CN108841747B - Protease-producing bacillus subtilis and method of use - Google Patents
Protease-producing bacillus subtilis and method of use Download PDFInfo
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- C12R2001/07—Bacillus
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Abstract
The invention provides a bacillus subtilis DB005 (B)Bacillus subtilis) The microbial inoculum is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.15646 and the preservation date of 2018, 4 months and 26 days. The invention also discloses an application of the bacillus subtilis, which is applied to stichopus japonicus culture and comprises two methods: (1) fermenting the stichopus japonicus feed by using the bacillus subtilis; (2) and sprinkling the bacterial liquid of the bacillus subtilis into the stichopus japonicus culture pond. The strain can secrete protease outside cells, the protease can decompose protein in feed and environment, growth speed of the stichopus japonicus is increased, water quality is purified, morbidity is reduced, and survival rate is increased.
Description
Technical Field
The invention belongs to the field of biology, and relates to a bacillus subtilis, in particular to a bacillus subtilis capable of producing protease and a using method thereof.
Background
With the rapid development of the aquaculture industry, the scale of intensive high-density aquaculture is gradually enlarged, and the economic loss of the aquaculture industry caused by the environmental deterioration and the disease damage of the aquaculture industry is more obvious. The traditional solution is to use antibiotics. It is well known that the use of antibiotics is prone to the problem of resistant strains and may remain in the body. Probiotics are living microorganisms that produce beneficial effects on the animal body over a range of concentrations. Probiotics function to promote growth of animals by enhancing the inhibitory effect of animals on intestinal harmful microflora or by preventing diseases by enhancing non-specific immune functions. Therefore, in recent years, probiotics have been rapidly developed as a partial field of substitution for antibiotics. At present, probiotics are widely applied to the culture of fishes, shellfishes, shrimps and stichopus japonicus.
Bagheri et al (2008) add the composite bacillus preparations with different concentrations into the rainbow trout feed for two months, and as a result, the composite probiotic preparation with high concentration can obviously promote the growth of the rainbow trout. Doeschate et al (2008) research proves that the indigenous probiotic flora separated from the Bao in south Africa can improve the digestive enzyme activity and promote the growth. Iehata et al (2009) found that lactic acid bacteria have certain effects on improving the intestinal digestive enzyme activity of abalone.
The invention patent application 201510896838.8 provides a strain of bacillus licheniformis and a feed additive containing the strain. The invention specifically provides a novel Bacillus licheniformis DN29(Bacillus licheniformis DN29), and the preservation number is CCTCCNO: m2015482. The bacillus licheniformis DN29 can effectively inhibit pathogenic bacteria such as vibrio splendidus and pseudoalteromonas, reduce the occurrence probability of diseases of cultured animals, and can be used as a feed additive, thereby obviously improving the utilization rate of the cultured animals to the feed and promoting the growth of the animals. The bacillus licheniformis DN29 can be used as a probiotic bacterium to be applied to the cultivation and production process of stichopus japonicus, has a remarkable promoting effect on the growth of stichopus japonicus, and can also effectively improve the immunity of the stichopus japonicus and the resistance to vibrio lautus.
Disclosure of Invention
The invention provides a Bacillus subtilis strain, which can secrete protease outside cells, the protease can decompose protein in feed and environment, the growth speed of stichopus japonicus is increased, the water quality is purified, the morbidity is reduced, and the survival rate is increased.
The technical scheme of the invention is as follows:
the Bacillus subtilis is named as Bacillus subtilis DB005 and is preserved in China general microbiological culture Collection center (CGMCC No. 15646), the preservation date is 2018, 4 and 26 days, and the preservation address is No. 3 of Navy district, Xilu No.1 of Beijing city, and North Cheng.
The Bacillus subtilis DB 00516S rDNA sequence is shown in SEQ NO. 1.
The activation method of the bacillus subtilis comprises the steps of inoculating the bacillus subtilis into an LB culture medium, and culturing for 8-10 hours at the temperature of 25-30 ℃. Wherein the LB culture medium comprises the following components in percentage by weight: 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl.
