Bacillus subtilis, microecological preparation thereof and application thereof

Abstract

This invention belongs to the field of microecology, specifically relating to a strain of Bacillus subtilis, its microecological preparations, and their applications. This invention provides a novel Bacillus strain with significant cellulase and IAA production capabilities, exhibiting a cellulase activity of 824 U / mL and an IAA production of 48.52 mg / L. This invention utilizes this Bacillus strain to ferment roe decanter powder. Through the cell wall-breaking effect of Bacillus cellulase, the release rate and amount of active ingredients in the roe decanter powder are accelerated, thereby enhancing the removal of unwanted fish. The fermented roe decanter root powder broth after fermentation with this Bacillus strain shows an increase in roe ketone content of approximately 30%–45%, resulting in enhanced impurity removal speed and effectiveness.

Description

Technical Field

[0001] This invention belongs to the field of microecology, and specifically relates to a strain of Bacillus subtilis, its microecological preparations, and applications. Background Technology

[0002] In recent years, the scale of shrimp and crab farming has been increasing year by year, reaching nearly 3 million mu in Jiangsu Province. For crustacean aquaculture, eliminating wild fish is a major problem for farmers. An increase in wild fish not only eats mother crayfish and small shrimp larvae in the water, leading to reduced crayfish production, but also muddies the pond bottom, causing turbidity in shrimp and crab ponds. Furthermore, they compete with shrimp and crabs for food, consume oxygen, and affect the growth and molting speed of shrimp and crabs. Currently, the two most commonly used and safe wild fish removers on the market are tea seed cake and roe derrick powder. Tea seed cake's main component is tea saponin, and it is used in large quantities (10-20 kg / mu). However, its use is inconvenient in large-scale shrimp and crab ponds, and it also has some side effects on aquatic plants in the ponds. Roe derrick powder is the powder of the root of the medicinal legume *Rhizophora stylosa*, which has been used as a fishing agent since ancient times. Its active ingredient is roe ketone. Rotenone powder is usually applied evenly to shrimp and crab ponds as a water-soaked solution to remove wild fish. However, rotenone is almost insoluble in water and is not easily released from the powder, resulting in inconsistent effectiveness. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention isolated a strain of Bacillus subtilis 3-2 from a crab farming pond. This Bacillus subtilis is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the preservation number CGMCC NO. 13011. The Bacillus subtilis has irregular edges, a rough surface, rod-shaped cells, and is Gram-positive.

[0004] Furthermore, the 16S rRNA gene sequence of the Bacillus subtilis is shown in SEQ ID No. 1 of the sequence listing.

[0005] The present invention also provides a rotenone root powder ferment, which is obtained by fermenting rotenone root powder using the aforementioned Bacillus subtilis strain.

[0006] Furthermore, the preparation method of the rotenone root powder ferment is as follows: The seed liquid of Bacillus subtilis is inoculated into the fermentation medium at an inoculation rate of 1-3%, the temperature is 35-37℃, the rotation speed is 180-300 r / min, the tank pressure is 0.05 MPa, the aeration ratio is 1:(0.4-0.5), and the fermentation time is 15-18 hours, until the spore formation rate is above 90% and the viable cell count reaches 10. 9 ~10 10The concentration of cfu / mL was obtained; wherein the fermentation medium consisted of the following components and contents: 50-60 g / L rotenone root powder, 0.8-1.5 g / L KH2PO4, 2-4 g / L sucrose, 8-12 g / L soybean meal, 0.25-0.5 g / L FeSO4, 1-3 g / L CaCO3, 2.5-3.5 g / L MgSO4, 3-5 g / L cornmeal, 6-10 g / L fishmeal, and 0.05-0.1% defoamer;

[0007] The present invention also provides a cleansing microecological preparation containing the aforementioned Bacillus subtilis strain.

[0008] Furthermore, the preparation method of the cleansing microecological preparation is as follows: the Bacillus subtilis powder, rotenone root powder, brown sugar, and water are mixed evenly in a mass or volume ratio of 1:10:0.5:200, and fermented in an outdoor pond in the dark for 24 hours to obtain the preparation.

[0009] Furthermore, the viable count of the Bacillus subtilis powder is 10. 9 ~10 10 cfu / ml.

