder, and the mixture was shaken at 200 rpm and 37°C for 24 hours to uniformly disperse the fine powder and prepare a bacterial suspension. The enrichment culture medium formula is: 5 g / L soluble starch, 5 g / L sorghum grain starch, 10 g / L peptone, 1 g / L K2HPO4, 1 g / L NaCl, 0.5 g / L MgSO4, 0.1-0.5% (w / v) activated carbon, 50 mg / L cycloheximide, and the pH was adjusted to 6.8.
[0031] Step 3, separation and purification. Use sterile water to dilute the bacterial suspension in a gradient manner, diluting 10 3 times, 10 4 times, 10 5 times, 10 6 times, and take 50 μL of the bacterial suspension at each dilution concentration, spread it evenly on LB solid medium, and culture it upside down at 37°C. 5 times, 10 6 A total of 21 single colonies with different morphological characteristics were observed on the LB solid culture medium after the bacterial suspension was spread with multiple-fold gradient dilutions. These single colonies were streaked onto the LB solid culture medium for purification.
[0032] Step 4: Initial strain screening. The 21 purified strains were individually inoculated onto starch screening medium and incubated inverted at 37°C for 24 hours. Then, 4 mL of Lugol's iodine solution was added to the starch screening medium, covering the entire surface. After standing for 1 minute, the iodine solution was poured off. Nine strains produced transparent zones around the colonies. The diameter of the transparent zone (D) and the colony diameter (d) were measured. The D / d ratios of all nine strains were greater than 1.5, indicating that all were amylase-producing strains.
[0033] Step 5: Rescreening of strains to determine the amylase activity of the primary screened strains. The 9 amylase-producing strains obtained from the primary screening were inoculated into the seed culture medium and cultured at 37°C with a shaking speed of 200 r / min. 600 =1, transfer the inoculum to the basic fermentation medium at a rate of 5% (v / v) and continue shaking culture for 24 hours. Take an appropriate amount of fermentation broth and centrifuge to collect the supernatant, which is the crude enzyme solution. The amylase activity in the crude enzyme solution is determined by 3,5-dinitrosalicylic acid (DNS) colorimetry. The amylase activity of 9 amylase-producing strains (B1-B9) is shown in Figure 1 The amylase activity of the strains was greater than 200 U / mL, indicating that they were all high-yielding amylase strains. The basic fermentation medium was formulated as follows: 20 g / L glucose, 20 g / L peptone, 1 g / L K2HPO4, 1 g / L NaCl, pH 7.0.
[0034] The above description is only for better explanation of the embodiments of the present invention and is not intended to limit the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention are intended to be within the scope of the present invention.
Claims
1. A method for efficiently isolating high-yielding amylase-producing endophytic bacteria from sorghum grains, characterized in that: The following steps are involved: Step 1, surface disinfection: Soak the sorghum kernels in ethanol, transfer the kernels to a chitosan-sodium hypochlorite compound disinfectant solution, gently stir during soaking, and finally rinse the kernels with sterile distilled water; take the last rinse solution and evenly spread it on LB solid medium for culture. If no colonies appear on the LB solid medium, the sorghum kernels are considered to have passed the surface disinfection; Step 2, targeted enrichment: Surface-sterilized sorghum grains are fully ground into fine powder, sieved, mixed with enrichment culture medium, and cultured with shaking to prepare a bacterial suspension; Step 3, separation and purification: dilute the bacterial suspension with sterile water in a gradient manner, diluting 10 3 times, 10 4 times, 10 5 times, 10 6 times, and evenly spread the bacterial suspension at each dilution concentration on LB solid medium, invert and culture, pick single colonies with different morphological characteristics, streak inoculate on LB solid medium for purification, and obtain purified strains; Step 4, initial screening of strains: The purified strains were inoculated onto starch screening medium and cultured in an inverted manner. After the culture was completed, Lugol's iodine solution was added to the starch screening medium to cover the surface of the medium. After standing, the iodine solution was poured out and the colonies were observed for the presence of a transparent zone. The diameter D of the transparent zone and the diameter d of the colony were measured. Strains with a D / d ratio greater than 1.5 were considered to be amylase-producing strains. Step 5, strain rescreening: inoculate the amylase-producing strain obtained in the initial screening into a seed culture medium for shaking culture. When the OD600 of the culture medium is 1, transfer it to the basic fermentation medium and continue shaking culture; take the fermentation liquid and centrifuge it to collect the supernatant, i.e., the crude enzyme liquid; use 3,5-dinitrosalicylic acid colorimetry to determine the amylase activity in the crude enzyme liquid; strains with amylase activity >200 U / mL are considered high-amylase-producing strains.
