CN116162566A - Low-temperature fungus bacillus mycoides 21 and application thereof - Google Patents

Abstract

The invention provides a low-temperature bacillus mycoides 21 and application thereof. The bacillus mycoides 21 is classified as bacillus mycoides Bacillus mycoides, the preservation number of the strain is CGMCC No.25259, and the strain has the characteristic of producing protease under the low-temperature condition and can be applied to the fields of aquaculture and the like. The microbial preparation provided by the invention can effectively reduce the protein content and COD content in the aquaculture water, effectively reduce the organic pollution of the water, and play a role in improving the water quality.

Description

A low-temperature fungus Bacillus mycoides 21 and its use

technical field

The invention belongs to the technical field of microbes, and in particular relates to a low-temperature bacterium Bacillus mycoides 21 and its use, in particular to the use of the strain in the fields of preparation of microecological preparations for aquaculture water environment improvement and the like.

Background technique

Intensive and high-density sea cucumber farming methods have led to increasingly serious organic pollution in farming water. While high-density breeding has brought considerable benefits, it has also triggered a new chain reaction: in the growing season, there is often a surplus of supplementary bait, poor adhesion of the feed, serious loss, and a large amount of residual bait during the breeding process. The residual bait is deposited at the bottom of the pond, attached to the artificial attachment base, and cannot be effectively discharged by changing the water. These accumulated organic pollutants release a large amount of harmful substances such as small molecular organic and inorganic substances to the breeding environment through slow decomposition, endangering the health and growth of farmed animals. It has also become a breeding ground for pathogenic bacteria. The indiscriminate use and abuse of chemical drugs may cause pathogenic bacteria to develop drug resistance under these conditions, triggering a secondary crisis. This not only threatens the stability of farming, but also the safety of farming products is worrying.

Probiotics are widely used in the aquaculture industry due to their many advantages, such as strong practicability, low cost, high income, simple operation, and not easy to form secondary pollution. But at the same time, there are also problems such as the specificity of the bacterial agent is not strong, it is not suitable for the mariculture environment, and the number of viable bacteria is low. In view of this, this patent has screened and obtained a strain that can tolerate low-temperature aquaculture environment, can still produce protease under low temperature conditions, effectively decompose organic matter in water, effectively purify the aquaculture water, and solve the problem of water in the sea cucumber cultivation process. pollution problem.

Contents of the invention

The present invention provides a Bacillus mycoides 21 with low temperature resistance and protease production and its application. The Bacillus mycoides 21 has the functions of salt tolerance, anaerobic resistance, medium and low temperature resistance, can specifically produce protease, and can Improve the water environment, suitable for both fresh water and salt water environments, especially conducive to improving the breeding environment of sea cucumbers.

To achieve the above-mentioned purpose of the invention, the present invention is achieved through the following technical solutions:

The invention provides a strain of Bacillus mycoides 21 with low temperature resistance and protease production, which is characterized in that: it is classified as Bacillus mycoides, and has been preserved in China Microorganisms Preservation Management on July 8, 2022 Committee General Microbiology Center, deposit number is CGMCC No.25259.

Further, the colony of the Bacillus mycoides strain 21 has a diameter of about 3 mm, is round, milky white, flat, slightly raised in the middle, dull and opaque, and has a short rod shape with terminal spores.

Further, the gene sequence of the Bacillus mycoides 21 is shown as SEQ ID No.1. Further, the Bacillus mycoides 21 biosynthesizes microbial enzymes in a low temperature environment, preferably, the microbial enzymes are proteases.

Another aspect of the present invention provides the use of Bacillus mycoides 21 in the preparation of microecological preparations for improving water quality.

Further, the probiotics include one or more of Bacillus mycoides 21, bacterial powder, bacterial liquid, microbial enzymes biosynthesized therein, and other metabolites.

Further, the bacterial liquid and its metabolites are prepared according to the following method, Bacillus mycoides 21 in the fermentation medium, 30-37°C, 200-400rpm, pressure 0.3-0.7kg, flow rate 10-25L/ Min, culture under the condition of initial pH 6-8 for more than 8 hours, keep the pH constant, and the cell density is greater than or equal to 10 ⁹ CFU/mL; the preparation of the bacterial powder and its metabolites is after the bacterial liquid is prepared and the protective agent is added Spray dry, the cell density is greater than or equal to 10 ¹⁰ CFU/g.

Further, the use lies in the preparation of microecological preparations for improving the aquaculture environment.

Further, the use lies in the preparation of microecological preparations for sea cucumber cultivation.

