CN106676029B

CN106676029B - Bacillus pumilus strain, and microecological preparation and feed thereof

Info

Publication number: CN106676029B
Application number: CN201610288317.9A
Authority: CN
Inventors: 付维来; 易敢峰; 卢俊; 王蕊; 张聪颖; 孙强; 王宁; 朱传忠
Original assignee: Fujian Dabeinong Fisheries Science & Technology Co ltd; Tianjin Changnong Science & Technology Co ltd; Beijing Dabeinong Technology Group Co Ltd
Current assignee: Fujian Dabei Nonghuayou Aquatic Technology Group Co ltd; Tianjin Dabeinong Biotechnology Co ltd; Beijing Dabeinong Biotechnology Co Ltd
Priority date: 2016-05-03
Filing date: 2016-05-03
Publication date: 2021-06-11
Anticipated expiration: 2036-05-03
Also published as: CN106676029A

Abstract

The invention belongs to the technical field of biology, and discloses a bacillus pumilus capable of inhibiting streptococcus agalactiae and application thereof. The screened metabolite of the bacillus pumilus can obviously inhibit the metabolism and growth of streptococcus agalactiae, reduce the number of streptococcus agalactiae in a tilapia culture environment and control the occurrence of streptococcicosis. The bacillus pumilus is bred for multiple times and can be subjected to liquid submerged fermentation to prepare aqua products and powder products. The powder product can be prepared into tilapia pellet feed according to a certain proportion and applied to aquaculture.

Description

Bacillus pumilus strain, and microecological preparation and feed thereof

Technical Field

The invention belongs to the technical field of biology, and discloses a strain of bacillus pumilus capable of inhibiting tilapia streptococcus, which is prepared into aqua products and powder products through fermentation optimization and can be applied to aquatic feeds.

Background

Tilapia (Tilapia) is one of the most main breeding varieties in the world aquatic industry, and has the advantages of high growth speed, poor feeding property, good meat quality, high nutritional value, strong reproductive capacity and the like, and is widely distributed in the light saline water areas of tropical and subtropical zones. The yield of tilapia cultivated in coastal areas of China, particularly in the provinces of Hainan, Guangdong, Fujian, Guangxi and the like accounts for more than half of the world. However, in recent years, the tilapia streptococcicosis caused by Streptococcus agalactiae (Streptococcus agalactiae) infection along with the increasing culture area and culture density of tilapia becomes a main factor for preventing the continuous healthy development of the tilapia culture industry in China. Particularly, tilapia 'streptococcicosis' shows a tendency of national outbreak since 2009, the incidence rate is more than 50% and the death rate can reach more than 80% at the highest in high-temperature seasons of each year, particularly in 7-9 months

At present, antibiotics are mostly adopted to control the occurrence and infection of tilapia streptococcus, and the problem of excessive use and overproof residue of the antibiotics in the culture process is caused by the culture mode, so that the export and the sale of finished tilapia are severely restricted, and the problem becomes a bottleneck problem limiting the development of tilapia industry.

Disclosure of Invention

According to the invention, a strain of bacillus pumilus is screened from the tilapia culture environment, and the bacillus pumilus can inhibit growth of streptococcus agalactiae causing tilapia streptococcicosis, can remarkably control generation and infection of tilapia streptococcicosis, reduce antibiotic use, reduce tilapia culture risk and improve tilapia culture benefit.

Firstly, the invention provides a new strain of bacillus pumilus capable of inhibiting tilapia streptococcicosis.

The bacillus pumilus is obtained by separating tilapia culture water, and performing directional primary screening and secondary screening. The vegetative cells are in a slender rod shape, spores of the vegetative cells are in an oval or long cylinder shape and are secondary or secondary, and cysts are slightly expanded. Isolated or in short chain, with semi-circular ends. The bacterial colony can be formed within 12-16 h, is round or irregular, has a hair-shaped edge, is white and opaque, and has no wrinkles. Is gram-positive.

