A granular preparation of clostridium butyricum for pond bottom improvement and a method for preparing the same

Abstract

The application discloses a Clostridium butyricum granular preparation for pond bottom improvement, which comprises the following components: 15-20 parts of Clostridium butyricum (CCTCC M 2018426) fermentation liquor, 5-17 parts of gallnut extract liquor, 35-50 parts of montmorillonite and 5-15 parts of corn starch, wherein the viable bacterial count of Clostridium butyricum in the fermentation liquor is above 10 8 CFU / ml, and the spore rate reaches above 90%. In the scheme, Clostridium butyricum is matched with traditional Chinese medicine gallnut, and the granular preparation is formed by taking montmorillonite and corn starch as carriers. In the application of pond bottom improvement, it is found that the granular preparation can greatly inhibit the reproduction of Vibrio alginolyticus and Vibrio parahaemolyticus in a short period, and is helpful to improve the pond bottom environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiological technology, in particular to a Clostridium butyricum granular preparation for improving the bottom of a pond and a preparation method thereof. BACKGROUND

[0002] With the rapid development of large-scale and intensive aquaculture industry, high-density and high-feeding aquaculture mode is widely used. Under this mode, the residual feed and feces accumulate rapidly at the bottom of the pond, forming a breeding ground for the rapid growth of pathogenic bacteria. For example, Vibrio parahaemolyticus can reach a high density of 10 4 CFU / g in the sediment, which easily leads to the death of a large number of farmed animals such as South American prawns.

[0003] In order to control the pathogenic bacteria hidden in the pond sediment, some strong chemical oxidants, antibiotics and the like are widely used in the improvement of the bottom of the pond. The disadvantages are obvious, such as environmental drug residues, stress reactions of farmed animals, emergence of more drug-resistant strains, and difficulty in maintaining the bactericidal efficiency for a long time due to the existence of half-life, which easily leads to the failure of antibiotics after 2-3 days and the recovery of the number of Vibrio to the level before use. The existing mode of adding antibiotics at a fixed time and quantity will bring potential risks to the water environment and the ecological system.

[0004] In addition to chemical preparations and antibiotics, the bacteriostatic mode of bacterial agents has become a research hotspot for antibiotic drug substitutes. Taking Clostridium butyricum as an example, it can produce Clostridium butyricum, which has an inhibitory effect on Vibrio. At the same time, it can adapt to the anaerobic environment in the sediment. For example, in the application of prawn culture, it can act on the sediment of the pond, and the species of Vibrio it can inhibit include Vibrio alginolyticus, Vibrio campbellii, Vibrio harveyi, and Vibrio parahaemolyticus. However, in the actual experiment, it is found that Clostridium butyricum has a slow effect, and the number of Vibrio is reduced after 72 hours of use. Therefore, it is necessary to improve the slow effect. SUMMARY

[0005] To solve at least one of the above technical defects, the present application provides the following technical solutions:

[0006] The present application discloses a Clostridium butyricum granular preparation for improving the bottom of a pond, which comprises the following components:

[0007]

[0008] The number of viable Clostridium butyricum in the fermentation broth is more than 10 8 CFU / ml, and the spore rate is more than 90%.

[0009] In the scheme, Clostridium butyricum is preferably combined with Chinese medicine gallnut, and a granular preparation is formed with montmorillonite and corn starch as carriers. In the application of improving the bottom of the pond, it is found that the granular preparation can greatly inhibit the reproduction of Vibrio alginolyticus and Vibrio parahaemolyticus in a short period of time, and help to improve the bottom environment of the pond.

[0010] Further, the Clostridium butyricum fermentation broth is formed by inoculating Clostridium butyricum (CCTCC M 2018426) into a culture medium and then fermenting.

[0011] Further, the components of the culture medium account for: 18-21 g of cornmeal, 8-11 g of soybean meal, 4-6 g of glucose, 4.5-5.5 g of yeast paste, 4-6 g of sodium chloride, 2-4 g of K2HPO4, 2-4 g of KH2PO4, 0.2-0.4 g of MnSO4, and 1 L of water.

[0012] Further, when the fermentation is to 16 h, the solution of 0.2-0.4 g / L of MnSO4 is added for induction under the condition of no spore production, spore production is induced, and the culture time is shortened.

[0013] Further, the gallnut extract is obtained by water extraction method, and the first extraction is carried out by mixing gallnut and water in a mass ratio of 1:12-17 and heating extraction, solid-liquid separation, and reserving the filtrate;

[0014] The second extraction is carried out by adding 6-10 times the volume of water to the filtrate after the first extraction, heating, and concentrating to 0.9-1.2 times the mass of the gallnut, and the gallnut extract is obtained. The twice heating and water extraction process can better extract the bacteriostatic components in the gallnut.