The application of the bacillus subtilis is to apply the bacillus subtilis to stichopus japonicus culture. The application of the bacillus subtilis in the cultivation of the stichopus japonicus comprises two methods: (1) fermenting the stichopus japonicus feed by using the bacillus subtilis; (2) and sprinkling the bacterial liquid of the bacillus subtilis into the stichopus japonicus culture pond.
Wherein the method (1) specifically comprises the following steps: (A) activating the Bacillus subtilis strain (bacteria content 1.0 x 10)8cfu/ml-1.0*109cfu/ml), then taking a proper amount, putting the proper amount into the initial stichopus japonicus breeding feed, and uniformly mixing to obtain a mixed stichopus japonicus breeding feed; the weight percentage of the bacillus subtilis liquid is 2-5%. (B) Adding a proper amount of water into the mixed stichopus japonicus breeding feed, wherein the amount of the water is 1.5-3 times of the weight of the initial stichopus japonicus breeding feed, and then fermenting for 12-32 hours at the temperature of 20-32 ℃ to obtain a final product, namely the fermented stichopus japonicus breeding feed. The obtained Stichopus japonicus feed is slurry liquid, and has fermentation fragrance. The stichopus japonicus feed fermented by the strain can decompose protein in the feed into peptides and free amino acid, and is more favorable for digestion and absorption of stichopus japonicus.
Wherein the method (2) specifically comprises the following steps: uniformly putting the activated bacterial liquid into a stichopus japonicus culture pond or a stichopus japonicus seedling protection pond, wherein the concentration of the bacterial liquid is 5-10 PPM; and periodically replenished to maintain that concentration based on consumption. The method comprises the following specific steps: the activated bacterial liquid (the bacterial content is 1.0 x 10)8cfu/ml-1.0*109cfu/ml) is diluted by 10 times, and then the diluted solution is uniformly put into an apostichopus japonicus seedling-protecting pond or an apostichopus japonicus culture pond. The use concentration is 5-10PPM, the supplementary feeding is carried out once in 5 days, and the supplementary feeding is carried out once after each pond is poured (namely, the stichopus japonicus in the pond and the attaching base of the stichopus japonicus are moved to a culture pond after being cleaned and disinfected). Splashing in the culture water bodyThe strain can obviously degrade the protein in residual bait and excrement in water, purify water quality, improve the survival rate of the stichopus japonicus and play a role in improving the culture benefit.
A fermented stichopus japonicus feed containing the bacillus subtilis is prepared by the method (1).
The using method of the fermented stichopus japonicus feed is characterized by comprising the following steps: uniformly mixing the fermented stichopus japonicus feed and sea mud (floating mud on the sea bottom, which is a mixture of silt, few animal and plant debris, benthic diatoms and the like in seawater) according to the weight ratio of 1:4-1:6, and then putting the mixture into stichopus japonicus culture water; the daily dosage is 1-3% of the total weight of the stichopus japonicus to be fed.
The invention has the beneficial effects that:
(1) the protease secreted by the bacillus subtilis DB005 can decompose the protein of the feed, improve the utilization rate of the feed and improve the growth speed of the stichopus japonicus, and the weight gain rate of 2 months is increased from 236.7 percent to 298.3 percent in average and increased by 61.6 percent;
(2) the stichopus japonicus feed fermented by the strain is used for cultivating stichopus japonicus, residual feed and excrement in the cultivated water can be decomposed, the water quality is purified, the morbidity is reduced, and after the stichopus japonicus feed is used for 2 months, the survival rate is improved from 84.7 percent of the original average survival rate to 90.7 percent of the original average survival rate and is improved by 5.97 percent.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1: isolation and selection of strains
1.1 isolation of beneficial bacteria
(1) Primary screening:
properly diluting the supernatant of the stichopus japonicus pond sediment in a super-clean workbench by using secondary distilled water, uniformly coating the supernatant on a 2216E solid culture medium, putting the culture medium in a constant-temperature incubator for upright culture for 1 hour at a constant temperature of 30 ℃, and then carrying out inverted culture until a single colony grows out. And (3) performing aseptic operation in a clean bench, picking a single colony by using an aseptic toothpick, putting the single colony in a 1.5ml EP tube filled with 1ml of LB liquid culture medium, immersing the single colony in the culture medium, discarding the toothpick, and covering the toothpick tightly to number. Culturing in a shaker at 30 ℃ and 180rpm until the bacterial liquid is turbid. Then mixing with 50% glycerol 1:1, and storing in a refrigerator at-80 deg.C for use.
