CN111676155B - Bacillus licheniformis and application thereof in aquaculture - Google Patents
Bacillus licheniformis and application thereof in aquaculture Download PDFInfo
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- CN111676155B CN111676155B CN202010481276.1A CN202010481276A CN111676155B CN 111676155 B CN111676155 B CN 111676155B CN 202010481276 A CN202010481276 A CN 202010481276A CN 111676155 B CN111676155 B CN 111676155B
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- bacillus licheniformis
- starch
- bacillus
- fish
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Abstract
The invention relates to the technical field of functional microorganism screening and application, in particular to a novel bacillus licheniformis and application thereof in aquaculture. The bacillus licheniformis is selected from intestinal tracts of crucian carps, has a preservation number of CCTCC NO: M2020090, can efficiently utilize starch, improves carbohydrate metabolism of fish bodies, and is beneficial to reducing feed cost. Meanwhile, the bacillus subtilis can obviously inhibit pathogenic bacteria, improve the immunity and disease resistance of aquatic animals, promote the growth of the aquatic animals, and can be widely applied to the field of aquatic feeds.
Description
Technical Field
The invention relates to the technical field of functional microorganism screening and application, and relates to bacillus licheniformis and application thereof in aquaculture.
Background
As one of the three major nutrients, carbohydrates are the major energy sources for the animal's body. In addition, the carbohydrate can be used as a precursor for synthesizing functional and structural macromolecules by an organism, influences the digestion and absorption functions of intestinal tracts of an animal organism and plays a key role in the growth and development of animals. The proper amount of carbohydrate in the fish feed can not only improve the growth performance of the fish, but also reduce the consumption of feed protein as energy, thereby playing the role of saving protein. With the increasing shortage and price rise of animal raw material resources such as fish meal and fish oil, the improvement of the utilization efficiency of feed raw materials becomes an important research direction for relieving the shortage of the feed raw materials. Carbohydrates, represented by starch, are important components in aquatic feeds and are widely available.
However, due to its physiological characteristics and the influence of factors such as living environment, the utilization capacity of the fish to carbohydrates is obviously different from that of terrestrial animals. The utilization degree of the carbohydrates in the feed by the fish is lower than that of the terrestrial animals, and the excessive carbohydrates easily cause the fish body to generate a nutrition stress effect and reduce the immunity. Therefore, the improvement of the digestion and utilization efficiency of the fish body to the carbohydrate not only is beneficial to reducing the dependence of the aquatic feed industry on fish meal and fish oil, increasing the cyclic utilization of agricultural resources and protecting natural resources, but also is beneficial to relieving nutritional diseases of the cultured fish and improving the health and the quality of aquatic animals.
The utilization rate of the fish to the carbohydrate in the feed can be improved by adjusting the proportion of the components in the feed formula, improving the culture environment and the like, but the effect is limited. Recent studies have shown that animal gut microorganisms can participate in host nutritional metabolism through a variety of mechanisms, including protein metabolism, lipid metabolism, and carbohydrate metabolism. The invention screens a strain of starch degrading bacteria from fish bodies, can improve the utilization rate of fish carbohydrates and improve the nutritional metabolism of the fish bodies.
Disclosure of Invention
The invention aims to provide a novel bacillus licheniformis and application thereof in aquaculture. The Bacillus licheniformis (Bacillus licheniformis) is screened from the intestinal tract of the crucian, can efficiently utilize starch, improves the carbohydrate metabolism of fish bodies, and is beneficial to reducing the feed cost. Meanwhile, the bacillus subtilis can obviously inhibit pathogenic bacteria, improve the immunity and disease resistance of aquatic animals, promote the growth of the aquatic animals, and can be widely applied to the field of aquatic feeds.
On one hand, the invention provides a strain of Bacillus licheniformis DN01(Bacillus licheniformis DN01), which is preserved in China center for type culture collection of Wuhan university in Wuhan, China in 4-29 th 2020, and the preservation number is CCTCC NO: M2020090.
In another aspect of the invention, there is provided a microbial preparation comprising the bacillus licheniformis.
The microbial preparation also comprises any one or the combination of two or more of bacillus subtilis, bacillus coagulans, lactobacillus plantarum, enterococcus faecalis, lactobacillus acidophilus, enterococcus faecium and bifidobacterium.
The viable bacteria amount of the Bacillus licheniformis in the microbial preparation is not less than 109CFU/g。
The invention also provides application of the microbial preparation in aquatic feeds.