Carrying out PCR identification on the screened strains by using the universal primers of the bacteria: extracting template DNA according to the operation instruction of the bacterial DNA extraction kit. The 16S rRNA gene fragment of the bacterium was amplified using an upstream primer 5'-AGAGTT TGA TCC TGG CTC AG-3' (SEQ ID No.2) and a downstream primer 5'-GGT TACCTT GTT ACG ACT T-3' (SEQ ID No.3) of a conserved sequence of 16S rRNA. The Bacillus pumilus belongs to Bacillus pumilus (Bacillus pumilus) through sequencing, and the sequencing result of the amplified fragment is shown in SEQ ID No. 1.

The obtained Bacillus pumilus CD6 was tested for its antibacterial activity, temperature resistance and fermentation characteristics using LB medium tryptone 10/L, yeast extract 5g/L and NaCl 10 g/L. During the plate test, CD6 was found to significantly inhibit the growth of streptococcus agalactiae, forming a significantly transparent zone of inhibition. The CD6 spore has good temperature resistance, the spore is in water bath at 85 ℃ for 10min, and the survival rate of the spore is more than 80%.

The screened Bacillus pumilus (Bacillus pumilus) is named as CD6 and is preserved in the China general microbiological culture Collection center at 2016, 3 months and 9 days, and the address is as follows: the microbial research institute of the national academy of sciences No.3, Xilu No.1, Beijing, Chaoyang, and the preservation numbers are as follows: CGMCC No. 12197.

The invention also provides a high-density fermentation culture medium of the bacillus pumilus, which comprises the following components: KH (Perkin Elmer)₂PO₄0.8-1.5 g/L, 3-6 g/L of cane sugar, 8-12 g/L of bean cake powder and FeSO₄0.25～0.5g/L，CaCO₃1～3g/L，MgSO₄2.5-3.5 g/L, 4-6 g/L of corn flour, 6-10 g/L of fish meal, 0.05-0.1% of defoaming agent and 7.0-7.5% of initial pH.

Preferably, the high-density fermentation medium contains KH₂PO₄1.0g/L, 3g/L of cane sugar, 8g/L of bean cake powder and FeSO₄0.30g/L，CaCO₃1.5g/L，MgSO₄3.0g/L of corn flour, 5g/L of corn flour, 8g/L of fish meal, 0.05% of antifoaming agent and initial pH of 7.5.

The invention also provides a high-density fermentation method of the bacillus pumilus, which comprises the following steps: inoculating the seed liquid into the fermentation culture medium according to the inoculation amount of 3-6%, wherein the temperature is 30-37 ℃, the rotating speed is 200-400 r/min, the tank pressure is 0.05Mpa, and the aeration ratio is 1: 0.4 to 0.6, hairFermenting for 12-16 h to obtain the bacillus with the spore generation rate of more than 90 percent and the viable count of 10⁹～10¹⁰CFU/ml of Bacillus pumilus fermentation broth.

The invention also provides a microecological preparation for inhibiting streptococcal disease of tilapia, which contains the fermentation liquor of Bacillus pumilus (Bacillus pumilus) CGMCC No. 12197.

In one embodiment of the invention, the microecological preparation for inhibiting streptococcal disease of tilapia further comprises NaCl with a final concentration of 12-15%.

The invention also provides another microecological preparation for inhibiting streptococcal disease of tilapia, which contains the bacterial powder of Bacillus pumilus (CGMCC No. 12197), and the viable count is 10¹⁰～10¹¹CFU/g。

Wherein, the preparation method of the fungus powder is as follows: concentrating the high-density fermentation liquor by using a ceramic composite membrane for 3 times, adding 10% starch as a carrier, and performing spray drying under the following drying conditions: the air inlet temperature is 200-400 ℃, the outlet temperature is 70-85 ℃, the feeding speed is 10-12L/min, and the atomizing turning speed is 20000-30000 r/min.

According to the developed microecological preparation, the water aqua is directly splashed to a culture water area to inhibit the growth of streptococcus agalactiae; the powder product is added into tilapia mossambica particle feed to improve the resistance of tilapia mossambica to streptococcal diseases.