(2) Re-screening:
activating strains: taking out the bacteria liquid preserved at-80 deg.c and thawing on ice. Aseptically processing in a clean bench, adding 800 μ l LB liquid culture medium and 200 μ l bacteria liquid into 1.5ml centrifuge tube, and mixing. Shaking at 30 deg.C and 180rpm until the liquid is cloudy.
Properly diluting the activated bacterial liquid by using secondary distilled water, sucking a proper amount of bacterial liquid, uniformly coating the bacterial liquid on a casein solid culture medium, putting the casein solid culture medium in a constant-temperature incubator, positively culturing for 1 hour at a constant temperature of 30 ℃, and then inversely culturing until a single bacterial colony generating a proteolytic loop grows out. Picking out single bacterial colony capable of generating proteolytic loop by using sterile toothpick, placing toothpick into 1.5ml EP tube filled with 1ml LB liquid culture medium, dropping single bacterial colony in culture medium, discarding toothpick, covering cap tightly and numbering again. Shaking culture at 30 deg.C and 180rpm until the bacterial liquid is turbid. Mixing with 50% glycerol 1:1, and storing in refrigerator at-80 deg.C.
Example 2: identification of strains
Taking out the bacteria liquid preserved at-80 deg.c, and thawing on ice. Aseptically processing in a clean bench, adding 800 μ l LB liquid culture medium and 200 μ l bacteria liquid into 1.5ml centrifuge tube, and mixing. The mixture was incubated in a shaker at 30 ℃ and 180rpm until the liquid became cloudy. Then, about 200. mu.l of the bacterial solution was put into a 1.5ml centrifuge tube, centrifuged at 5000rpm for 5min, the supernatant was discarded, 30. mu.l of DEPC sterilized water was added thereto, boiled for 10min, and rapidly put into ice to cool for 5 min. Centrifuging at 12000rpm for 5min at 4 ℃, taking the supernatant as a bacterial liquid PCR template, and carrying out PCR amplification by using primers 27F and 1492R. PCR reaction (10. mu.l): mu.l cDNA, 10 XBuffer 1. mu.l, 2.5mM dNTP 0.8. mu.l, 10. mu.M forward and reverse primers 0.5. mu.l each, 0.05. mu.l rTaq enzyme, 2H2O 6.15.15. mu.l.
PCR primers:
the PCR reaction conditions are as follows:
after the whole reaction was completed, the reaction product was detected by electrophoresis on 1.0% agarose gel. The target band was cut back under a gel imaging system, and DNA was recovered and purified according to the instructions of the Shanghai Biotech SanPrep column kit. DNA was recovered and stored at-20 ℃ or used for transformation ligation. And connecting the recovered product with a PMD18-T cloning vector, and connecting overnight at 16 ℃ to obtain a gene sequence PMD18-T recombinant vector of the target strain.
The linking system is as follows:
the product recovered was 4.5ul
PMD18-T 0.5ul
Solution I 5ul
The ligation products were all transferred into the prepared E.coli competent DH5 alpha, washed with crushed ice for 30min, heat-shocked at 42 ℃ for 30s, and placed on ice. 650. mu.L of LB medium which had been preheated to 37 ℃ were added and shake-cultured at 37 ℃ for 1 hour. Then, the mixture was centrifuged at 5000rpm for 3min, most of the supernatant was discarded, and the remaining 150. mu.L of the supernatant was mixed with the precipitate by pipetting, spread on an LB plate containing 100mg/mL ampicillin sodium (Amp), and cultured at 37 ℃ for 1 hour in an upright manner, followed by culturing for 12 to 16 hours in an inverted manner.
Single colonies growing well on the plate were picked, cultured at 37 ℃ for 6 hours, screened for positive clones with specific primers 27F and 1492R, and the correctly ligated bacterial solution was sent to Hipposony Biometrics for sequencing. And finally, performing Blast comparison on the cDNA sequence of the target strain to determine the strain species.