Further, according to the use, the concentration of Bacillus mycoides 21 in the sea cucumber culture water environment is greater than 10 ³ CFU/mL. Compared with the prior art, the present invention has the following advantages and technical effects:

1) The present invention provides a strain of Bacillus mycoides 21 with low temperature resistance and protease production. The strain is salt-tolerant, low temperature resistant, anaerobic, and capable of specifically producing protease.

2) The present invention provides the use of Bacillus mycoides 21 in the preparation of microecological preparations for improving the water quality environment, the water body includes fresh water and seawater, further, the use of Bacillus mycoides 21 in improving sea cucumber cultivation, specifically It can effectively improve the protein degradation rate and COD degradation rate, thereby reducing the organic pollution of the water body and improving the water quality.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. Those skilled in the art can also obtain other drawings based on these drawings without going beyond the scope of protection of the present invention.

Figure 1 shows the colony morphology and photomicrograph of Bacillus mycoides 21. Among them, (a) the colony morphology on LB medium; (b) Gram staining photomicrograph

Fig. 2 is a phylogenetic tree of Bacillus mycoides 21.

Figure 3 is the growth curve and enzyme production curve of Bacillus mycoides 21.

Figure 4 is the effect of temperature on the growth of Bacillus mycoides 21.

Figure 5 is the effect of NaCl on the growth of Bacillus mycoides 21.

Figure 6 shows the liquid submerged fermentation of Bacillus mycoides 21 - monitoring of cell density and OD ₆₀₀ .

Fig. 7 is the degradation curve of sea cucumber bait lysate protein by Bacillus mycoides 21.

Figure 8 shows the effect of Bacillus mycoides 21 on the COD of sea cucumber bait dissolves.

Figure 9 shows the changes in the number and pH of sea cucumber breeding water in the control group.

Figure 10 shows the effect of Bacillus mycoides 21 on the number and pH of sea cucumber culture water.

Figure 11 is the degradation curve of Bacillus mycoides 21 on protein in sea cucumber culture water.

Figure 12 shows the effect of Bacillus mycoides 21 on COD in sea cucumber culture water.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts belong to the protection scope of the present invention.

Unless defined otherwise, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of the invention or the field to which such terms are applied. The test methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used can be obtained from commercial sources unless otherwise specified.

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific examples. It should be understood that the specific embodiments described below are only used to explain the present invention, not to limit the present invention.

The formula of the medium required in the present invention is as follows:

(1) LB medium (1L): Tryptone 10g, yeast extract 5g, NaCl 10g, pH7.0, dilute to 1L, sterilize at 121°C for 20min.

(2) Skim milk powder culture medium (1 L): 20 g of skim milk powder, 20 g of agar, natural pH, constant volume to 1 L, sterilized at 115° C. for 20 min.

(3) Starch medium (1L): tryptone 10g, yeast extract 5g, NaCl 10g, soluble starch 2g, agar 16-18g, pH natural, constant volume to 1L, sterilized at 121°C for 20min.

(4) Cellulose medium (1L): 4.72g sodium succinate, 10g sodium carboxymethylcellulose, 10g peptone, 10g NaCl, 5g yeast extract, 1g potassium dihydrogen phosphate, 0.2g magnesium sulfate, 16-18g agar , with a natural pH, set the volume to 1L, and sterilize at 121°C for 20min.

(5) Sea cucumber bait medium (1L): Take 20g of juvenile ginseng seedling bait, boil it with 1000mL sterilized water for 30min, soak it for 48h, centrifuge to get the supernatant, add 3.5% seawater, adjust the pH to 7.0, and sterilize at 121℃ for 20min spare.

(6) Seed culture medium (1L): tryptone 10g, yeast extract 5g, NaCl 10g, pH7.0, dilute to 1L, sterilize at 121°C for 20min.

(7) Fermentation medium (1L): 20g sucrose, 9g peptone, 1g yeast powder, 2.5g sodium chloride, 2g magnesium sulfate, dilute to 1L, sterilize at 121°C for 20min.

The Bacillus mycoides 21 described in the present invention is obtained by screening from the bottom mud of fish ponds. Through methods such as morphology and molecular biology, the strain is identified as Bacillus mycoides. The strain has been preserved on July 8, 2022 at General Microbiology Center of China Committee for the Collection of Microbial Cultures, collection number: CGMCC No.25259, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

The identification of embodiment 1 Bacillus mycoides 21

1. Morphological identification

Strain 21 was streaked on the LB plate and cultured at 15°C for 48 hours to observe its colony morphology. The colony of strain 21 on the LB plate had a diameter of about 3mm, nearly round, milky white, flat, slightly raised in the middle, dull and opaque (Figure 1a). Gram staining was carried out on strain 21, and the results of Gram staining showed that strain 21 was a Gram-positive bacterium, which was straight rod-shaped and had terminal spores (Fig. 1b).

