CN110669693A - Bacillus halophilus for improving salt tolerance of nile tilapia and application thereof - Google Patents
Bacillus halophilus for improving salt tolerance of nile tilapia and application thereof Download PDFInfo
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Abstract
The invention discloses a halophilic bacillus for improving the salt tolerance of nile tilapia and application thereof. Activating a common seawater liquid culture medium of the bacillus halophilus under an aseptic condition; and performing amplification culture on the activated bacterial liquid, wherein the cultured culture liquid is the inoculation liquid. Washing twice with sterilized phosphate buffer, re-suspending, making into cell suspension, and uniformly spraying on tilapia mossambica basic feed to make its concentration reach 108cfu/g feed. The bacillus halophilus 221 is added into the tilapia basic feed, can obviously improve the digestion and absorption capacity of nile tilapia living in an environment with salinity of 16 per mill, can improve the oxidation resistance and the antibacterial capacity of nile tilapia,lays a favorable foundation for the quality of tilapia. And has important significance for perfecting the tilapia formula feed.
Description
Technical Field
The invention belongs to the technical field of biology, relates to bacillus halophilus, and particularly relates to bacillus halophilus for improving the salt tolerance of nile tilapia and application thereof.
Background
The aquaculture industry in China develops rapidly, the total amount of aquaculture is the first in the world for many years, and among all cultured fishes, tilapia is the freshwater fish which is mainly cultured in the world. Nile tilapia is an economic fish in China and is an excellent variety for aquaculture with development potential. With the enlargement of intensive culture scale and the change of culture environment, higher requirements are put forward on the growth performance and disease prevention of the intensive culture. In the past, the water changing frequency is usually increased, and the purpose of high yield and disease prevention is achieved by using a large amount of antibiotics, but the quality of aquatic products is greatly influenced, and the economic value of Nile tilapia is seriously influenced.
Under the culture conditions of intensification, scale and environmental change, animals generate stress reactions due to feeding density, virus infection, frightening and the like, various adverse physiological reactions occur, the absorption of nutrient substances is influenced, the growth performance is further influenced, and great loss is caused to the production benefits of the culture farm. The method has the advantages that factors such as poor management and the like exist, pathogenic bacteria can generate drug resistance due to overuse of antibiotics, chemical drug residues interfere intestinal flora of aquatic animals and continuously destroy ecological environment, health of the aquatic animals is affected, drug content of aquatic products is excessive, quality of the aquatic products is reduced, and great hidden danger is brought to food safety problems. The problem of drug residue frequently becomes a bottleneck restricting the development of aquatic products in China to the world and causes huge economic loss to aquaculture in China. In order to improve the water environment, reduce the drug residue in aquatic products and effectively promote the healthy growth of aquatic animals, people begin to transfer the eyesight to probiotics.
From the last 60 s foreign scholars put forward the concept of probiotics to the modern definition of probiotics: under the condition of a proper amount, live microorganisms which bring health benefits to the host by improving the in vivo microbial balance can better colonize and adapt to the internal environment of the digestive tract of animals due to the animal-derived probiotics, thereby being more beneficial to the function of the protozoan flora. Modern scholars have a deeper understanding of probiotics through continuous experiments and practices. In order to inhibit the growth of pathogenic microorganisms and the invasion of pathogenic microorganisms to aquatic animals, promote the digestion and absorption of nutrient substances by the intestinal tracts of the aquatic animals, strengthen the natural immunity of the aquatic animals and improve water with poor water quality, the probiotics and the probiotics applied to the aquatic animals for promoting the protozoa colonized by the digestive tracts play a role, so that modern highly-intensive aquaculture can be continuously developed towards the direction of green, ecology and environmental protection.