[0010] The present invention also provides the application of the aforementioned rotenone root powder fermentation product in the removal of wild fish in shrimp and crab ponds.

[0011] This invention also provides the application of the aforementioned microecological preparation in eliminating wild fish in shrimp and crab ponds.

[0012] Preferably, the amount of the microecological preparation used in the crab pond is 5-10 L / mu.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] This invention provides a novel Bacillus strain that exhibits significant cellulase and IAA production capabilities, with cellulase activity reaching 824 U / mL and IAA production at 48.52 mg / L.

[0015] This invention utilizes a Bacillus strain to ferment roe decanter powder. Through the cellulase produced by the Bacillus, the release rate and amount of active ingredients in the roe decanter powder are accelerated, thereby enhancing the removal of unwanted fish. The fermented roe decanter powder broth after fermentation with this Bacillus strain exhibits a roe ketone content increased by approximately 30%–45%, resulting in enhanced impurity removal speed and effectiveness.

[0016] This invention allows for the direct fermentation of rotenone root powder using Bacillus subtilis at pond sites. This avoids the transportation costs associated with liquid fermentation products and enhances the impurity removal effect compared to water soaking solutions.

[0017] The microecological preparation provided by this invention not only does not harm aquatic plants, but the selected Bacillus can produce plant growth hormones and also has a certain growth-promoting effect on aquatic plants, phytoplankton, and algae in crab ponds. Attached Figure Description

[0018] Figure 1 This is a preliminary screening image of cellulase activity for CGMCC NO.13011;

[0019] Figure 2 This is the IAA standard curve graph;

[0020] Figure 3 The standard curve for the detection of rotenone;

[0021] Figure 4 Comparison of rotenone concentration in aqueous extract and fermentation broth of rotenone root powder;

[0022] Figure 5 The effect of rotenone powder water soak solution (control) and rotenone powder fermentation solution (experimental group B) on the removal of wild fish. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments.

[0024] Example 1: Isolation and identification of strains, determination of cellulase activity and determination of IAA secretion capacity.

[0025] 1. Strain isolation and hemolytic assay

[0026] (1) Take 10g of mud sample from a crab farming pond in Yancheng, Jiangsu Province, and place it in a small bottle containing 90mL of sterile water with glass beads. Shake at 37℃ and 180rpm for 30min to disperse the mud sample, and heat in a water bath at 85℃ for 15min. Dilute the sample using a 10-fold serial dilution method, and spread 100μL onto LB agar plates. Incubate upside down in a 37℃ incubator for 24h. After colonies grow, select colonies with different appearances based on colony size, color, and presence or absence of wrinkles for streak purification 2-3 times to obtain 23 candidate strains.

[0027] (2) The purified candidate strains were inoculated on fresh rabbit blood plates and cultured at 37°C for 24 hours to observe hemolysis. Sixteen strains without hemolysis zones were obtained and subsequent cellulase activity assays were performed.

[0028] 2. Cellulase activity detection

[0029] (1) Culture medium: 0.5g yeast powder, 1g peptone, 1g sodium chloride, 2g CMC-Na, 2g agar, pH 7.2.

[0030] (2) Initial screening method: Non-hemolytic strains were inoculated onto this medium and incubated upside down at 37°C for 1-2 days. The culture was then stained with 1 mg / mL Congo red solution for 1 hour, the stain was discarded, and the strains were washed with 1 mol / L sodium chloride for 1 hour. The cellulase activity of the strains was measured by the ratio of the diameter of the hydrolysis zone to the colony diameter. See the appendix for preliminary screening of strains with cellulase activity. Figure 1 Among them, strain 3-2 produced cellulase with a hydrolysis zone of about 2.5 cm.