The bacterial powder prepared by the product can be used as a feed additive to prepare tilapia mossambica pellet, and can improve the resistance of tilapia mossambica to streptococcicosis.

Drawings

FIG. 1: bacillus pumilus CD6 zone of inhibition.

Detailed Description

The screening method, the preparation process and the use of the present invention are illustrated in detail by the following examples, which are illustrative and do not limit the scope of the present invention.

Example 1 method for testing the inhibition of Bacillus pumilus CD6

1) Method for culturing streptococcus agalactiae

Liquid culture medium: 10.0g/L of peptone, 12.5g/L of dehydrated calf brain extract powder, 5.0g/L of dehydrated calf heart extract powder, 5.0g/L of sodium chloride, 2.0g/L of glucose, 2.5g/L of disodium hydrogen phosphate and pH 7.5.

Solid medium: 10.0g/L of peptone, 12.5g/L of dehydrated calf brain extract powder, 5.0g/L of dehydrated calf heart extract powder, 5.0g/L of sodium chloride, 2.0g/L of glucose, 2.5g/L of disodium hydrogen phosphate, pH7.5 and 1.5% of agar powder.

2) Screening plate fabrication

The single colony of Streptococcus agalactiae on the plate was inoculated to a liquid medium and cultured at 25 ℃ for 16 hours. Pouring the cultured fermentation liquor into a sterilized solid culture medium according to the proportion of 10% under the aseptic condition, cooling to 40-45 ℃, mixing uniformly, pouring into an aseptic plate with 30ml per plate, and cooling and solidifying for later use.

3) Screening of bacillus pumilus by Oxford cup method

Placing a sterile oxford cup on the flat plate prepared by the method 2), adding 200 mu L of the fermentation liquor of the CD6LB culture medium into the oxford cup, culturing for 16h at 30 ℃, and observing the situation of the inhibition zone.

By the above method, the inhibitory effect of CD6 on streptococcus agalactiae was seen, as shown in fig. 1, with a significant clear zone of inhibition around the oxford cup containing CD 6.

Example 2 determination of minimum inhibitory concentration of fermentation supernatant of Bacillus pumilus CD6

1) Preparation method of bacillus pumilus CD6 fermentation broth supernatant

Inoculating CD6 into 100ml triangular sample containing 25ml LB culture medium, culturing at 37 deg.C for 24h, taking 10ml fermentation liquid, centrifuging at 5000r/min for 15min, and collecting supernatant containing no thallus.

2) Preparation of supernatants of fermentation broths of CD6 at various concentrations

Taking 5 glass test tubes, respectively marking as 0, 1, 2, 3 and 4, adding 1ml of supernatant which does not contain thalli after centrifugation into each test tube, adding 1ml of purified water into the test tube No.1, adding 2ml of water into the test tube No.2, adding 3ml of purified water into the test tube No.3, and adding 4ml of water into the test tube No. 4. The concentrations of the test tube supernatants No.1, 2, 3 and 4 were diluted 2 times, 3 times, 4 times and 5 times respectively.

3) Determination of minimum inhibitory concentration of fermentation supernatant of Bacillus pumilus CD6

The inhibition of Streptococcus agalactiae was determined at various dilution concentrations according to the method of example 1, and the results are as follows.

TABLE 1 determination of minimum concentration of CD6 fermentation supernatant

Numbering	0	1	2	3	4
						Concentration of dilution	1 times of	2 times of	3 times of	4 times of	5 times of
Bacteriostatic effect	+++++	++++	++	+

As can be seen from Table 1, the fermentation supernatant of CD6 has excellent effect of inhibiting Streptococcus agalactiae, and still has bacteriostatic effect after being diluted 4 times.