Because the endospores of the bacillus can resist severe environmental conditions, the bacillus is convenient to store and difficult to lose efficacy, a stable microbial preparation product can be prepared and formed, and the bacillus can be mixed with feed to feed cultivated animals, can resist acidic and alkaline conditions of the digestive tract to show a plurality of advantages which other bacteria which can not produce spores do not have, and is greatly concerned. In addition, the bacillus is easy to obtain, is easy to propagate, grows quickly, has low nutrient requirement, is high-heat resistant, is easy for industrial production, can secrete various extracellular enzymes and bacteriocins, has no pathogenicity and the like, and thus becomes a good candidate strain for intestinal probiotics.
Disclosure of Invention
The invention aims to provide a halophilic bacillus strain 221 capable of improving the growth performance, the immune function and the antioxidant function of nile tilapia in saline water and effectively improving the intestinal health.
In order to achieve the purpose, the technical scheme of the invention is as follows: provides a bacillus halophilus for improving the salt tolerance of Nile tilapia, wherein: the bacillus is halophilic bacillus 221, and is preserved in China center for type culture collection in 2019, 8 and 26 months, wherein the preservation number of the strain is CCTCC NO: m2019669 deposit address: wuhan university in China.
Preferably, the halophilus 221 belongs to gram-positive bacteria, and the colony is light yellow, round and slightly convex, and has a smooth surface and neat edges.
Preferably, the bacillus halophilus 221 comprises per liter of medium: 1-3 g of yeast powder, 2-5 g of beef extract, 3-8 g of peptone and 25-35 g of sodium chloride, and the pH value is 7-8.
Preferably, the bacillus halophilus 221 comprises per liter of medium: 1g of yeast powder, 3g of beef extract, 5g of peptone and 30g of sodium chloride, and the pH value is 7.6-7.8.
Preferably, the culture condition of the bacillus halophilus 221 is 28-35 ℃ and the culture lasts 20-24 hours.
Preferably, the Bacillus halophilus 221 is cultured at 30 ℃ for 24 hours.
The invention also aims to provide application of the bacterial liquid prepared by activating the bacillus halophilus 221 to basal feed of tilapia.
Preferably, the bacillus halophilus 221 is activated in a common seawater liquid culture medium under aseptic conditions; carrying out amplification culture on the activated bacterial liquid; washing with sterilized phosphate buffer, and resuspending to obtain cell suspension; the cell suspension is uniformly sprayed on the basal feed of tilapia.
Preferably, the content of the bacillus halophilus 221 in the basal feed of the tilapia is 108cfu/g feed.
The bacillus halophilus 221 provided by the invention is obtained by screening nile tilapia intestinal tracts stressed by 16 per thousand salinity: taking a plurality of intestinal tracts of Nile tilapia, crushing the sample by using a sample crusher, and diluting the sample to a concentration of 10 by using sterile phosphate buffer solution-1The suspension is diluted 10 times, 200 μ L of suspension with different dilutions is absorbed, coated on common seawater culture medium, and cultured (aerobically or anaerobically) at 30 deg.C for 3 d. After the colonies with different characters are picked for dyeing microscopic examination, further purification culture is carried out; and (5) preserving and culturing. And inoculating the purified strain to a common seawater culture medium, culturing at 30 ℃ for 200r/min overnight for 20-24 hours to obtain a bacterial liquid.
The bacillus halophilus 221 is added into the tilapia basic feed, so that the digestion and absorption capacity of nile tilapia living in an environment with salinity of 16 per mill can be remarkably improved, the oxidation resistance and the antibacterial capacity of nile tilapia can be improved, and a favorable foundation is laid for the quality of tilapia. And simultaneously has important significance for perfecting the matched feed of the tilapia.
Detailed Description
Example 1
Taking a plurality of intestinal tracts of Nile tilapia, and grinding the sample to obtain homogenateDiluted to 10 concentration with sterile phosphate buffer-1The suspension was diluted 10-fold in succession, 200. mu.L of each suspension was aspirated, and the suspension was applied to a common seawater medium and cultured (aerobically or anaerobically) at 30 ℃ for 3 days. After selecting colony stains with different characters and microscopic examination, further performing purification culture; and (5) preserving and culturing. And inoculating the purified strain to a common seawater culture medium, culturing at 30 ℃ for 200r/min overnight for 20-24 hours to obtain a bacterial liquid.