[0015] The second aspect of the present application provides a preparation method of Clostridium butyricum granular preparation for improving the bottom of a pond, comprising the following steps:

[0016] First, 15-20 parts of Clostridium butyricum (CCTCC M 2018426) fermentation broth, 5-17 parts of gallnut extract, 35-50 parts of montmorillonite, and 5-15 parts of corn starch are mixed according to the proportion;

[0017] Second, the mixed material in the first step is prepared into granules;

[0018] Third, the obtained granules are dried to less than 10% moisture, and the granular preparation is obtained.

[0019] The forming process is simple, the granular preparation is convenient to use, and the obtained granular preparation can be packaged and sold.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] 1. The preferred Clostridium butyricum is matched with Chinese herbal medicine Chinese gallnut, and the matched Clostridium butyricum is matched with montmorillonite and corn starch to form granules, which can be directly applied in pond bottom improvement, effectively inhibits the reproduction of Vibrio alginolyticus and Vibrio parahaemolyticus, and improves the pond bottom environment. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0023] Figure 1 is the identification result diagram of Clostridium butyricum in Example 1;

[0024] Figure 2 is the granule preparation stacking morphology diagram in Example 1; DETAILED DESCRIPTION

[0025] The present application will be further described below in combination with the drawings and specific embodiments.

[0026] Example 1

[0027] The preparation of Clostridium butyricum granule preparation for pond bottom improvement is as follows:

[0028] First, preparation of Clostridium butyricum fermentation broth

[0029] First, prepare the culture medium, and the culture medium component ratio is: cornmeal 20g, soybean meal 10g, glucose 5g, yeast extract 5g, sodium chloride 5g, K2HPO4 3g, KH2PO4 3g, MnSO4 0.3g, and distilled water 1L.

[0030] Cultivation conditions: 200L fermentation tank with 120L liquid, Clostridium butyricum from China Center for Type Culture Collection, preservation number CCTCC M2018426, inoculated after conventional expansion, inoculation amount about 4%, fermentation temperature 36℃, rotation speed 100rpm, nitrogen gas in the tank, tank pressure stabilized at 0.02Mpa, fermentation to spore conversion of vegetative cells, end of fermentation, ready for use, mirror examination at 16h during fermentation, if no spore is produced, do not add 0.3g / L MnSO4 solution for induction. After expansion of Clostridium butyricum, the strain is sent to Guangdong Microbial Germplasm Bank for detection, the homology of the expanded strain with Clostridium butyricum reaches 99%, and the identification result confirms that it is Clostridium butyricum, and the sequence alignment is as shown in Figure 1

[0031] Second, preparation of Chinese gallnut extract

[0032] ​Single extraction tank feeding 150 kg of raw material, the first extraction: add 10 times the volume of drinking water, extraction temperature 100°C, when the temperature reaches 100°C, start timing, constant temperature time is 1.5 h, through the double filter liquid input tank. The second extraction: add 8 times the volume of drinking water, extraction temperature is 100°C, reaches 100°C, start timing, constant temperature time is 1.5 h, through the double filter liquid input tank.

[0033] Centrifugal section: centrifuge with centrifuge, when the flow rate valve to the full amount of 1 / 2.

[0034] Concentration: the supernatant liquid into the concentrator, concentrated, to the concentrated liquid and medicinal materials quality is 1:1.

[0035] Discharge: the concentrated extract, into the transfer bucket placed in the freezer for standby.

[0036] Third, the preparation of granules

[0037] According to the following proportion of each raw material and sent to the three mixed machine mixing.

[0038]

[0039] This process needs to be premixed first montmorillonite and corn starch, butyric acid Clostridium fermentation broth, extract of gall-nut premix, then according to the order of solid premix, liquid premix one by one into the three-dimensional mixing machine, mixed for one hour. After stirring evenly take out the soft material, the soft material is added to the roll mill granulator, using the tray to collect the prepared granules (stacking morphology as shown in Figure 2 The prepared Clostridium butyricum granules (moisture content less than 10%), 1000 g / bag of aluminum foil bag, bag sealing, the product is obtained.

[0040] Comparative example 1

[0041] Compared with example 1, the difference is that: in this example, no gall-nut extract, the same quality of Clostridium butyricum fermentation broth instead of gall-nut extract.

[0042] Comparative example 2

[0043] Compared with example 1, the difference is that: in this example, no Clostridium butyricum fermentation broth, the same quality of gall-nut extract instead of Clostridium butyricum fermentation broth.

[0044] Comparative example 3

[0045] Compared with Example 1, the difference lies in that: in the present example, commercially available Clostridium butyricum (Henan Jinbai Biotechnology Co., Ltd.) is used to replace Clostridium butyricum CCTCC M 2018426 in Example 1, and the fermentation process is consistent.

[0046] Verification of the antibacterial effect of the bottom-modified granule product

[0047] Purpose of the experiment: construct a triangular flask model to verify the inhibitory effect of Clostridium butyricum bottom-modified granules on Vibrio.

[0048] LB culture was used to prepare mixed bacterial solution containing Vibrio alginolyticus and Vibrio parahaemolyticus, with a bacterial count ratio of 1:1.