2. Molecular biological identification

Pick a single colony from the LB plate and place it in a 1.5mL EP tube filled with 50μL sterile water, cook at 100°C for 10min, take 1μL as a template, and use bacterial universal primers 27F and 1492R to amplify the 16S rRNA gene sequence of strain 10 . The PCR amplification system is 25 μL, 1 μL of bacterial liquid as a template, 1 μL of upstream and downstream primers, 12.5 μL of 2×Mix, and 9.5 μL of ddH ₂ O. PCR reaction program: pre-denaturation at 95°C for 5min; denaturation at 94°C for 30s; annealing at 55°C for 1min and extension at 72°C for 40s, after 30 cycles, extension at 72°C for 10min. The PCR product was subjected to DNA sequencing. The sequence shown in SEQ ID No.1 was submitted to the NCBI database for Blast comparison. The phylogenetic tree was constructed using the type strains (Figure 2), and strain 21 was identified as Bacillus mycoides.

Example 2 Bacillus mycoides 21 Enzyme Production Activity Research

Prepare skimmed milk powder medium plates, starch medium plates, and cellulose medium plates respectively, inoculate a single colony of Bacillus mycoides 21 on the plates, and culture them upside down at 15°C to observe the types and activities of enzymes produced. It has been verified that Bacillus mycoides 21 has good protease activity, but does not have amylase and cellulase activities. The ratio of the diameter of the transparent circle to the diameter of the colony (R/r) is shown in Table 1.

Table 1 Types and activities of enzymes produced by Bacillus mycoides 21

Example 3 Study on Growth Characteristics of Bacillus mycoides 21

1. Low temperature growth curve and low temperature enzyme production curve

Inoculate the strain into LB liquid medium, set 3 parallels for each gradient, culture in a shaker at 15°C and 100 rpm, take samples every 4 hours, measure the growth curve by OD ₆₀₀ value, and measure the enzyme production curve by the folin phenol method .

The growth curve and enzyme production curve of Bacillus mycoides 21 at 15°C are shown in Figure 3. The strain grew slowly at low temperature for 0-40h, grew rapidly after 40h, and entered a stable phase at 56h, with an OD ₆₀₀ value of 2.13. Similarly, when the strain grew for 40 hours, the enzyme production increased rapidly, and the enzyme activity reached the maximum at 56 hours, which was 9.84U/mL.

2. Determination of growth temperature

Set 3 temperature gradients of 15°C, 28°C, and 37°C, inoculate the strains into LB liquid medium, set 3 parallels for each gradient, culture them in a shaker at 15°C, 100rpm, take samples every 8h, and measure their OD ₆₀₀ .

From the results (Fig. 4), the higher the temperature within the designed range, the faster the strain grows and the earlier it enters the logarithmic phase. Under the condition of 37 ℃, the growth rate of the strain is the fastest, and the bacterial activity reaches the peak first, which is more conducive to the pilot production. Therefore, it is selected to carry out the preparation of microbial preparations at this temperature.

3. Determination of NaCl tolerance

Set 5 NaCl concentration gradients of 0, 2%, 4%, 6%, and 8%, inoculate the strains into LB liquid medium, set 3 parallels for each gradient, and culture them in a shaker at 15°C and 100rpm. Samples were taken at 8 hours, and the OD ₆₀₀ was measured.

The NaCl concentration tolerance of the strain at 15°C is shown in Figure 5. When the NaCl concentration was 0, the OD ₆₀₀ value was 2.13 at 64 hours; when the NaCl concentration was 2%, the growth of the strain was hardly affected by the salt concentration, and at 64 hours The OD ₆₀₀ value was 2.07; when the NaCl concentration was 4%, the OD ₆₀₀ value decreased slightly, and the OD ₆₀₀ was 1.899 at 64 hours, the difference was not significant; when the NaCl concentration was higher than 4%, the growth of the strain was significantly affected. The seawater salt concentration is 3.5%, which shows that the bacterial strain is completely applicable to freshwater and seawater aquaculture.

4. Anaerobic growth performance

The puncture test was used to verify the anaerobic growth performance of the strain. Pick Bacillus mycoides 21 with an inoculation needle and puncture it into the LB test tube culture medium containing 0.8% agar, the puncture depth is about 5 cm, and culture it statically at 15°C to observe its growth.