And (3) detecting hemolytic activity: purifying the bacterial liquid, and performing streak culture on a common seawater agar culture medium. And (4) selecting a single colony obtained by separation and purification, dibbling the single colony on a blood plate by using a sterile toothpick, and observing whether hemolytic rings appear around the colony after culturing for 48 hours. Bacteria capable of producing hemolysin can dissolve blood cells away, and the colony edge of the animal blood plate can form a transparent ring.
And (3) detecting the antagonistic activity of pathogenic bacteria: selecting aeromonas schubertii stored in the laboratory as an indicator bacterium, culturing the indicator bacterium in a common seawater culture solution for 12h, and adjusting to OD by using a sterile PBS buffer solution600About 0.8, sucking 0.1mL of the solution, coating the solution on a fresh common seawater agar plate, and naturally drying the plate. And (3) dipping a single bacterial colony by using a sterile toothpick, dibbling the single bacterial colony on a common seawater agar plate containing an indicator bacterium, culturing for 24 hours in a constant-temperature incubator at 28 ℃, observing whether a bacteriostatic ring exists around the bacterial colony, measuring the diameter (Di) of the bacteriostatic ring and the diameter (Dc) of the bacterial colony, and preliminarily judging the antagonistic capacity of the bacterial strain according to the ratio (Di/Dc) of the diameter of the bacteriostatic ring and the diameter of the bacterial colony. The results are shown in Table 1.
And (3) detecting the activity of extracellular enzymes: the single colony which is not hemolyzed in the hemolytic test is picked by a sterile toothpick and spotted on a common seawater agar plate containing 1 percent of soluble starch and a common seawater agar plate containing 2 percent of casein for enzyme production determination. For the starch agar plate, the plate is cultured in a constant temperature incubator at 28 ℃ for 24h, and after adding Lu's iodine solution, if a colorless transparent ring appears around the colony, the soluble starch is decomposed. The protein medium was incubated at 28 ℃ for 24 hours in a constant temperature incubator, and a colorless transparent ring appeared around the colony, indicating that casein was decomposed. The hydrolysis loop diameter (Dh) and the colony diameter (Dc) were measured and recorded, and the enzyme-producing ability was preliminarily judged on the basis of the ratio (Dh/Dc) of the hydrolysis loop diameter and the colony diameter. The results are shown in Table 1.
Potential probiotic tolerance test:
artificial intestinal juice screening potential probiotic bacterial strain
The method referred to Manhar et al is slightly modified. An artificial intestinal juice having a pH of 8.0 was prepared by adding trypsin (0.1mg/mL) and bovine bile salt (0.3%) to sterile Phosphate Buffered Saline (PBS) (pH7.4), adjusting the pH to 8.0 with 1mol/L NaOH, and then sterilizing the artificial intestinal juice through a 0.22 μm filter. The test bacterial solution cultured overnight at 30 ℃ was centrifuged at 3000rpm for 15min, washed 2 times with sterile Phosphate Buffered Saline (PBS) (pH7.4), and then the bacterial pellet was resuspended in artificial intestinal fluid. Culturing at 28 deg.C for 4 hr. And (4) taking out 100uL of the culture medium in 2h and 4h respectively, carrying out gradient dilution by 10 times, counting viable bacteria on a flat plate, and calculating the survival rate of the bacteria to be detected. The results are shown in Table 1.
Artificial gastric juice screening potential probiotic bacterial strain
Reference is made to the method of Manhar et al with minor modifications. Artificial gastric juice was prepared at pH 2.0, NaCl (0.5%) pepsin (0.3mg/mL) was added to sterile Phosphate Buffered Saline (PBS) (pH7.4) and pH adjusted to 2.0 with 1mol/L HCl, and then the artificial gastric juice was sterilized through 0.22 μ filter. The bacterial liquid to be tested, which is cultured overnight at 28 ℃, is centrifuged for 15min at 3000rpm, washed 2 times with sterile Phosphate Buffer Solution (PBS) (pH7.4), then the bacterial precipitate is resuspended with artificial gastric juice, and cultured for 4h in a constant temperature incubator at 30 ℃. 100uL of the strain is taken out for 10 times of gradient dilution in 2h and 4h respectively, plate viable bacteria count is carried out, and the survival rate of the strain is calculated. The results are shown in Table 1.