2. The method for efficiently isolating high-amylase-producing endophytic bacteria from sorghum grains according to claim 1, characterized in that: In step 1, 75% ethanol is used to soak the sorghum grains for 5 minutes. After the soaking, the sorghum grains are transferred to a chitosan-sodium hypochlorite composite disinfectant and soaked for 10 minutes, gently stirred 2-3 times during the soaking, and finally the sorghum grains are washed 5 times with sterile distilled water.
3. The method for efficiently isolating high-amylase-producing endophytic bacteria from sorghum grains according to claim 1, characterized in that: The chitosan-sodium hypochlorite composite disinfectant in step 1 is prepared by: slowly adding the acetic acid solution to the chitosan powder at a ratio of 100 mL of 1% acetic acid solution for every 0.5 g of chitosan powder, and stirring until the chitosan powder is completely dissolved to prepare a chitosan solution with a concentration of 0.5%; then slowly adding the sodium hypochlorite stock solution to the chitosan solution until the available chlorine concentration is 0.5-1%, stirring while adding to avoid a violent reaction; and finally adjusting the pH value of the mixed solution to 5.9±0.1 with NaHCO3 to maintain the stability of the sodium hypochlorite.
4. The method for efficiently isolating high-amylase-producing endophytic bacteria from sorghum grains according to claim 1, characterized in that: The sieving in step 2 is through a 100-200 mesh standard sieve; the rotation speed of the shaking culture is 200 r / min, the temperature is 37° C., and the time is 24 h.
5. The method for efficiently isolating high-amylase-producing endophytic bacteria from sorghum grains according to claim 1, characterized in that: In step 2, the ratio of the fine powder to the enrichment culture medium is 1 g / 100 mL.
6. The method for efficiently isolating high-amylase-producing endophytic bacteria from sorghum grains according to claim 1, characterized in that: The formula of the enrichment culture medium in step 2 is: 5 g / L soluble starch, 5 g / L sorghum grain starch, 10 g / L peptone, 1 g / L K2HPO4, 1 g / L NaCl, 0.5 g / L MgSO4, 0.1-0.5% (w / v) activated carbon, 50 mg / L cycloheximide, and the pH is adjusted to 6.
8.
7. The method for efficiently isolating high-amylase-producing endophytic bacteria from sorghum grains according to claim 1, characterized in that: The temperature of the inverted culture in step 3 is 37° C. and the time is 2-5 days.
8. The method for efficiently isolating high-amylase-producing endophytic bacteria from sorghum grains according to claim 1, characterized in that: The inverted culture temperature in step 4 is 37° C. and the time is 24 h.
9. The method for efficiently isolating high-amylase-producing endophytic bacteria from sorghum grains according to claim 1, characterized in that: The rotation speed of the shaking culture in step 5 is 200 r / min.
10. The method for efficiently isolating high-amylase-producing endophytic bacteria from sorghum grains according to claim 1, characterized in that: The formula of the basic fermentation medium in step 5 is: glucose 20 g / L, peptone 20 g / L, K2HPO4 1 g / L, NaCl 1 g / L, pH 7.0.