The invention has the advantages of
The bacillus licheniformis DN01 screened by the invention has strong amylase producing capability under the liquid culture condition. The amylase activity can reach 2417.5U/ml at the highest after 40h of culture. The strain can efficiently degrade starch to generate short-chain fatty acids such as acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and the like, wherein the content of acetic acid is up to 1.54g/L, and the content of butyric acid is also up to 1.34 g/L. The apparent digestibility of crucian carp fed with bacillus licheniformis DN01 is improved by 22.5%, the storage amount of liver glycogen and myoglycogen is reduced by 42.3% and 40.5%, and the contents of blood sugar and insulin are reduced by 43.7% and 27.8%; the addition of the bacillus licheniformis DN01 enables key enzymes of sugar metabolism of fish bodies to be active, obviously enhances the utilization efficiency of the fish bodies to carbohydrate, can be widely applied to aquatic feeds as feed additives, and promotes the growth of aquatic animals. Compared with a control group, the weight gain rate and the survival rate of crucian carps fed with the probiotic group of the bacillus licheniformis DN01 provided by the invention are respectively improved by 28.3% and 114.3%.
In addition, the bacillus licheniformis DN01 can be well co-aggregated with pathogenic bacteria such as staphylococcus aureus, aeromonas hydrophila, pseudomonas fluorescens, edwardsiella and the like and inhibit the growth of the pathogenic bacteria, particularly has the strongest inhibition effect on the aeromonas hydrophila and the edwardsiella, is favorable for improving the immunity and disease resistance of aquatic animals, and has wide application prospect in the field of aquaculture.
Drawings
FIG. 1 is a graph showing amylase production by Bacillus licheniformis DN 01;
FIG. 2 is a diagram of analysis of relative expression of sugar metabolism related genes of crucian carp.
Detailed Description
The equipment and reagents used in the examples of the present invention may be selected from any commercially available ones. For the specific methods or materials used in the embodiments, those skilled in the art can make routine alternatives based on the existing technologies based on the technical idea of the present invention, and not limited to the specific descriptions of the embodiments of the present invention.
The invention is further illustrated with reference to specific examples.
Example 1 isolation, screening and identification of strains
1. Sample (I)
The intestinal canal of the crucian is collected in a fresh water culture test field in the south of jiao Nan of Qingdao.
2. Culture medium
1) LB culture medium: 10g of peptone, 10g of sodium chloride and 1000ml of distilled water, pH7.0-7.2, preparing a solid medium, adding 20g of agar, and sterilizing at 121 ℃ for 20 min.
2) Starch screening culture medium: 20g of soluble starch, 1g of potassium nitrate, 0.5g of sodium chloride, 0.5g of dipotassium phosphate, 0.5g of magnesium sulfate, 0.01g of ferric sulfate, 1000ml of distilled water and pH7.0-7.2, preparing a solid culture medium, adding 20g of agar, and sterilizing at 121 ℃ for 20 min.
3) Starch utilization medium: 10g of soluble starch, 5g of peptone, 10g of sodium chloride and 1000ml of distilled water, and the pH value is 7.0-7.2.
4) Starch-bromocresol purple medium: 10g of soluble starch, 5g of peptone, 10g of sodium chloride, 4m1 of 1.6% bromocresol purple and 1000ml of distilled water, wherein the pH value is 7.0-7.2.
3. Separation and screening
Taking out intestinal tract after crucian carp is hungry for 24 hours, shearing about 1cm intestinal tract near the stomach end, repeatedly washing the inner cavity with sterile water, collecting washing liquid, coating starch screening culture medium plate, and culturing at 30 deg.C for 24-48 h. Selecting uniform and clear single colony, inoculating a starch-bromocresol purple culture medium, culturing at 30 ℃ for 24h, selecting target bacteria which can change the bromocresol purple from purple to yellow, and streaking purified bacteria, wherein the names are DN01, DN02 and DN03 … … DN 12. The 12 strains are strains capable of degrading starch to produce acid.
4. Relative amylase activity assay
And measuring the relative amylase activity in the bacterial liquid by adopting a starch iodine solution color development method. 5m 10.5% soluble starch diluent and 0.5ml bacterial liquid are taken to react for 5min at the temperature of 40 ℃ and the pH value is 6.0, and 5m 10.1mo 1/L sulfuric acid is added to stop the reaction. 0.5m1 reaction solution and 5ml 0.4mmol/L potassium iodide solution were taken for color development, and the absorbance at 620nm was measured. Relative amylase activity was calculated using sterile water as a control, and the specific results are shown in table 1.