And (3) detecting the adhesion capability of the potential probiotics, wherein the detection of the adhesion capability of the potential probiotics comprises the evaluation of the hydrophobic activity on the cell surface and the detection of the self-coagulation capability:
evaluation of cell surface hydrophobic activity of potential probiotic strain
Reference is made to the test method of Rosenberg. Centrifuging the bacterial solution to be tested cultured at 28 deg.C overnight at 3000rpm for 15min, washing with sterile Phosphate Buffer Solution (PBS) (pH7.4) for 1-3 times, and re-suspending with sterile Phosphate Buffer Solution (PBS) (pH7.4) to light absorption OD600Is about 1.000. Mixing equal volume of the suspension with carbohydrate (xylene, chloroform, ethyl acetate), turbine oscillating for 2min, standing at room temperature for 1 hr, separating water phase from liquid phase, sucking water phase, and measuring OD600Lower absorbance (xylene in water, chloroform in water, and ethyl acetate in water). And calculating the cell surface hydrophobic percentage of the strain to be detected: the hydrophobicity (%) - (At-a0)/a0 × 100At is an absorbance value of an aqueous phase after mixing with a carbohydrate and standing, and a0 is an absorbance value of an aqueous phase after mixing with an organic solvent. The results are shown in Table 1.
Potential probiotic strain self-agglutination assay
Reference is made to the method of Del Re et al. Centrifuging the bacterial liquid to be detected which is cultured at 28 ℃ overnight at 3000rpm for 15min, washing the bacterial liquid with sterile Phosphate Buffer Solution (PBS) (pH7.4) for 1-3 times, then resuspending the bacterial liquid with sterile Phosphate Buffer Solution (PBS) (pH7.4), carrying out turbine oscillation for 10s, incubating the bacterial liquid at room temperature for 24h, absorbing 0.1mL of upper liquid at 0h, 1h, 2h, 3h and 24h respectively, adding the upper liquid into 0.9mLPBS to measure the light absorption value of the bacteria at the wavelength of 600 nm. The percent of self-agglutination was 1-At/a0 × 100At for absorbance values of 1h, 2h, 3h and 24h, and a0 for absorbance value of 0 h. The results are shown in Table 1.
TABLE 1 isolation data for screening bacteria
Under the aseptic condition, activating the common seawater liquid culture medium of the bacillus halophilus 221; carrying out amplification culture on the activated bacterial liquid; washing with sterilized phosphate buffer, and resuspending to obtain cell suspension; the cell suspension is uniformly sprayed on the basal feed of the tilapia mossambica to ensure that the concentration of the cell suspension reaches 108cfu/g feed.
Example 2
The bacillus halophilus 221 of the invention is added to the basic feed of tilapia to influence the growth performance of tilapia:
100 tilapia mossambica domesticated for 14 days and 200 tilapia mossambica juvenile fish with similar weight (1.8g +/-0.03 g) through 16 per thousand saline water are randomly and uniformly distributed in 4 and 8 plastic barrels of 100L. Wherein the fresh water control group and the 16 per mill salinity control group are fed with basic daily ration, and the 16 per mill salinity experimental group is fed with the feed added with the bacillus halophilus 221. All groups were fed at 7:00 am and 17:00 pm, and after 56 days of the experiment, 15 fish were randomly selected from each group to measure body length and body weight and their intestinal and liver pancreas were examined for antioxidant capacity, and the results are shown in tables 2 and 3.