Example 3: conditions for activation of the strains
Inoculating the bacillus subtilis strain into an LB culture medium, and culturing for 8-10 hours at the temperature of 25-30 ℃ to obtain activated bacillus subtilis. The LB culture medium comprises the following components in percentage by weight: 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl.
Example 4: the bacillus subtilis is adopted to ferment the stichopus japonicus feed and feed the stichopus japonicus
(1) 200g of activated bacillus subtilis liquid (the bacteria content is 1.0 x 10)8cfu/ml-1.0*109cfu/ml), adding into 10kg of initial stichopus japonicus breeding feed, and uniformly mixing to obtain the mixed stichopus japonicus feed.
(2) Adding 22kg of water into 10kg of the mixed stichopus japonicus feed, and then fermenting for 12 hours at the temperature of 32 ℃ to obtain the fermented stichopus japonicus feed. The fermented stichopus japonicus feed is a slurry liquid and has fermentation fragrance.
(3) Feeding the stichopus japonicus: about 500 heads per 500g of stichopus japonicus with basically the same size are randomly put into 3 water body pools with the size of 15 cubic meters, and 5.0kg is put into each pool. The fermented stichopus japonicus feed and sea mud (floating mud on the sea bottom, which is a mixture of silt, few animal and plant debris, benthic diatoms and the like in seawater) are uniformly mixed according to the weight ratio of 1:5, and then the mixture is put into stichopus japonicus culture water.
The daily dosage is 3% of the total weight of the stichopus japonicus to be fed. Namely, 0.15kg of feed is fed to the pond at the beginning of the experiment, the condition of the residual feed is checked in the morning of the next day, and the feeding amount of the feed is adjusted according to the ingestion condition. After feeding for 60 days, weighing and counting the whole pool, and calculating the weight gain rate and the survival rate. The three pools weighed 20.6kg, 19.2kg and 18.8kg, respectively, with an average of 19.5 kg. The weight gains were 312%, 284% and 276%, respectively, and the average was 290%. The survival rates were 90%, 89% and 92%, respectively, with an average of 90.3%.
Control group 1 (not fed stichopus japonicus with the strain feed of the invention):
about 500 heads per 500g of stichopus japonicus with basically the same size are randomly put into 3 water body pools with the size of 15 cubic meters, and 5.0kg is put into each pool. Feeding common commercial feed at a feeding rate of 3% per day of Stichopus japonicus, i.e. feeding 0.15kg feed/pond at the beginning of the experiment, checking the residual material condition in the morning of the next day, and adjusting the feeding amount according to the ingestion condition. After feeding for 60 days, the whole pond is weighed, and the weights of the three ponds are respectively 15.2kg, 17.1kg and 16.6kg, and the average weight is 16.3 kg. The weight gain rates are 204%, 242% and 232% respectively, and the average is 226%; the survival rates were 87%, 85% and 82%, with an average of 84.7%.
Example 5: the bacillus subtilis is adopted to ferment the stichopus japonicus feed and feed the stichopus japonicus
In contrast to the embodiment 4, the process of the invention,
(1) adding 300g of activated bacillus subtilis liquid into 10kg of initial stichopus japonicus culture feed, and uniformly mixing to obtain a mixed stichopus japonicus feed;
(2) adding 30kg of water into 10kg of the mixed stichopus japonicus feed, and fermenting for 32 hours at the temperature of 20 ℃ to obtain the fermented stichopus japonicus feed. The fermented stichopus japonicus feed is a slurry liquid and has fermentation fragrance.
(3) Feeding the stichopus japonicus: the fermented stichopus japonicus feed and sea mud (floating mud on the sea bottom, which is a mixture of silt, few animal and plant debris, benthic diatoms and the like in seawater) are uniformly mixed according to the weight ratio of 1:6, and then the mixture is put into stichopus japonicus culture water.