After static culture at 15°C for 48 hours, colonies grew from the puncture surface to a depth of 5cm, indicating that the strain has better anaerobic performance

The safety experiment of embodiment 4 Bacillus mycoides 21

Zebrafish with a body length of 2-4cm, 20 zebrafish per tank, set up 5 parallels, and the experimental period was 7 days. Spray the bacterial solution with a final concentration of 1×10 ⁵ CFU/mL, and observe the vigor and death of the zebrafish. After the experiment, the zebrafish in all experimental groups had good vigor and no death.

Example 5 Evaluation of Bacillus mycoides 21 on bait degradation ability

Bacillus mycoides 21 was inoculated into LB liquid medium at 2%, cultured at 15°C and 100 rpm for 48 hours, and then transferred to sterile bait medium so that the final concentration of the bacteria was 1.0×10 ⁵ CFU/mL. The inoculated treatment was used as the control, and each treatment was set up in 3 replicates, cultured on a shaker at 15°C and 100 rpm, and samples were taken at 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, and 8h, and centrifuged at 8000rpm for 10min. The protein and COD content in the supernatant were determined. The protein content was determined by the Folin phenol method, and the COD content was determined by the alkaline potassium permanganate method.

It was determined that after 8 hours of inoculation, the degradation rate of Bacillus mycoides 21 to the protein in the bait medium could reach (40.30±0.55)%, and the degradation rate of COD could reach (20.92±0.45)% (Figure 6 and Figure 7).

The preparation of embodiment 6 bacterial strain liquid fermented bacterial agents

The scale of the pilot test fermentation is 50L fermenter, the initial liquid volume is 20L, and the fermentation conditions are set as temperature 37°C, rotation speed 350rpm, pressure 0.6kg, flow rate 13.5L/min, initial pH is set to 7.0, fermentation 16-20h according to Cans on case cans.

During the fermentation process, the constant pH value and the sufficient carbon source are realized by supplementing alkali and feed to ensure the growth of bacteria. Among them, the pH of the ammonia water is kept constant at 6.0 during the alkali supplementation process, and the ammonia water is automatically added when the pH is lower than 6.0 during the fermentation process; 2kg of glucose is fixed in 5L of water during the feeding process, and the feeding program is set to delay feeding at the 6th hour after the fermentation starts Time 360min.

During the fermentation process, the indicators for the monitoring and determination of the bacteria were determined by the OD ₆₀₀ value and the density of the bacteria, and samples were taken every 2 hours. Among them, the measurement of bacterial cell density was performed by 10-fold dilution coating method, and then counted after being inverted in a 37°C incubator for 12 hours, and each gradient coating was repeated three times.

The pilot test fermentation results of the strains are shown in Figure 8. The cell density of Bacillus mycoides 21 increased rapidly after fed-batch fermentation for 8 hours, and the cell density was the highest at 16 hours, with a cell density of 1.32×10 ¹⁰ CFU/mL and an OD ₆₀₀ of 11.2.

The preparation of embodiment 7 Bacillus mycoides 21 solid microbial preparations

Take the preserved Bacillus mycoides 21 and activate it in LB liquid medium, culture it on a shaker at 37°C and 180-200rpm for 12-18 hours, and inoculate it into a 50L fermenter with an inoculum amount accounting for 1% of the total mass of the medium. Under culture for 16-18 hours, as seed liquid, and then inoculated in a 1000L fermenter with an inoculation amount of 1% of the total mass of the medium, cultivated for more than 8 hours, and added diatomaceous earth accounting for 8% to 10% of the total mass of the medium Or zeolite powder and then spray dry to obtain the bacteria powder and metabolites of Bacillus mycoides 21, and the bacteria density is greater than or equal to 10 ¹⁰ CFU/g.

Application of embodiment 8 bacterial strains in sea cucumber culture

In this experiment, a sea cucumber seedling room with a water body of ^7m3 was selected, and the water temperature was 12-13°C. A control group and an experimental group were designed. The control group did not add any microbial products and drugs, and the experimental group was sprinkled with the above-mentioned liquid fermentation bacteria every 3 days. The final concentration of bacteria is about 10 ⁴ CFU/mL. The specific bacteria and feeding time are as follows:

Each group was sampled at the surface of the supercharger vortex and at a water depth of 50 cm, and each sample was taken for 3 replicates. Sampling time: 1st, 4th, 7th day: before using bacteria, after using bacteria, before feeding, after feeding, after feeding for 2 hours, after feeding for 4 hours; Day 2, 5, 8: before feeding, feeding After feeding, 2 hours after feeding, and 4 hours after feeding; on the 3rd and 6th day: 14:00 sampling once. After sampling, the number of low-temperature bacteria, pH value of water, protein degradation rate, and COD degradation rate were measured respectively.