TABLE 1 screening of strains relative to amylase Activity
| Bacterial strains | Relative amylase activity |
| DN01 | 0.48±0.01 |
| DN02 | 0.08±0.02 |
| DN03 | 0.12±0.02 |
| DN04 | 0.23±0.01 |
| DN05 | 0.16±0.03 |
| DN06 | 0.26±0.02 |
| DN07 | 0.14±0.03 |
| DN08 | 0.25±0.01 |
| DN09 | 0.29±0.03 |
| DN10 | 0.31±0.01 |
| DN11 | 0.22±0.03 |
| DN12 | 0.26±0.02 |
As can be seen from the data in Table 1, among the 12 strains screened by the present invention, which can degrade starch and produce acid, the bacterial solution of DN01 strain has the highest relative amylase activity.
5. Identification of strains
(1) And (3) colony morphology characteristics: the DN01 strain is re-streaked and appears white, opaque, convex colonies and neat edges, and microscopic examination shows that the cells are short rod-shaped.
(2) The genomic DNA of the strain DN01 is extracted, a 16SrRNA sequence is amplified by utilizing a PCR technology, the similarity of the sequence and the published 16S rRNA sequence of a plurality of strains of Bacillus licheniformis is higher than 99 percent through sequencing BLAST comparison analysis, and the identification proves that the strain DN01 is the Bacillus licheniformis (Bacillus licheniformis) and is consistent with the biochemical identification result.
(3) The DN01 strain is named as Bacillus licheniformis DN01(Bacillus licheniformis DN01), which is preserved in China center for type culture Collection of Wuhan university in Wuhan, China at 29 months 4 in 2020, and the preservation number is CCTCC NO: M2020090.
EXAMPLE 2 determination of the Amylase-producing ability of Bacillus licheniformis DN01
Inoculating activated bacillus licheniformis DN01, taking 5ml of activated bacterial liquid, inoculating the activated bacterial liquid into a liquid fermentation culture medium, culturing at 37 ℃, sampling once every 8h, and determining the amylase activity in the fermentation supernatant by referring to the GB/T24401-. The amylase activity is defined as that 1g of soluble starch is liquefied in 1 hour under the conditions of pH7.0 and 37 ℃, namely an enzyme activity unit (U). The amylase production curve of Bacillus licheniformis DN01 is shown in figure 1.
As can be seen from FIG. 1, the Bacillus licheniformis DN01 provided by the invention has strong amylase producing capability under liquid culture conditions. After the strain is cultured for 8 hours, the amylase activity produced by fermentation of the strain is increased rapidly, the highest amylase activity can be 2417.5U/ml at 40 hours, the amylase activity is within the range of 32 hours-80 hours, the amylase producing capacity of bacillus licheniformis DN01 is continuously high, and unexpected technical effects are achieved.
Example 3 degradation of starch by Bacillus licheniformis DN01 to produce short chain fatty acids
Short chain fatty acids generated by decomposing probiotics can influence the physiological function and the growth of the intestinal tract of an organism, can provide energy for intestinal epithelial cells, plays a role in maintaining the balance of water and electrolyte in the intestinal tract, has a maintenance role on mucosal immune cells, reduces the generation of proinflammatory factors, and is beneficial to the repair of mucosal inflammation.
Inoculating bacillus licheniformis DN01 into a starch utilization culture medium, culturing for 24h, precooling a bacterial liquid sample, acidifying concentrated sulfuric acid, extracting with diethyl ether, and detecting a sample of a supernatant. Testing with gas chromatography, with a sample inlet temperature of 180 deg.C, a column temperature of 80 deg.C, a preliminary test time of 2min, a temperature rise of 5 deg.C/min to 150 deg.C, a detector temperature of 180 deg.C, and a sample loading amount of 1 μ l. The results are shown in Table 2.
TABLE 2 production of short chain fatty acids by Bacillus licheniformis DN01
| Short chain fatty acids | Content g/L |
| Acetic acid | 1.54 |
| Propionic acid | 0.23 |
| Butyric acid | 1.34 |
| Isobutyric acid | 0.09 |
| Valeric acid | 0.06 |
| Isovaleric acid | 0.12 |
From the results in Table 2, the Bacillus licheniformis DN01 provided by the invention can effectively degrade starch to produce short-chain fatty acids such as acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and the like, wherein the content of acetic acid is up to 1.54g/L, and the content of butyric acid is also up to 1.34 g/L. The short-chain fatty acids generated by degrading starch by bacillus licheniformis DN01 can participate in body energy metabolism in a high-grade manner, regulate body energy balance and promote body health.
Example 4 effect of bacillus licheniformis DN01 on crucian carbohydrate utilization
Activating Bacillus licheniformis DN01, culturing, fermenting in liquid state, and spray drying to obtain Bacillus licheniformis with a viable bacteria amount of about 1010CFU/g of bacterial powder.