TABLE 2 influence of Bacillus halophilus 221 of the present invention on the growth performance of juvenile tilapia
| Fresh water control group | Control group with 16% salinity | 16% salinity test group | |
| Specific growth Rate (%) | 484±2a | 465±3b | 501±13a |
| Relative growth Rate (%) | 1257.03±142.51ab | 1105.67±26.62b | 1351.78±80.99a |
| Coefficient of feed | 1.31±0.03a | 1.35±0.03b | 1.2±0.02a |
| Weight gain (%) | 1314.93±101.69b | 1105.5±24.36b | 1371.83±84.61a |
| Specific for liver | 1.52±0.02a | 1.27±0.08b | 1.25±0.07b |
| Fullness of fertilizer | 1.81±0.03a | 1.81±0.01a | 1.85±0.02b |
In the table, the same row notes different letters to indicate significant difference (P <0.05)
From table 2, it can be seen that the addition of bacillus halophilus 221 to the tilapia basic feed of the present invention significantly improves the growth performance of juvenile tilapia at 16% salinity, and can significantly improve the economic benefit.
TABLE 3 Effect of Bacillus halophilus 221 of the present invention on the antioxidant capacity of Tilapia mossambica larvae
In the table, the same row notes different letters to indicate significant difference (P <0.05)
As can be seen from Table 3, when the Bacillus halophilus 221 is added to the basal feed of tilapia, the contents of liver superoxide dismutase (SOD) and intestinal malondialdehyde are reduced remarkably (P <0.05), and the basal feed has no significant difference (P >0.05) from the basal feed of the tilapia and also reduces the content of liver MDA (P >0.05), but the liver MDA content is not significantly different.
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, therefore, the present invention is not limited by the appended claims.
Claims (9)
1. A Bacillus halophilus for improving the salt tolerance of Nile tilapia is characterized in that: the bacillus is halophilic bacillus 221, and is preserved in China center for type culture collection in 2019, 8 and 26 months, wherein the preservation number of the strain is CCTCC NO: m2019669.
2. The bacillus halophilus for improving the salt tolerance of nile tilapia according to claim 1, wherein: the bacillus halophilus 221 belongs to gram-positive bacteria, and bacterial colonies are light yellow, round and slightly convex, smooth in surface and neat in edge.
3. The bacillus halophilus for improving the salt tolerance of nile tilapia according to claim 1, wherein: the bacillus halophilus 221 comprises per liter of culture medium: 1-3 g of yeast powder, 2-5 g of beef extract, 3-8 g of peptone and 25-35 g of sodium chloride, and the pH value is 7-8.
4. The bacillus halophilus for improving the salt tolerance of nile tilapia according to claim 3, wherein: the bacillus halophilus 221 comprises per liter of culture medium: 1g of yeast powder, 3g of beef extract, 5g of peptone and 30g of sodium chloride, and the pH value is 7.6-7.8.
5. The bacillus halophilus for improving the salt tolerance of nile tilapia according to claim 1, wherein: the culture condition of the bacillus halophilus 221 is 28-35 ℃, and the bacillus halophilus is cultured for 20-24 hours.
6. The Bacillus halophilus for improving the salt tolerance of Nile tilapia according to claim 5, characterized in that: the Bacillus halophilus 221 was cultured at 30 ℃ for 24 hours.
7. The bacillus halophilus for improving the salt tolerance of nile tilapia according to claim 1, wherein: the bacterium liquid prepared by activating the bacillus halophilus 221 is applied to a tilapia feed additive.
8. The bacillus halophilus for improving the salt tolerance of nile tilapia according to claim 7, wherein: under the aseptic condition, activating the common seawater liquid culture medium of the bacillus halophilus 221; carrying out amplification culture on the activated bacterial liquid; washing with sterilized phosphate buffer solution, and resuspending to obtain cell suspension; the cell suspension is uniformly sprayed on the basal feed of tilapia.
9. The bacillus halophilus for improving the salt tolerance of nile tilapia according to claim 8, wherein: the content of bacillus halophilus 221 in the basal feed for tilapia is 108cfu/g feed.
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| CN111543363A (en) * | 2020-05-08 | 2020-08-18 | 海南大学 | Method for breeding and raising seedlings of red swamp crayfish |
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