[0031] (3) Secondary screening of cellulase-producing strains: The strains screened in the initial screening were inoculated into LB medium and cultured at 37℃ and 180 rpm for 24 h on a shaker. 4 mL of the bacterial solution was transferred to a 5 mL centrifuge tube and centrifuged at 12000 rpm for 2 min. The supernatant was transferred to a new 5 mL centrifuge tube for enzyme activity determination. 1.5 mL of 1% CMC-Na solution was added to a 25 mL colorimetric tube. 0.5 mL of fermentation supernatant was added to the experimental group, and 0.5 mL of deionized water was added to the blank group. The mixture was reacted in a 50℃ water bath for 30 min. After reaction, 1.5 mL of DNS solution was added, and the mixture was boiled in a water bath for 10 min. After cooling to room temperature, the volume was adjusted to 25 mL, and the absorbance was measured at a wavelength of 540 nm. Enzyme activity definition (U / mL): Under certain conditions, the amount of enzyme required to hydrolyze the substrate to produce 1 μg of glucose per minute using 1 mL of crude enzyme solution. The results are shown in Table 1. Strain 3-2 had the highest cellulase activity of 824.35 U / mL, and the cellulase hydrolysis zone reached 2.5 cm.

[0032] Table 1 Comparison of cellulase activity among rescreened strains

[0033]

[0034]

[0035] 3. Determination of IAA production capacity of the strain:

[0036] (1) Construction of standard curve

[0037] Prepare standard solutions with concentrations of 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL. Take 500 μL of each solution and add it to an equal volume of the detection solution. Let stand for 20 min, and measure the absorbance at 530 nm. Construct a standard curve based on the relationship between absorbance and concentration (see Appendix). Figure 2 .

[0038] (2) Preparation of IAA detection solution: Weigh 1.2g FeCl3, dissolve it in 30mL of deionized water, add 42.97mL of 98% concentrated sulfuric acid while stirring, let it stand and cool, and then dilute to 100mL.

[0039] (3) IAA detection: A single colony of strain 3-2 was picked and inoculated into LB medium and cultured on a shaker at 180 rpm for 24 h. 1 mL of the fermentation broth was centrifuged at 12,000 rpm for 1 min, and 500 μL of the supernatant was transferred to a new centrifuge tube. An equal volume of IAA detection solution was added, and the mixture was allowed to stand for 20 min. The absorbance was measured at 530 nm. Based on the IAA standard curve and the absorbance measured from the 3-2 fermentation broth, the IAA production of this bacterium was determined to be 48.52 mg / L.

[0040] 4. Identification of strain 3-2

[0041] Colony PCR was performed using the universal primers 27F / 1492R for the bacterial 16S rRNA gene. The obtained target sequence was sequenced, and the NCBI sequence alignment showed the highest similarity to *Bacillus subtilis*. The 16S rRNA gene sequence of *Bacillus subtilis* 3-2 is shown in SEQ ID No. 1 of the sequence listing. *Bacillus subtilis* 3-2 was deposited on September 19, 2016, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCCNO. 13011.

[0042] Example 2: Preparation of Impurity-Clearing Microecological Preparation

[0043] (1) Preparation of high-density fermentation broth of Bacillus subtilis 3-2;

[0044] Fermentation medium composition and content: KH2PO4 1g / L, sucrose 3g / L, soybean meal 10g / L, FeSO4 0.3g / L, CaCO3 2g / L, MgSO4 3g / L, cornmeal 4g / L, fish meal 8g / L, defoamer 0.05%, initial pH 7.0.

[0045] Fermentation method: Each component was weighed according to the fermentation medium method and sterilized at 121℃ for 20 min. An inoculum of 3% was used, with a temperature of 37℃, a rotation speed of 220 rpm, a tank pressure of 0.05 MPa, an aeration ratio of 1:0.5, and a fermentation time of 18 h to obtain a high-density fermentation broth of Bacillus thuringiensis fermented rotenone powder. The viable cell count in the fermentation broth was 2 × 10⁻⁶. 9 cfu / mL. (2) Preparation of Bacillus subtilis 3-2 bacterial powder:

[0046] 1) Fermentation broth concentration: Add 1% calcium chloride and 1% disodium hydrogen phosphate to the above fermentation broth, mix well, let it settle for 15 minutes, then add 10% diatomaceous earth and stir well. Filter the mixture using a ceramic membrane plate and frame to obtain a filter cake with a water content of 50%.

[0047] 2) Flash drying: Inlet air temperature 115℃, outlet air temperature 75℃. The outlet air temperature is adjusted by controlling the feed rate. The powder fineness is: over 85% passing through a 200-mesh sieve, resulting in a powder with a moisture content of 6% and a viable count of 10. 10 cfu / g.

