CN116162566A

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

Info

Publication number: CN116162566A
Application number: CN202211271773.4A
Authority: CN
Inventors: 何增国; 汤伟; 王祺; 张军; 孙晓雯; 唐涛; 乔晓妮
Original assignee: Qingdao Bioantai Biotechnology Co ltd; Qingdao Marine Biomedical Research Institute Co Ltd
Current assignee: Qingdao Bioantai Biotechnology Co ltd; Qingdao Marine Biomedical Research Institute Co Ltd
Priority date: 2022-10-18
Filing date: 2022-10-18
Publication date: 2023-05-26

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

Low-temperature fungus bacillus mycoides 21 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, in particular relates to a low-temperature fungus bacillus mycoides 21 and application thereof, and in particular relates to application of the strain in the fields of preparation of microecologics for improving aquaculture water environment and the like.

Background

The intensive high-density sea cucumber culture mode causes the increasingly serious organic pollution of the culture water body. While bringing considerable benefits, high density cultivation also initiates new chain reactions: excess bait is usually supplied in the growing season, the adhesiveness of the bait is poor, the loss is serious, and a large amount of residual bait can be generated in the cultivation process. Residual bait is deposited at the bottom of the pond and attached to the artificial attachment base, and cannot be effectively discharged through water exchange. The accumulated organic pollutants are slowly decomposed to continuously release a large amount of harmful substances such as micromolecular organic matters, inorganic matters and the like to the breeding environment, so that the health and the growth of the breeding animals are endangered. And also becomes a warm bed of pathogenic bacteria. The misuse and abuse of chemical drugs can cause pathogenic bacteria to generate drug resistance under the condition, thereby triggering secondary crisis. This threatens not only the stability of the cultivation, but also the safety of the cultivated products.

The microecological preparation has the advantages of strong practicability, low cost, large benefit, simple operation, difficult secondary pollution formation and the like, and is widely applied to the breeding industry. But also has the problems of low specificity of the microbial inoculum, inapplicability to mariculture environment, low viable count and the like. In view of the above, the screening of the patent obtains the water body pollution problem in the sea cucumber culture process, which can resist the low-temperature culture environment, can still produce protease under the low-temperature condition and effectively decompose organic matters in the water body, and effectively plays a role in purifying the culture water body.

Disclosure of Invention

The invention provides a bacillus mycoides 21 with low temperature resistance and protease production and application thereof, wherein the bacillus mycoides 21 has the functions of salt resistance, anaerobism and medium and low temperature resistance, can specifically produce protease, can improve water environment, is suitable for fresh water and salt water environments, and is particularly beneficial to improving the culture environment of sea cucumbers.

In order to achieve the aim, the invention is realized by the following technical scheme:

the invention provides a bacillus mycoides 21 with low temperature resistance and protease production, which is characterized in that: the classification of the bacillus is named as bacillus mycoides Bacillus mycoides, and the bacillus mycoides is preserved in China general microbiological culture Collection center (CGMCC) No.25259 in the

year

7 and 8 of 2022.

Furthermore, the bacterial colony diameter of the bacillus mycoides strain 21 is about 3mm, and the bacillus mycoides strain is round, milky white, flat, slightly convex in the middle, matt and opaque, and the thallus is in a short rod shape and is produced at the end of spores.

Furthermore, the gene sequence of the bacillus mycoides 21 is shown as SEQ ID No. 1. Further, the bacillus mycoides 21 is used for biologically synthesizing a microbial enzyme in a low-temperature environment, preferably, the microbial enzyme is a protease.

In another aspect of the invention there is provided the use of bacillus mycoides 21 in the manufacture of a probiotic formulation for improving an aqueous environment.

Further, the microecological preparation comprises one or more of bacillus mycoides 21, fungus powder, fungus liquid, microbial enzymes for biosynthesis thereof and other metabolites.

Further, the bacterial liquid and the metabolite thereof are prepared by culturing the bacillus mycoides 21 in a fermentation medium for more than 8 hours under the conditions of 30-37 ℃ and 200-400 rpm, 0.3-0.7kg of pressure, 10-25L/min of flow and 6-8 initial pH, keeping the pH constant and the thallus density more than or equal to 10 ⁹ CFU/mL; the preparation of the bacterial powder and the metabolite thereof is that after bacterial liquid is prepared, the protective agent is continuously added and then sprayed to dryness, and the bacterial density is more than or equal to 10 ¹⁰ CFU/g。

Further, the use is in the preparation of a micro-ecological preparation for improving the aquaculture environment.