The daily feeding amount is 2 percent of the total weight of the stichopus japonicus to be fed, namely 0.05kg of feed/pool is fed at the beginning of the experiment, the condition of residual materials is checked in the morning of the next day, and the feeding amount of the feed is adjusted according to the ingestion condition. After feeding for 60 days, weighing and counting the whole pool, and calculating the weight gain rate and the survival rate. The three pools weighed 19.1kg, 21.0kg and 20.5kg, respectively, with an average of 20.2 kg. The weight gains were 282%, 320% and 310%, respectively, and the average was 304%. The survival rates were 91%, 92% and 90%, respectively, with an average of 91.0%.
Control group 2 (not fed stichopus japonicus with the strain feed of the invention):
feeding common commercial feed at a feeding rate of 1%/day of Stichopus japonicus, i.e. feeding 0.05kg feed/pond at the beginning of the experiment, checking the residual material condition in the morning of the next day, and adjusting the feeding amount according to the ingestion condition. After feeding for 60 days, the whole pond is weighed, and the weights of the three ponds are respectively 17.4kg, 17.9kg and 16.1kg, and the average weight is 17.1 kg. The weight gains were 248%, 258% and 222%, respectively, and the average was 242.7%. The survival rates were 86%, 88% and 83%, with an average of 85.7%.
Example 6: the bacillus subtilis is adopted to ferment the stichopus japonicus feed and feed the stichopus japonicus
In contrast to the embodiment 4, the process of the invention,
(1) and (3) adding 500g of activated bacillus subtilis liquid into 10kg of initial stichopus japonicus culture feed, and uniformly mixing to obtain the mixed stichopus japonicus feed.
(2) Adding 15kg of water into 10kg of the mixed stichopus japonicus feed, and then fermenting for 26 hours at the temperature of 24 ℃ to obtain the fermented stichopus japonicus feed. The fermented stichopus japonicus feed is a slurry liquid and has fermentation fragrance.
(3) Feeding the stichopus japonicus: the fermented stichopus japonicus feed and sea mud (floating mud on the sea bottom, which is a mixture of silt, few animal and plant debris, benthic diatoms and the like in seawater) are uniformly mixed according to the weight ratio of 1:4, and then the mixture is put into stichopus japonicus culture water.
The daily feeding amount is 2 percent of the total weight of the stichopus japonicus to be fed, namely 0.1kg of feed/pool is fed when the experiment is started, the condition of residual materials is checked in the morning on the next day, and the feeding amount of the feed is adjusted according to the ingestion condition. After feeding for 60 days, weighing and counting the whole pool, and calculating the weight gain rate and the survival rate. The three pools weighed 20.6kg, 19.8kg and 19.7kg, respectively, with an average of 20.0 kg. The weight gain was 312%, 296% and 294%, respectively, and the average was 300.7%. The survival rates were 93%, 90% and 89%, respectively, with an average of 90.7%.
Control group 3 (not fed stichopus japonicus with the strain feed of the invention):
feeding common commercial feed at a feeding rate of 1%/day of Stichopus japonicus, i.e. feeding 0.05kg feed/pond at the beginning of the experiment, checking the residual material condition in the morning of the next day, and adjusting the feeding amount according to the ingestion condition. After feeding for 60 days, the whole pond is weighed, and the weights of the three ponds are respectively 17.5kg, 16.8kg and 16.9kg, and the average weight is 17.1 kg. The weight gains were 250%, 236%, and 238%, respectively, and averaged 241.3%. The survival rates were 83%, 82% and 86%, with an average of 83.7%.
Example 7: the bacillus subtilis is adopted to ferment the stichopus japonicus feed and feed the stichopus japonicus
(1) Taking 400g of activated bacillus subtilis liquid (the bacteria content is 1.0 x 10)8cfu/ml-1.0*109cfu/ml), adding into 10kg of initial stichopus japonicus breeding feed, and uniformly mixing to obtain the mixed stichopus japonicus feed.
(2) Adding 25kg of water into 10kg of the mixed stichopus japonicus feed, and fermenting for 18 hours at the temperature of 28 ℃ to obtain the fermented stichopus japonicus feed. The fermented stichopus japonicus feed is a slurry liquid and has fermentation fragrance.