[0048] (3) Preparation of microecological preparation for cleaning: Take 100g of the above-prepared bacterial powder, 1kg of fish roe root powder and 50g of brown sugar, dissolve them in 20L of water and stir evenly. Ferment in an outdoor pond in the dark for 18-24 hours to obtain the preparation (the outdoor temperature is best above 25℃).

[0049] When using, dilute the impurity-removing microecological agent with water and evenly sprinkle it into the shrimp and crab ponds that need impurity removal.

[0050] Example 3: Preparation of rotenone powder fermentation product

[0051] The roe drupe ferment was prepared according to the following culture medium and method:

[0052] (1) Composition and content of fermentation medium: 50 g / L rotenone root powder, 1 g / L KH2PO4, 3 g / L sucrose, 10 g / L soybean meal, 0.3 g / L FeSO4, 2 g / L CaCO3, 3 g / L MgSO4, 4 g / L cornmeal, 8 g / L fishmeal, 0.05% defoamer, initial pH 7.0

[0053] (2) Fermentation method: Each component was weighed according to the fermentation medium method and sterilized at 121℃ for 20 min. A 3% inoculum was prepared at 37℃, 220 rpm, 0.05 MPa, an aeration ratio of 1:0.5, and a fermentation time of 18 h to obtain a high-density fermentation broth of Bacillus thuringiensis fermented with rotenone powder. The viable cell count in the fermentation broth was 10-1. 9 cfu / mL.

[0054] When using, the above-mentioned rotenone powder fermentation liquid can be directly diluted with water and sprinkled into the shrimp and crab ponds that need to be cleaned.

[0055] Example 4: Detection of Rotenone Concentration in Bacillus subtilis 3-2 Ferment and Rotenone Root Powder Aqueous Extract

[0056] (1) Experimental treatment: The experiment was divided into a control group and an experimental group. The control group was 5g of rotenone powder dissolved in 100mL of tap water and soaked for 24h. The experimental group was 100mL of fermented rotenone root powder fermented with Bacillus subtilis 3-2.

[0057] (2) Extraction of active ingredients

[0058] The fermentation broth and the aqueous extract were extracted with 100 mL of chloroform for 30 min. After the liquid level stabilized, the chloroform-extracted sample was concentrated by distillation at 70 °C to obtain an extract, which was then dried in an oven.

[0059] (3) Detection of effective ingredients

[0060] The dried sample obtained in step 2 was dissolved in 5 mL of methanol and filtered through a 0.45 μm organic filter membrane. Detection was performed by high-performance liquid chromatography (HPLC). The mobile phase was methanol:water (65:35), the sample loading volume was 20 μL, the column temperature was 30 °C, and the detection wavelength was 495 nm.

[0061] A standard curve is prepared based on the peak area of ​​the standard and its known concentration (see...). Figure 3 The concentrations of rotenone in the fermentation broth and soaking solution were calculated based on the standard curve. The rotenone concentration in the aqueous extract was 42 μg / mL, and the rotenone concentration in the fermentation broth was 60 μg / mL, representing a 42.82% increase in the effective component (see...). Figure 4 ).

[0062] Example 5: Impurity Removal Effect of the Fish Remover

[0063] (1) Indoor testing

[0064] The experiment was set up as a control group, experimental group A, and experimental group B, with three replicates in each group. Each breeding box contained 4L of water and 10 crucian carp with a body length of about 10cm.

[0065] The control group was prepared by weighing 1.0 g of rotenone powder into a centrifuge tube containing 50 mL of water, shaking well (rotenone powder concentration 20 mg / mL), and soaking for 24 hours. Then, using a pipette tip with the tip removed, 20 mL of the soaking solution was transferred to an Erlenmeyer flask containing 180 mL of water (rotenone powder concentration 2 mg / mL).

[0066] The specific method for Experiment Group 1 is as follows: Take 0.1g of Bacillus powder, 1g of rotenone powder, and 0.05g of brown sugar obtained in Example 2, add 50mL of tap water to an Erlenmeyer flask, ferment at 30℃ for 24h, stirring 4-5 times during the fermentation period, and dilute 10 times before use to obtain a rotenone powder concentration of 2mg / mL.