Further, the application is in preparing a sea cucumber culture microecological preparation.

Further, the application has the thallus concentration of the bacillus mycoides 21 in the sea cucumber culture water environment of more than 10 ³ CFU/mL. Compared with the prior art, the invention has the following advantages and technical effects:

1) The invention provides a bacillus mycoides 21 with low temperature resistance and protease production, and the bacillus mycoides has the purposes of salt resistance, low temperature resistance, anaerobism and capability of specifically producing protease.

2) The invention provides application of bacillus mycoides 21 in preparing a microecological preparation for improving water quality environment, wherein water bodies comprise fresh water and sea water, and further, application of bacillus mycoides 21 in improving sea cucumber culture can specifically and effectively improve protein degradation rate and COD degradation rate, thereby reducing organic matter pollution of the water bodies and playing a role in improving water quality.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it will be apparent that the drawings in the following description are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings by those skilled in the art without departing from the scope of the claimed invention.

FIG. 1 is a colony morphology and a micrograph of Bacillus mycoides 21. Wherein, (a) colony morphology on LB medium; (b) Gram staining photomicrographs

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

FIG. 3 shows the growth curve and the enzyme production curve of Bacillus mycoides 21.

FIG. 4 shows the effect of temperature on the growth of Bacillus mycoides 21.

FIG. 5 shows the effect of NaCl on the growth of Bacillus mycoides 21.

FIG. 6 is a diagram ofLiquid submerged fermentation of bacillus mycoides 21-cell density and OD ₆₀₀ And (5) monitoring.

FIG. 7 is a degradation curve of Bacillus mycoides 21 against sea cucumber bait lysate protein.

FIG. 8 shows the effect of Bacillus mycoides 21 on COD of sea cucumber bait lysate.

FIG. 9 shows the change of the bacterial population and pH of the sea cucumber culture water body in the control group.

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

FIG. 11 is a degradation curve of the protein of the sea cucumber aquaculture water by the Bacillus mycoides 21.

FIG. 12 shows the effect of Bacillus mycoides 21 on COD in sea cucumber aquaculture water.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains or to which this term applies. The test methods used in the examples described below are conventional methods unless otherwise specified, and materials, reagents and the like used are commercially available.

The present invention will be described in further detail with reference to the following specific examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the following detailed description is merely illustrative of the invention, and is not intended to limit the invention.

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

(1) LB medium (1L): 10g of tryptone, 5g,NaCl 10g,pH7.0 of yeast extract, and sterilizing for 20min at 121 ℃ to 1L.

(2) Skimmed milk powder medium (1L): skim milk powder 20g, agar 20g, natural pH, constant volume to 1L, and sterilizing at 115 deg.C for 20min.

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

(4) Cellulose medium (1L): sodium succinate 4.72g, sodium carboxymethylcellulose 10g, peptone 10g, naCl 10g, yeast extract 5g, monopotassium phosphate 1g, magnesium sulfate 0.2g, agar 16-18 g, natural pH, constant volume to 1L, and sterilization at 121 ℃ for 20min.

(5) Sea cucumber bait culture medium (1L): taking 20g of young ginseng seedling bait, boiling with 1000mL of sterilized water for 30min, soaking for 48h, centrifuging to obtain supernatant, adding 3.5% seawater, adjusting pH to 7.0, and sterilizing at 121 ℃ for 20min for later use.

(6) Seed medium (1L): 10g of tryptone, 5g,NaCl 10g,pH7.0 of yeast extract, and sterilizing for 20min at 121 ℃ to 1L.

(7) Fermentation medium (1L): 20g of sucrose, 9g of peptone, 1g of yeast powder, 2.5g of sodium chloride and 2g of magnesium sulfate, and sterilizing for 20min at 121 ℃ to 1L.

The bacillus mycoides 21 is obtained by screening from bottom mud of a fish pond, and is identified as Bacillus mycoides by morphological, molecular biological and other methods, and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 7 months and 8 days of 2022, wherein the preservation number is: CGMCC No.25259, address: beijing, chaoyang area, north Chen Xili No.1, 3, china academy of sciences, microbiological institute.

EXAMPLE 1 identification of Bacillus mycoides 21

1. Morphological identification

After streaking on LB plates, strain 21 was cultured at 15℃for 48 hours, and then the colony morphology was observed. The colony of strain 21 on LB plate had a diameter of about 3mm, was nearly round, milky white, flat, slightly convex in the middle, matt and opaque (FIG. 1 a). Gram staining of strain 21 showed that strain 21 was a gram positive bacterium, straight bar, and sporulated (FIG. 1 b).