(3) Feeding the stichopus japonicus: about 500 heads per 500g of stichopus japonicus with basically the same size are randomly put into 3 water body pools with the size of 15 cubic meters, and 5.0kg is put into each pool. The fermented stichopus japonicus feed and sea mud (floating mud on the sea bottom, which is a mixture of silt, few animal and plant debris, benthic diatoms and the like in seawater) are uniformly mixed according to the weight ratio of 1:3.5, and then the mixture is put into stichopus japonicus culture water.
The daily dosage is 3% of the total weight of the stichopus japonicus to be fed. Namely, 0.15kg of feed is fed to the pond at the beginning of the experiment, the condition of the residual feed is checked in the morning of the next day, and the feeding amount of the feed is adjusted according to the ingestion condition. After feeding for 60 days, weighing and counting the whole pool, and calculating the weight gain rate and the survival rate. The three pools weighed 20.1kg, 19.8kg and 18.9kg, respectively, with an average of 19.6 kg. The weight gains were 302%, 296% and 278%, respectively, and the average was 292%. The survival rates were 90%, 89% and 92%, respectively, with an average of 90.3%.
Control group 4 (not fed stichopus japonicus with the strain feed of the invention):
about 500 heads per 500g of stichopus japonicus with basically the same size are randomly put into 3 water body pools with the size of 15 cubic meters, and 5.0kg is put into each pool. Feeding common commercial feed at a feeding rate of 3% per day of Stichopus japonicus, i.e. feeding 0.15kg feed/pond at the beginning of the experiment, checking the residual material condition in the morning of the next day, and adjusting the feeding amount according to the ingestion condition. After feeding for 60 days, the whole pond is weighed, and the weights of the three ponds are respectively 15.4kg, 16.9kg and 16.3kg, and the average weight is 16.2 kg. The weight gain rates were 208%, 238% and 226%, respectively, with an average of 224%; the survival rates were 86%, 85% and 83%, with an average of 84.7%.
TABLE 1 examples 4-7 Effect of beneficial strains fermented feed on growth and survival of Stichopus japonicus
| Initial weight, kg | Terminal weight, kg | Weight gain ratio,% | Survival rate% | |
| Example 4 | 5.0 | 19.5 | 290.7 | 90.3 |
| Control group 1 | 5.0 | 16.3 | 226.0 | 84.7 |
| Example 5 | 5.0 | 20.2 | 304.0 | 91.0 |
| Control group 2 | 5.0 | 17.1 | 242.7 | 85.7 |
| Example 6 | 5.0 | 20.0 | 300.1 | 90.7 |
| Control group 3 | 5.0 | 17.1 | 241.3 | 83.7 |
| Example 7 | 5.0 | 19.6 | 292.0 | 90.3 |
| Control group 4 | 5.0 | 16.2 | 224.0 | 84.7 |
Example 8: sprinkling the bacterial liquid of the bacillus subtilis into a stichopus japonicus culture pond
(1) The Bacillus subtilis strain was activated as described in example 3.
(2) About 500 heads per 500g of stichopus japonicus with basically the same size are randomly put into 3 water body pools with the size of 15 cubic meters, and 5.0kg is put into each pool. The activated bacterial liquid (the bacterial content is 1.0 x 10)8cfu/ml-1.0*109cfu/ml) is firstly diluted by 10 times by seawater in the culture pond, and then is evenly put into the stichopus japonicus culture pond. The use concentration is 5-10PPM, the supplementary feeding is carried out once every 5 days, and the supplementary feeding is carried out once after each pond pouring (namely, the stichopus japonicus in the pond and the attaching base of the stichopus japonicus are moved to a culture pond after being cleaned and disinfected). After being fed by common unfermented feed for 60 days, the weight is respectively 18.3kg, 17.6kg and 18.1kg, and the average weight is 18.0 kg; the weight gain rates were 266%, 252%, and262%, average 260%; the survival rates were 89%, 91% and 90%, respectively, with an average of 90.0%.