Example 3 preparation of a high-Density fermentation broth of Bacillus pumilus

1) Plate culture rejuvenation: inoculating a bacillus pumilus strain on an LB (lysogeny broth) plate culture medium, culturing at 30 ℃ for 24 hours to rejuvenate the bacillus pumilus and form a single colony, selecting the single colony on the inoculation culture medium, and culturing at 37 ℃ for 24 hours;

2) preparing first-class seeds: transferring the Bacillus pumilus strain cultured in the step 1) to an LB slant culture medium of an eggplant bottle, and culturing at 37 ℃ for 24 hours to enable the strain to be in the late logarithmic phase to obtain a first-class seed;

3) preparing secondary seeds: preparing the primary seeds prepared in the step 2) into bacterial suspension by using sterile water, inoculating the bacterial suspension into a 100L seed tank filled with 60L LB seed culture medium, wherein the temperature is 30 ℃, the rotating speed is 200r/min, the tank pressure is 0.05MPA, and the ventilation ratio is as follows: 1: culturing for 14h at 0.6 to obtain secondary seed liquid.

4) Preparing a bacillus pumilus fermentation liquid: inoculating the secondary seed liquid prepared in the step 3) to 1m of 600L fermentation medium according to the inoculation amount of 5 percent³In a fermentation tank, the temperature is 30 ℃, the rotating speed is 200r/min, the tank pressure is 0.05Mpa, and the ventilation ratio is 1: 0.5, culturing for 16h to obtain spore production rate of more than 90%, viable count of 5 × 10⁹cfu/ml of Bacillus pumilus fermentation liquor;

the fermentation medium comprises the following components in percentage by mass: KH (Perkin Elmer)₂PO₄1.0g/L, 3g/L of cane sugar, 8g/L of bean cake powder and FeSO₄0.30g/L，CaCO₃ 1.5g/L,MgSO₄3.0g/L of corn flour, 5g/L of corn flour, 8g/L of fish meal, 0.05% of antifoaming agent and initial pH of 7.5.

EXAMPLE 4 preparation of aqueous Microecology

Sodium chloride accounting for 15% of the weight of the fermentation broth is added into the fermentation broth prepared in example 2, so that an aqueous product with a stable shelf life can be prepared, and the aqueous product is a plastic packaging bottle made of 1L or 5L PET (polyethylene terephthalate).

EXAMPLE 5 preparation of the powder Microecology

1) And (3) concentrating fermentation liquor: the produced fermentation liquor is filtered by adopting a ceramic membrane with the aperture of 1-10 microns, so that the separation of thalli and liquid is realized, and a triple concentrated solution is prepared.

2) Adding a carrier: and (4) installing the volume of the concentrated solution, adding 10% of corn starch, and uniformly stirring for later use.

3) Spray drying conditions: the air inlet temperature is 160-180 ℃, the air outlet temperature is 70-80 ℃, the rotation speed of the atomizer is 20000-30000 r/min, and the stability of the air outlet temperature is realized by controlling the feeding speed.

4) Preparing fungus powder: spraying the mixed solution prepared by the method 2) according to the method 3) to prepare the bacterial powder with the water content of 5 percent and the viable count content of 10 percent¹⁰CFU/g。

Example 6 application of aqueous Microecology

The aqueous microecology prepared according to example 3 was applied to a tilapia mossambica pond at a water depth of 1.5 meters per mu/liter. The number of the bacillus brevis in the water body can reach 10 through sampling detection³And the concentration of the CFU/L is far higher than that of natural colonies in the water body, so that the colony growth advantage is formed, and the aim of inhibiting the growth of the streptococcus agalactiae can be fulfilled.

TABLE 2 survival of Bacillus pumilus in water within one week after product use

As can be seen from Table 2, after the aqueous microecology of the present invention was applied, a higher level of viable Bacillus pumilus could be detected in the pond within one week, and the cell concentration reached the highest level on day 3.

Example 7 application of Tilapia feed containing CD6 powder in cultivation

The control pond and the test pond have two mouths, the area is 20 mu, and the water depth is 1.5 m. The tilapia variety is lucky, 3000 tails per mu, and the experimental period is 60 days.