2. Molecular biological identification

Single colonies were picked from LB plates and boiled at 100℃for 10min in 1.5mL EP tubes containing 50. Mu.L of sterile water, 1. Mu.L was used as template, and the 16S rRNA gene sequence of strain 10 was amplified using bacterial universal primers 27F and 1492R. The PCR amplification system was 25. Mu.L, 1. Mu.L of the bacterial liquid as a template, and 1. Mu.L of each of the upstream and downstream primers, 2 XMix 12.5. Mu.L, ddH ₂ O9.5. Mu.L. PCR reaction procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s; annealing at 55℃for 1min and extension at 72℃for 40s, after 30 cycles, extension at 72℃for 10min. The PCR products were DNA sequenced, the sequence of the sequencing was shown as SEQ ID No.1, and submitted to NCBI database for Blast comparison, and phylogenetic tree construction was performed using model strain (FIG. 2), identifying strain 21 as Bacillus mycoides.

EXAMPLE 2 enzyme production Activity Studies of Bacillus mycoides 21

Preparing a skim milk powder culture medium plate, a starch culture medium plate and a cellulose culture medium plate respectively, inoculating single bacterial colonies of bacillus mycoides 21 on the plates, culturing in an inverted mode at 15 ℃, and observing the enzyme production type and activity of the bacillus mycoides. Bacillus mycoides 21 was shown to have good protease activity, no amylase and cellulase activity, and the ratio (R/R) of clear circle diameter to colony diameter is shown in Table 1.

TABLE 1 enzyme production type and Activity of Bacillus mycoides 21

EXAMPLE 3 research on growth characteristics of Bacillus mycoides 21

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

Inoculating the strain into LB liquid culture medium, arranging 3 parallel gradients, culturing at 15deg.C in shaking table at 100rpm, sampling every 4 hr, and passing the growth curve through OD ₆₀₀ The values were determined and the enzyme production curve was determined by the Fu Lin Fen method.

The growth curve and enzyme production curve of Bacillus mycoides 21 at 15deg.C are shown in figure 3, and the strain is under low temperatureSlowly grow for 0-40h, quickly grow after 40h until 56h enters the stationary phase, OD ₆₀₀ The value was 2.13. Similarly, when the strain grows for 40 hours, the enzyme yield increases rapidly, and the enzyme activity reaches the maximum value for 56 hours, which is 9.84U/mL.

2. Determination of growth temperature

Setting 3 temperature gradients at 15deg.C and 28deg.C and 37deg.C, inoculating strain into LB liquid medium, setting 3 parallel gradients, culturing in shaking table at 15deg.C and 100rpm, sampling every 8 hr, and measuring OD ₆₀₀ 。

From the results (fig. 4), the higher the temperature within the designed range, the faster the strain grows and the earlier the log phase time is entered. The growth speed of the strain is fastest at 37 ℃, the bacterial activity reaches the peak at first, and the pilot plant test production is facilitated. The preparation of the microbial preparation is therefore carried out at this temperature.

3. Determination of NaCl tolerance

Setting

0, 2%, 4%, 6%, 8%5 NaCl concentration gradients, inoculating strain into LB liquid medium, setting 3 parallel gradients, culturing in shaking table at 15deg.C and 100rpm, sampling every 8 hr, and measuring OD ₆₀₀ 。

The NaCl concentration tolerance of the strain under 15 ℃ culture condition is shown in figure 5, and OD at 64h is when the NaCl concentration is 0 ₆₀₀ A value of 2.13; at a NaCl concentration of 2%, the strain growth is hardly affected by the salt concentration, OD at 64h ₆₀₀ A value of 2.07; OD at NaCl concentration of 4% ₆₀₀ The value is slightly reduced, OD at 64h ₆₀₀ 1.899, the difference was not significant; when the NaCl concentration is higher than 4%, the strain growth is significantly affected. The seawater salt concentration is 3.5%, which shows that the strain is completely applicable to fresh water and seawater culture.

4. Anaerobic growth Property

Puncture tests were used to verify the anaerobic growth performance of the strain. The Bacillus mycoides 21 was picked up by an inoculating needle and was then inoculated into LB test tube medium containing 0.8% agar, the inoculated strain was subjected to stationary culture at 15℃to a depth of about 5cm, and the growth was observed.