Control group 5: (e.g., control for fermented feed)
About 500 heads per 500g of stichopus japonicus with basically the same size are randomly put into 3 water body pools with the size of 15 cubic meters, and 5.0kg is put into each pool. Feeding common commercial feed at a feeding rate of 2%/day of Stichopus japonicus, i.e. feeding 0.1kg feed/pond at the beginning of the experiment, checking the residual material condition in the morning of the next day, and adjusting the feeding amount according to the ingestion condition. After feeding for 60 days, weighing the whole pond, wherein the weights of the three ponds are respectively 16.2kg, 17.2kg and 17.1kg, and the average weight is 16.8 kg; the weight gain rates were 224%, 244% and 242%, respectively, with an average of 236.7%; the survival rates were 86%, 84% and 83%, with an average of 84.3%.
Taking out water samples from the culture pond sprayed with beneficial bacteria and the control pond respectively, measuring the COD values of the water samples by using an alkaline potassium permanganate method in 3 days, 5 days and 7 days respectively, and recording experimental data. And analyzing the capability of the composite microbial inoculum for degrading organic matters in the culture water through the measured experimental data. Degradation rate is (initial COD value-final COD value)/initial COD value. The degradation rates of the organic matters are respectively 7.4%, 6.8% and 6.2% in 3 days, and the average degradation rate is 6.8%; 19.3 percent, 16.5 percent and 17.4 percent at 5 days respectively, and the average is 17.7 percent; at 7 days, 38.6%, 37.5% and 36.9%, respectively, averaged 37.7%.
TABLE 2 influence of beneficial strains splashing on water body on growth, survival rate and degradation rate of organic matter in water body
As can be seen from Table 1, Stichopus japonicus feed was fermented with Bacillus subtilis and fed with Stichopus japonicus; the weight gain rate is 290.7-304.0% after 60 days, and the survival rate is 90.3-91.7%; the weight gain rate of the stichopus japonicus in the control group is 224.0-242.7%, and the survival rate is 83.7-85.7%. Compared with a control group, the weight gain rate of the stichopus japonicus fed by the stichopus japonicus feed fermented by the bacillus subtilis is increased by 61.6 percent, and the survival rate is increased by nearly 6 percent. As can be seen from table 2, the bacterial liquid of bacillus subtilis is sprinkled in the stichopus japonicus culture pond, and the stichopus japonicus is fed by adopting a common culture feed, after 60 days, the weight gain rate of the stichopus japonicus is 260%, and the survival rate is 90%; the weight gain rate of the stichopus japonicus in the control group (without splashing the bacterial liquid) is 236.7%, and the survival rate is 84.3%. Compared with a control group, the stichopus japonicus culture pond has the advantages that the weight gain rate is increased by 23.3 percent and the survival rate is increased by 5.7 percent by sprinkling the bacillus subtilis liquid to the stichopus japonicus in the stichopus japonicus culture pond.
In conclusion, the bacillus subtilis is used for raising the stichopus japonicus, can accelerate the growth speed of the stichopus japonicus, and improves the survival rate of the stichopus japonicus.
In addition, the bacterial liquid of the bacillus subtilis is splashed into the stichopus japonicus culture pond, and the degradation rate of organic matters is 6.8% when 3 days occur; 17.7% at 5 days; 37.7% in 7 days; while the control group could not degrade the organic substances. The bacillus subtilis liquid sprayed into the stichopus japonicus culture pond can degrade residual feed and protein in excrement in water and purify water quality, so that the survival rate of stichopus japonicus is improved, good economic benefit is generated, and the stichopus japonicus culture pond has a good promotion effect on the development of stichopus japonicus industry.