Figures 9 and 10 show the changes in the number and pH of sea cucumber culture water in the control group and the experimental group on days 1-8. The real-time bacterial activity in the pond after using bacteria in the experimental group can basically reach (0.63-0.92)×10 ⁴ CFU/ mL, according to the colony shape on the plate, it can be determined as the target strain. The number of viable spores in the control group was stable at about 10 ² CFU/mL before and after the start of the experiment. The pH of the two groups was relatively stable, basically maintained at about 8.2.

Before the start of the experiment, the initial protein content of each group was about 0.8 mg/mL, and after feeding, the protein content was between 1.5-1.7 mg/mL, which gradually showed a downward trend with the extension of time, and reached the lowest value before the next feeding. Compared with the control group, the protein and COD degradation rates of the experimental group were significantly higher than those of the control group, and the protein degradation rate of the experimental group was between (48.71±2.52)% and (60.50±0.81)% after feeding for 24 hours. The degradation rates are all in the range of (25.09±2.62)% to (31.97±1.37)%. For the data, see Table 2, Figure 11 and Figure 12 for details. It can be seen that the reduction of protein and COD is mainly due to the accelerated decomposition of protein and other organic matter by Bacillus mycoides 21, in addition to the intake of sea cucumbers.

Table 2 Effect of Bacillus mycoides 21 on protein and COD in sea cucumber culture water

COD is an important indicator of water environment control. The microecological preparation obtained by the present invention includes one or more of Bacillus mycoides 21, bacterial powder, bacterial liquid, microbial enzymes of its biosynthesis and other metabolites, except It can obviously improve the aquatic environment of sea cucumber cultivation, and can also improve other aquatic environments, including the environment of fish and shrimp cultivation and other aquaculture.

The above embodiments only illustrate the technical solutions of the present invention, but are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still describe the contents of the foregoing embodiments. Modifications to the technical solutions, or equivalent replacement of some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed in the present invention.

Claims (10)

1. A strain of Bacillus mycoides 21 with low temperature resistance and protease production, characterized in that: its classification name is Bacillus mycoides, and it has been preserved in the China Microorganism Culture Collection Management Committee on July 8, 2022. Microbiology Center, the deposit number is CGMCC No.25259. 2. the Bacillus mycoides bacterial strain 21 according to claim 1, is characterized in that: described Bacillus mycoides bacterial strain 21 colony diameter is about 3-5mm, nearly round, milky white, flat, slightly raised in the middle, without Glossy and opaque; the bacteria are short rod-shaped, with terminal spores. 3. The Bacillus mycoides 21 according to claim 1, characterized in that the 16SrRNA gene sequence of the Bacillus mycoides 21 is shown in SEQ ID No.1. 4. The Bacillus mycoides 21 according to claim 1, characterized in that, the Bacillus mycoides 21 biosynthesizes microbial enzymes in a low temperature environment, preferably, the microbial enzymes are proteases. 5. the purposes of the Bacillus mycoides 21 described in claim 1 in the preparation of the probiotics of improving water quality environment. 6. The use according to claim 5, characterized in that, the probiotics comprise one or more of Bacillus mycoides 21, bacterial powder, bacterial liquid, microbial enzymes of its biosynthesis and other metabolites kind. 7. The use according to claim 6, characterized in that, the bacterial liquid and its metabolites are prepared as follows, Bacillus mycoides 21 in the fermentation medium, 30-37 ° C, 200 ~ 400rpm , pressure 0.3-0.7kg, flow rate 10-25L/min, and initial pH 6-8 under the conditions of cultivating for more than 8 hours, keeping the pH constant; the preparation of the bacterial powder and its metabolites is to continue to add the protective agent after the bacterial liquid is prepared obtained after spray drying. 8. The use according to claim 7, characterized in that the cell density in the bacterial liquid and its metabolites is greater than or equal to 10 ⁹ CFU/mL, and the cell density in the bacterial powder and its metabolites is Greater than or equal to 10 ¹⁰ CFU/g. 9. The use according to claim 5, characterized in that, the use is for preparing a microecological preparation for improving the aquaculture environment, preferably, the use is for preparing a microecological preparation for sea cucumber cultivation. 10. The use according to claim 9, characterized in that the bacterial cell concentration of Bacillus mycoides 21 in the sea cucumber culture water environment is greater than 10 ³ CFU/mL.

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