The experimental feed is a high-starch formula, the addition amount of starch is 45%, a control group and a probiotic group are arranged, three groups are respectively arranged in parallel, and the tail of each parallel crucian carp is 40%. Wherein, the control group is fed with basic feed, and the probiotic group is fed with Bacillus licheniformis DN01 powder in a ratio of 2 ‰ by mass, and is mixed uniformly. During the culture experiment, the bait is thrown three times a day, the dissolved oxygen is more than or equal to 7mg/l, the temperature is 28 +/-1 ℃, and the pH is 7.5 +/-0.2.
The experimental feed is added with yttrium oxide for marking, excrement is collected in the experimental process, the content of yttrium element is measured by ion chromatography, and the apparent digestibility is calculated. After the culture experiment is finished, random sampling is carried out, the contents of liver glycogen, muscle glycogen, blood sugar and insulin of the fish body are respectively measured, and the specific results are shown in a table 3.
And (3) dissecting and taking the liver and the intestinal tract, and measuring the expression condition of the genes related to the glycometabolism by fluorescent quantitative PCR. The following genes were selected for detection: glycolytic pathway Pyruvate Kinase (PK) and Glucokinase (GK); glucose uptake-associated glucose transporter 4(Glut 4); glycogen Synthase Kinase (GSK), Glycogen Synthase (GS) and Insulin Receptor (IR) of the insulin signal transduction pathway are shown in fig. 2.
TABLE 3 crucian apparent digestibility and sugar metabolism related indexes
As can be seen from the data in table 3, compared with the control group, the apparent digestibility of the crucian in the experimental group fed with the bacillus licheniformis DN01 provided by the invention is improved by 22.5%; at the same time, the storage of liver glycogen and muscle glycogen was reduced by 42.3% and 40.5%, and the blood glucose and insulin contents were also reduced by 43.7% and 27.8%.
Fig. 2 shows the effect of bacillus licheniformis DN01 on expression of crucian sugar metabolism related genes:
(1) on the glycolytic pathway, GK promotes the conversion of glucose to glucose-6-phosphate. The low GK expression level is probably the reason that the utilization efficiency of the starch is low for the fish, and the addition of the bacillus licheniformis DN01 obviously improves the gene expression of the GK;
(2) in terms of glucose uptake, Glut4 is responsible for transporting glucose into the cytoplasm. The expression level of the Glut4 gene is increased in a bacillus licheniformis DN01 addition group;
(3) in the insulin signal transduction pathway, the IR expression is increased and insulin binds to more receptors, thereby lowering blood glucose levels. At the same time, GSK expression decreased and GS expression increased.
In conclusion, the addition of the bacillus licheniformis DN01 enables key enzymes of sugar metabolism of fish bodies to be active, obviously enhances the utilization efficiency of the fish bodies to carbohydrates, and achieves unexpected technical effects.
Example 5 determination of bacteriostatic ability of Bacillus licheniformis DN01
5.1 ability to co-collect bacteria and pathogenic bacteria
Respectively selecting Bacillus licheniformis DN01 and pathogenic bacteria single colony for activation, inoculating, culturing, collecting thallus, and regulating bacterial amount to 107CFU/ml, mixing 500 mul of Bacillus licheniformis DN01 with the same volume of pathogenic bacteria, standing and incubating, measuring OD600 value of the upper layer bacterial liquid of the mixed sample and the control group after 6h, and calculating the copolymerization capacity of the bacteria, wherein the result is shown in Table 4.
Copolymerization ability [ [ (A)Probiotics+ A diseaseOriginal bacterium) -A mixingSample combination]/(AProbiotics+APathogenic bacteria)×100％。
TABLE 4 comparison of the ability of Bacillus licheniformis DN01 to co-aggregate with pathogenic bacteria
| Pathogenic bacteria | Copolymerization ability (%) |
| Staphylococcus aureus | 66.31±2.13 |
| Aeromonas hydrophila | 73.28±3.12 |
| Pseudomonas fluorescens | 70.54±3.02 |
| Edwardsiella sp | 83.75±4.65 |
The results in Table 4 show that Bacillus licheniformis DN01 provided by the invention can well co-aggregate with pathogenic bacteria such as staphylococcus aureus, aeromonas hydrophila, pseudomonas fluorescens, Edwardsiella and the like, and has the strongest binding ability with Edwardsiella. This high binding capacity of bacillus licheniformis DN01 contributes to its effective binding and killing or inhibiting the growth of pathogenic bacteria.