Sequence listing
<110> Qingdao agricultural university
<120> protease-producing Bacillus subtilis and methods of use
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1573
<212>DNA
<213> Bacillus subtilis
<400>1
tgcagcttgc tgcctgcagg tcgacgatta gagtttgatc ctggctcagg acgaacgctg 60
gcggcgtgcc taatacatgc aagtcgagcg gacagatggg agcttgctcc ctgatgttag 120
cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa 180
accggggcta ataccggatg gttgtttgaa ccgcatggtt caaacataaa aggtggcttc 240
ggctaccact tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc 300
aaggcaacga tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 360
cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg 420
agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa 480
caagtaccgt tcaaataggg cggtaccttg acggtaccta accagaaagc cacggctaac 540
tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt 600
aaagggctcg caggcggttt cttaagtctg atgtgaaagc ccccggctta accggggagg 660
gtcattggaa actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg 720
tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact 780
gacgctgagg agcgaaagcg tggggagcga acaggaatag ataccctggt agtccacgcc 840
gtaaacgatg agtgctaagt gttagggggt ttccgcccct tagtgctgca gctaacgcat 900
taagcactcc gcctggggag tacggtcgca agactgaaac tcaaagggat tgacgggggc 960
ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 1020
ttgacatcct ctgacaatcc tagagatagg acgtcccctt cgggggcaga gtgacaggtg 1080
gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1140
acccttgatc ttagttgcca gcattcagtt gggcactcta aggtgactgc cggtgacaaa 1200
ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg 1260
tgctacaatg gacagagcaa agggcagcga aaccgcgagg ttaagccaat cccacaaatc 1320
tgttctcagt tcggatcgca gtctgcaact cgactgcgtg aagctggaat cgctagtaat 1380
cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1440
cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt ttaggagcca gccgccgaag 1500
gtgggacaga tgattggggt gaagtcgtaa caaggtaacc aatctctaga ggatccccgg 1560
taccgagctc gaa 1573
Claims (10)
1. A strain of Bacillus subtilis producing protease is characterized in that: the Bacillus subtilis is named as Bacillus subtilis DB005(Bacillus subtilis) The microbial inoculum is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.15646 and the preservation date of 2018, 4 months and 26 days.
2. The Bacillus subtilis of claim 1, wherein: the sequence of the Bacillus subtilis DB 00516 SrDNA is shown in SEQ NO. 1.
3. The method for activating Bacillus subtilis according to claim 1, wherein: the Bacillus subtilis of claim 1, which is inoculated into a culture medium and cultured at a temperature of 25 to 30 ℃ for 8 to 10 hours.
4. The use of bacillus subtilis according to claim 1 wherein: the bacillus subtilis is applied to the cultivation of stichopus japonicus.
5. The use of Bacillus subtilis according to claim 4, wherein: the application of the bacillus subtilis in the cultivation of the stichopus japonicus comprises two methods: (1) fermenting the stichopus japonicus feed by using the bacillus subtilis; (2) and sprinkling the bacterial liquid of the bacillus subtilis into the stichopus japonicus culture pond.
6. The use of Bacillus subtilis according to claim 5, wherein: the method (1) specifically comprises the following steps: (A) activating the bacillus subtilis strain, then taking a proper amount of the bacillus subtilis strain, putting the bacillus subtilis strain into the initial stichopus japonicus breeding feed, and uniformly mixing to obtain a mixed stichopus japonicus breeding feed; (B) adding a proper amount of water into the mixed stichopus japonicus breeding feed, and fermenting for a period of time under a proper temperature condition to obtain a final product, namely the fermented stichopus japonicus feed.
7. The use of Bacillus subtilis according to claim 6, wherein: the weight fraction of the bacillus subtilis strain in the step (A) is 2-5%; the adding amount of water in the step (B) is 1.5-3 times of the weight of the initial stichopus japonicus breeding feed; the temperature is 20-32 ℃, and the fermentation time is 12-32 hours.
8. The use of Bacillus subtilis according to claim 5, wherein: the method (2) specifically comprises the following steps: uniformly putting the activated bacterial liquid into a stichopus japonicus culture pond, wherein the concentration of the bacterial liquid is 5-10 PPM; and periodically replenished to maintain that concentration based on consumption.
9. A Stichopus japonicus cultivation feed comprising the Bacillus subtilis of claim 1, wherein: the stichopus japonicus breeding feed is prepared by the method (1) of claim 6 or claim 7.
10. The use method of the stichopus japonicus breeding feed as claimed in claim 9, wherein the feed comprises the following steps: uniformly mixing the stichopus japonicus breeding feed and sea mud according to the weight ratio of 1:4-1:6, and then putting the mixture into stichopus japonicus breeding water; the daily dosage is 1-3% of the total weight of the sea cucumber to be fed.
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