Control ponds were fed basal diet. The test pond is feed containing CD6 bacterial powder,the powder product according to example 4 was added to the basal ration of tilapia at a rate of 0.1%. The tilapia mossambica pellet feed prepared by the method has the live bacterial count of CD6>10⁶CFU/g。

The tilapia feeding method comprises the following steps: the feed is fed once a day in the morning and evening, and the feed amount fed in the control pond and the test pond is the same.

The sample making and measuring method comprises the following steps: 10 samples are taken from each pond, the weight is measured, the measurement is carried out once every 15 days, and the death condition of the fishes is counted.

TABLE 3 Tilapia mossambica weight gain statistics

As shown in Table 2, the feed containing CD6 is used for feeding tilapia, so that the weight gain of the tilapia in the culture process can be improved, the feed conversion rate can be improved, and the feed conversion rate can be reduced.

In the mortality statistics, the mortality of tilapia is found to be obviously reduced compared with the control group after the control feed is fed. The mortality rate of tilapia in the test pond was reduced by 10% compared to the control pond during the 60 day test.

Therefore, the microecological preparation product is safe, can obviously promote the individual weight gain of aquatic animals, and improves the yield and the total amount per unit area, thereby increasing the income of aquaculture and improving the benefit.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A new strain of Bacillus pumilus (Bacillus pumilus) with the preservation number of CGMCC No. 12197.

2. The Bacillus pumilus of claim 1, wherein the 16sRNA gene sequence is set forth in SEQ ID No. 1.

3. The method for high-density fermentation of Bacillus pumilus of claim 1, which comprises the steps of: inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 3-6%, wherein the temperature is 30-37 ℃, the rotating speed is 200-400 r/min, the tank pressure is 0.05Mpa, and the aeration ratio is 1: 0.4-0.6, 12-16 h of fermentation time, more than 90% of spore generation rate and 10 viable count⁹～10¹⁰CFU/ml of Bacillus pumilus fermentation liquor; wherein the fermentation medium contains KH₂PO₄0.8-1.5 g/L, 3-6 g/L of cane sugar, 8-12 g/L of bean cake powder and FeSO₄ 0.25～0.5 g/L，CaCO₃ 1～3 g/L，MgSO₄2.5-3.5 g/L, 4-6 g/L of corn flour, 6-10 g/L of fish meal, 0.05-0.1% of defoaming agent and 7.0-7.5% of initial pH.

4. The method for high-density fermentation of Bacillus pumilus of claim 3, wherein the fermentation medium comprises KH₂PO₄1.0g/L, 3g/L of cane sugar, 8g/L of bean cake powder and FeSO₄ 0.30 g/L，CaCO₃ 1.5 g/L，MgSO₄3.0g/L, 5g/L of corn flour, 8g/L of fish meal, 0.05% of antifoaming agent and 7.5 of initial pH.

5. A microecological formulation which inhibits streptococcal disease in tilapia mossambica comprising a fermentation broth of the bacillus pumilus of claim 1.

6. The microecological preparation for inhibiting streptococcal disease in tilapia according to claim 5, further comprising NaCl at a final concentration of 12-15%.

7. Restraining deviceA microecological preparation for treating streptococcal disease of tilapia, which contains the bacterial powder of the new strain of the bacillus pumilus as claimed in claim 1, wherein the viable count of the bacillus pumilus is 10¹⁰～10¹¹ CFU/g。

8. The microecological formulation for inhibiting streptococcal disease in tilapia according to claim 7, wherein said bacterial powder is prepared by: concentrating the high-density fermentation liquor by using a ceramic composite membrane for 3-5 times, adding 10-30% of starch as a carrier, and performing spray drying under the following drying conditions: the air inlet temperature is 200-400 ℃, the outlet temperature is 70-85 ℃, the feeding speed is 10-12L/min, and the atomizing turning speed is 20000-30000 r/min.

9. The tilapia feed containing the bacillus pumilus powder of claim 1, wherein the addition amount of the bacillus pumilus powder is 0.5-5 per mill.