5.2 bacteriostatic Effect-plate antagonism method
Common aquatic pathogenic bacteria are taken as indicator bacteria, and the plate antagonism method is adopted to respectively determine the bacteriostatic effect of the bacillus licheniformis DN 01. Inoculating Bacillus licheniformis DN01 bacterial liquid on a flat plate coated with indicator bacteria, culturing at constant temperature of 28 ℃, observing whether a bacteriostatic transparent area or a bacteriostatic covered area appears around the inoculation area within 48 hours, and measuring the size of a bacteriostatic circle, wherein the result is shown in Table 5. The inhibition is expressed as the ratio of the diameter of the zone of inhibition to the diameter of the colony, 1 means no inhibition, 1-2 means general inhibition, and >2 means strong inhibition.
TABLE 5 bacteriostatic effect of Bacillus licheniformis DN01 on aquatic pathogenic bacteria
| Bacterial strains | Staphylococcus aureus | Aeromonas hydrophila | Pseudomonas fluorescens | Edwardsiella sp |
| DN01 | 1 | 3.04±0.11 | 2.98±0.07 | 3.29±0.08 |
The results in Table 5 show that Bacillus licheniformis DN01 screened by the invention has stronger inhibition effect on aquatic pathogenic bacteria Aeromonas hydrophila, Pseudomonas fluorescens and Edwardsiella and especially has strongest inhibition effect on Aeromonas hydrophila and Edwardsiella.
Example 6 influence of Bacillus licheniformis DN01 on growth performance and immunity of crucian carp
The experimental feed is a high-starch formula, the addition amount of starch is 45%, a control group and a probiotic group are arranged, three groups are respectively arranged in parallel, and the tail of each parallel crucian carp is 40%. Wherein the control group is fed with basal feed, and the probiotic group is prepared by adding Bacillus licheniformis DN01 powder (10) into the basal feed at a mass ratio of 3 ‰10CFU/g), mixing uniformly and feeding. Cultivation experimentDuring the period, bait is thrown three times a day, the dissolved oxygen is more than or equal to 7mg/l, the temperature is 28 +/-1 ℃, and the pH is 7.5 +/-0.2.
And (5) after the culture experiment is finished, randomly sampling, weighing and recording, and carrying out statistical analysis on the growth performance. Selecting 20 fish and carrying out 108And (3) performing a CFU/ml aeromonas hydrophila toxicity attacking experiment, performing intraperitoneal injection according to 0.02m1/g, observing and recording survival conditions of tilapia, wherein specific results are shown in a table 6.
TABLE 6 influence of crucian growth performance and immunity
| The weight gain rate% | Survival rate% | |
| Control group | 274.54 | 35 |
| Probiotic group | 352.10 | 75 |
As can be seen from the data in table 6, the weight gain rate and the survival rate of the crucian carp in the probiotic group fed with bacillus licheniformis DN01 provided by the present invention are respectively improved by 28.3% and 114.3% compared with the control group. Therefore, the bacillus licheniformis DN01 provided by the invention can obviously promote the growth of crucian and greatly improve the survival rate of crucian after being attacked by toxin.
In conclusion, the bacillus licheniformis DN01 screened by the invention can efficiently utilize starch, generate short-chain fatty acid, regulate the expression of genes related to carbohydrate metabolism of fish bodies, improve the utilization efficiency of carbohydrate of fish bodies, obviously inhibit pathogenic bacteria, improve the immunity and disease resistance of aquatic animals, and promote the growth of the aquatic animals, can be independently used as a feed additive, can be combined with any one or two or more of bacillus subtilis, bacillus coagulans, lactobacillus plantarum, lactobacillus acidophilus, enterococcus faecium and bifidobacterium to prepare a microbial preparation, can be further compounded with polysaccharide immunopotentiators, vitamins and the like, and can be applied to feeds of aquatic animals such as fishes, shrimps, crabs and the like, and has wide application prospects.
Claims (6)
1. The bacillus licheniformis is characterized in that the preservation number of the bacillus licheniformis is CCTCC NO: M2020090.
2. Use of a bacillus licheniformis bacterium according to claim 1 for the preparation of an aquaculture feed.
3. A microbial preparation comprising the bacillus licheniformis of claim 1.
4. The microbial preparation of claim 3, further comprising any one or a combination of two or more of Bacillus subtilis, Bacillus coagulans, Lactobacillus plantarum, enterococcus faecalis, Lactobacillus acidophilus, enterococcus faecium, and Bifidobacterium.
5. The microbial preparation of claim 3 or 4, wherein the viable bacteria count of the Bacillus licheniformis bacteria in the microbial preparation is not less than 109CFU/g。
6. Use of a microbial preparation according to any one of claims 3 to 5 in the preparation of an aquaculture feed.
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