[0067] The specific method for Experiment Group 2 is as follows: Take 2 mL of the roe drupe fermentation liquid described in Example 3 and add it to 48 mL of tap water. The roe drupe mass concentration is 2 mg / mL.

[0068] Two mL of each of the three roe vine powder aqueous solutions prepared by the above three methods was added to a 4L aquaculture box to achieve a final concentration of 1 mg / L. The entire experiment lasted for 24 hours. The results showed that the efficiency of the three treatments in removing unwanted fish was ranked as follows: experimental group B (70%) > experimental group A (50%) > control group (30%).

[0069] (2) Pond cleaning test

[0070] Due to practical needs, a cleanup experiment was also conducted at the aquaculture ponds.

[0071] The experiment was conducted in nine crab farming ponds in Xizhou Village, Funing County, Yancheng City, Jiangsu Province, each with an area of ​​approximately 10 mu (about 6,667 square meters). The selected ponds used the same farming methods and had similar stocking times. The water depth was 50–60 cm, the pH was 7.2–7.5, and the water temperature was 25–28℃. Ponds 1, 2, and 3 served as control ponds, and the dosage for each pond was 300 g / mu (about 1,333 square meters). Specifically, 3 kg of rotenone root powder was soaked in 60 L of pond water in the dark for 24 hours, stirring 4–5 times during this period, and then diluted and evenly sprinkled.

[0072] Ponds 4, 5, and 6 were experimental group A, with a dosage of 300g / mu for each pond. The specific method was as described in Example 2: 3kg of rotenone root powder, 300g of the bacterial powder described in Example 2, and 150g of brown sugar were dissolved in 60L of water and stirred evenly. The mixture was fermented outdoors in the dark for 24 hours. After dilution with pond water, it was evenly sprinkled on the mixture.

[0073] Ponds 7, 8, and 9 were experimental group B, specifically using the fish roe root powder fermentation liquid described in Example 3, with a usage of 6L / mu, diluted with pond water and evenly sprinkled.

[0074] One hour after application, small fish were the first to float to the surface in experimental groups A and B. Two hours after application, the number of small fish in groups A and B was significantly greater than that in the control group. The small fish were 10-15 cm in size. The actual effect of application in group B was as follows... Figure 5 As shown in the table below, the fish that were collected and weighed were as follows. The results show that the removal effect was: Experimental Group B > Experimental Group A > Control Group.

[0075] Table 2 Comparison Experiment After 2 Hours of Use (Table 2)

[0076]

Claims

1. A method for preparing rotenone root powder fermentation product, characterized in that, The roe vine root powder ferment was obtained by fermenting roe vine root powder with Bacillus subtilis strain. Bacillus subtilis The strain's preservation number is CGMCC No. 13011. The composition and content of the fermentation medium are as follows: 50-60 g / L rotenone root powder, 0.8-1.5 g / L KH2PO4, 2-4 g / L sucrose, 8-12 g / L soybean meal, 0.25-0.5 g / L FeSO4, 1-3 g / L CaCO3, 2.5-3.5 g / L MgSO4, 3-5 g / L cornmeal, 6-10 g / L fishmeal, and 0.05-0.1% defoamer.

2. The method for preparing the fermented roe decanter as described in claim 1, characterized in that, The seed culture of Bacillus subtilis was inoculated into the fermentation medium at an inoculation rate of 1-3%. The temperature was 35-37 °C, the rotation speed was 180-300 r / min, the pressure was 0.05 MPa, the aeration ratio was 1:(0.4-0.5), and the fermentation time was 15-18 h. Fermentation continued until the spore formation rate was above 90% and the viable cell count reached 10⁻⁶. 9 ~10 10 cfu / ml, obtained.

3. A method for preparing a cleansing microecological preparation, characterized in that, Bacillus subtilis CGMCC No. 13011 bacterial powder, rotenone root powder, brown sugar, and water are mixed evenly in a mass or volume ratio of 1:10:0.5:200 and fermented in an outdoor pond in the dark for 24 hours to obtain the cleansing microecological preparation used to remove wild fish in shrimp and crab ponds.

4. The method for preparing the microecological preparation according to claim 3, characterized in that, The viable count of Bacillus subtilis powder is 10. 9 ~10 10 cfu / ml.

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