After static culture for 48 hours at 15 ℃, bacterial colonies grow on the puncture surface to the depth of 5cm, which proves that the strain has better anaerobic performance

EXAMPLE 4 safety test of Bacillus mycoides 21

The body length of the zebra fish is 2-4cm, 20 zebra fish are arranged in each cylinder, 5 zebra fish are arranged in parallel, and the experimental period is 7 days. At a final concentration of 1X 10 ⁵ And (3) sprinkling CFU/mL bacterial liquid, and observing the activity and death condition of the zebra fish. After the experiment is finished, the zebra fish in all experimental groups have good vitality and no death condition.

EXAMPLE 5 evaluation of bait degradation ability by Bacillus mycoides 21

Inoculating Bacillus mycoides 21 at 2% into LB liquid medium, culturing at 15deg.C and 100rpm for 48 hr, transferring into sterile bait medium to obtain final concentration of 1.0X10 ⁵ CFU/mL, with non-inoculation treatment as control, 3 replicates were set for each treatment, shaking culture was performed at 15deg.C with shaking table at 100rpm, samples were taken at 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, respectively, centrifugation was performed at 8000rpm for 10min, and the protein and COD content in the supernatant was determined. The protein content is determined by using Fu Lin Fenfa and the COD content is determined by using an alkaline potassium permanganate method.

After 8h inoculation, the degradation rate of the bacillus mycoides 21 on the protein in the bait culture medium can reach (40.30 +/-0.55)%, and the degradation rate of COD reaches (20.92+/-0.45)% (figures 6 and 7).

EXAMPLE 6 preparation of Strain liquid fermentation inoculant

The pilot scale fermentation scale is 50L of the fermentation tank, and the initial liquid loading amount is 20L; the fermentation conditions were set at 37℃and 350rpm, at 0.6kg pressure and 13.5L/min flow, the initial pH was set at 7.0, and the fermentation was carried out for 16-20 hours depending on the conditions on the tank.

The pH value is constant and the carbon source is sufficient in the fermentation process through alkali and feed supplement, so that the growth of thalli is ensured. Wherein, the pH value of ammonia water is constant at 6.0 in the alkali supplementing process, and ammonia water is automatically fed in the fermentation process when the pH value is lower than 6.0; in the feeding process, 2kg of glucose is fixed in 5L of water, and the feeding program is set to be 360min after the 6 th time of starting fermentation.

During fermentation, the monitoring and measurement index of the thallus passes through OD ₆₀₀ The values and cell densities were determined and sampled every 2 h. Wherein the cells areDensity determination after dilution coating by 10-fold dilution coating, counting was performed after incubation in an incubator at 37℃for 12 hours, 3 replicates were performed for each gradient coating.

As shown in FIG. 8, the strain has a pilot fermentation result, and the strain density of Bacillus mycoides 21 is increased rapidly after 8h of fed-batch fermentation, wherein the strain density is 1.32X10% at the highest 16h ¹⁰ CFU/mL，OD ₆₀₀ 11.2.

EXAMPLE 7 preparation of solid microbial preparation of Bacillus mycoides 21

Activating the preserved bacillus mycoides 21 in LB liquid medium, shaking at 37 deg.C and 180-200 rpm for 12-18 hr, inoculating to 50L fermenter in the amount of 1% of the total mass of the medium, culturing for 16-18 hr under aeration to obtain seed liquid, inoculating to 1000L fermenter in the amount of 1% of the total mass of the medium, culturing for 8 hr or more, adding diatomite or zeolite powder in the amount of 8-10% of the total mass of the medium, spray drying to obtain bacillus mycoides 21 powder and metabolite, and culturing to obtain bacillus mycoides with thallus density of 10 or more ¹⁰ CFU/g。

Example 8 application of Strain in sea cucumber culture

The experiment is carried out by selecting 7m ³ The sea cucumber seedling room of the water body has the water temperature of 12-13 ℃, and a control group and an experimental group are designed, wherein the control group is not added with any microorganism products and medicines, and the experimental group is sprayed with the liquid fermentation inoculant once every 3 days, so that the final concentration of the low-temperature thalli is about 10 ⁴ CFU/mL. The specific bacteria use and feeding time are as follows:

each group was sampled at the turbocharger swirl surface and 50cm water depth, respectively, with 3 replicates per sample. Sampling time:

day

1, 4, 7: before using bacteria, after using bacteria, before feeding, after feeding for 2 hours and after feeding for 4 hours;

day

2, 5, 8: before feeding, after 2 hours of feeding and after 4 hours of feeding; day 3, 6: 14:00 samples were taken once. And after sampling, respectively measuring the viable count of the low-temperature bacteria, the pH value of the water body, the protein degradation rate and the COD degradation rate.