[0049] A 250ml triangular flask was used, and 100mL of aquaculture water was added to each flask, followed by the addition of 0.1g of South American prawn feed (to simulate the real pond bottom environment). The mixture was sterilized at 121 degrees Celsius. After sterilization, 10 2 CFU / mL of the mixed bacterial solution was added to each flask according to the table below, and 0.1g of the above-prepared Clostridium butyricum granular preparation was added.

[0050] After inoculation, the flask was shaken for 10 minutes, and then the number of viable bacteria in the 10 flasks was counted using TCBS medium as the 0h data. The flasks were then placed on the test bench for static culture at room temperature, and the number of viable bacteria was counted again at 48h to observe the change in the number of Vibrio and verify the inhibitory effect of Clostridium butyricum bottom-modified product on Vibrio alginolyticus, as shown in Tables 1, 2, and 3.

[0051] Table 1: Change in the number of Vibrio (unit: 10 2 CFU / mL)

[0052]

[0053] Table 2: Change in the number of Vibrio (unit: 10 2 CFU / mL)

[0054]

[0055]

[0056] Table 3: Change in the number of Vibrio (unit: 10 2 CFU / mL)

[0057]

[0058] Table 4: Change in the number of Vibrio (unit: 10 2 CFU / mL)

[0059]

[0060] And increase the positive control group, the positive control group also uses the above triangular flask model, the difference is that only access to the butyric acid Clostridium fermentation broth used in the scheme and commercially available butyric acid Clostridium (Henna Jin Baihe Biological Technology Co., Ltd.) fermentation broth, the same amount and fermentation process, observe the number of vibrio changes, as shown in Table 5.

[0061] Table 5

[0062]

[0063] From Table 1, Table 2, Table 3, Table 4, Table 5 can be seen, the butyric acid Clostridium granular preparation prepared in the scheme can achieve at least 95% or more sterilization effect when 10 ppm is put, the inhibition effect on Vibrio alginolyticus, Vibrio parahaemolyticus and the like is obvious, while the bottom modified granules prepared in Comparative Example 1, Comparative Example 2, Comparative Example 3 have poor sterilization effect when 10 ppm is put, and the commercially available conventional butyric acid Clostridium has slow effect, and has poor sterilization effect in low concentration 48h culture.

[0064] The above is only the preferred embodiment of the present application, the protection scope of the present application is not limited to the above examples, any technical scheme falling within the idea of the present application belongs to the protection scope of the present application. It should be pointed out that for ordinary skilled in the art, some improvements and decorations without departing from the principles of the present application, these improvements and decorations should also be considered as the protection scope of the present application.

Claims

1. A Clostridium butyricum granule preparation for improving the bottom of a pond, characterized by, The following components are included: Clostridium butyricum CCTCC NO: M2018426 fermentation broth 15-20 parts Gallnut extract 5-17 parts Montmorillonite 35-50 parts Corn starch 5-15 parts The viable cell count of Clostridium butyricum in the fermentation liquor is above 10 8 CFU / ml, and the spore rate is above 90%; and the gallnut extract is prepared by water extraction.

2. The Clostridium butyricum granule preparation for improving the bottom of a pond according to claim 1, characterized by: The Clostridium butyricum fermentation broth is fermented from Clostridium butyricum CCTCC NO: M2018426 inoculated in a culture medium.

3. The Clostridium butyricum granule preparation for improving the bottom of a pond according to claim 2, characterized by: The culture medium components account for: cornmeal 18-21g, soybean meal 8-11g, glucose 4-6g, yeast extract 4.5-5.5g, sodium chloride 4-6g, K2HPO4 2-4g, KH2PO4 2-4g, MnSO4 0.2-0.4g, water 1L.

4. The Clostridium butyricum granule preparation for improving the bottom of a pond according to claim 3, characterized by: At 16h of fermentation, 0.2-0.4g / L of MnSO4 solution is added for induction without spores.

5. The Clostridium butyricum granule preparation for improving the bottom of a pond according to claim 1, characterized by: The gallnut extract is prepared by water extraction method, first extraction: gallnut and water are mixed in a mass ratio of 1:12-17 and heated for extraction, solid-liquid separation, and the filtrate is ready for use; Second extraction: add 6-10 times the volume of water to the filtrate after the first extraction, heat and concentrate to 0.9-1.2 times the mass of gallnut, and stop, i.e. gallnut extract is obtained.

6. A method for preparing a Clostridium butyricum granule preparation for pond bottom improvement, characterized by: The following steps are included: First, mix Clostridium butyricum CCTCC NO: M2018426 fermentation broth 15-20 parts, gallnut extract 5-17 parts, montmorillonite 35-50 parts, and corn starch 5-15 parts according to the proportion, and the gallnut extract is prepared by water extraction method; Second, prepare granules from the mixed materials of the first step; Third, dry the obtained granules to a moisture content of less than 10%, and the granular preparation is obtained.

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