The results of the change of the bacterial population quantity and pH of the sea cucumber culture water body of the control group and the experimental group on days 1-8 are shown in figures 9 and 10, and the real-time bacterial activity in the post-bacterial pond of the experimental group can basically reach (0.63-0.92) x 10 ⁴ CFU/mL, the target strain can be determined according to the colony morphology of the flat plate, and the number of the bacillus bacterial activities before and after the start of the experiment of the control group is stabilized at 10 ² CFU/mL. The pH of both groups was relatively stable, essentially maintaining around 8.2.

The initial protein content of each group before the start of the experiment is about 0.8mg/mL, the protein content after feeding is between 1.5 and 1.7mg/mL, the protein gradually shows a descending trend along with the extension of time, and the protein reaches the minimum value before the next feeding. Compared with the control group, the protein degradation rate and the COD degradation rate of the experimental group are obviously higher than those of the control group, the protein degradation rate of the experimental group after 24 hours of feeding is (48.71 +/-2.52)% (60.50+/-0.81)%, the degradation rate of the COD is (25.09+/-2.62)% (31.97+/-1.37)%, and the data are shown in Table 2, FIG. 11 and FIG. 12. From this, it can be seen that the reduction of protein and COD is mainly due to the acceleration of the decomposition of protein and other organic matters by the Bacillus mycoides 21 in addition to the ingestion of sea cucumber.

TABLE 2 influence of Bacillus mycoides 21 on protein and COD in sea cucumber culture Water

COD is an important index for water environment control, and the obtained microecological preparation contains one or more of bacillus mycoides 21, fungus powder, fungus liquid, biosynthesized microbial enzymes and other metabolites thereof, so that the aquatic environment of sea cucumber culture can be obviously improved, and other aquatic environments including fish and shrimp culture and other aquatic culture environments can be improved.

The above embodiments are merely illustrative of the technical solution of the present invention, and are not limiting thereof; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims

1. A strain of bacillus mycoides 21 having low temperature tolerance and protease production, characterized in that: the classification of the bacillus is named as bacillus mycoides Bacillus mycoides, and the bacillus mycoides is preserved in China general microbiological culture Collection center (CGMCC) No.25259 in the year 7 and 8 of 2022.

2. The bacillus mycoides strain 21 according to claim 1, characterized in that: the diameter of the bacterial colony of the bacillus mycoides strain 21 is about 3-5mm, and the bacillus mycoides strain is nearly round, milky white, flat, slightly convex in the middle, matt and opaque; the thallus is in a short rod shape and the spores are grown at the end.

3. The bacillus mycoides 21 of claim 1, wherein the 16S rRNA gene sequence of the bacillus mycoides 21 is shown in SEQ ID No. 1.

4. The bacillus mycoides 21 of claim 1, wherein said bacillus mycoides 21 is a biosynthetic microbial enzyme in a low temperature environment, preferably wherein said microbial enzyme is a protease.

5. Use of bacillus mycoides 21 as defined in claim 1 for the preparation of a micro-ecological formulation for improving an aqueous environment.

6. The use according to claim 5, wherein the microecological preparation comprises one or more of bacillus mycoides 21, bacterial powder, bacterial liquid, biosynthetic microbial enzymes and other metabolites thereof.

7. The use according to claim 6, wherein the bacterial liquid and the metabolite thereof are prepared by culturing bacillus mycoides 21 in a fermentation medium at 30-37 ℃ and 200-400 rpm under the conditions of 0.3-0.7kg, 10-25L/min flow and 6-8 initial pH for more than 8 hours, and keeping the pH constant; the preparation of the bacterial powder and the metabolite thereof is that after bacterial liquid is prepared, the protective agent is continuously added and then sprayed and dried.

8. The use according to claim 7, wherein the bacterial liquid and the metabolite thereof have a bacterial density of 10 or more ⁹ CFU/mL, the bacterial powder and the bacterial density in the metabolite thereof are more than or equal to 10 ¹⁰ CFU/g。

9. Use according to claim 5, characterized in that it consists in the preparation of a microecological preparation for improving the aquaculture environment, preferably in the preparation of a microecological preparation for sea cucumber cultivation.

10. The use according to claim 9, wherein the concentration of the bacterial cells of bacillus mycoides 21 in the sea cucumber aquaculture water environment is greater than 10 ³ CFU/mL。