CN105368749B - One bacillus subtilis and feed addictive comprising the bacterial strain - Google Patents
One bacillus subtilis and feed addictive comprising the bacterial strain Download PDFInfo
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- CN105368749B CN105368749B CN201510891728.2A CN201510891728A CN105368749B CN 105368749 B CN105368749 B CN 105368749B CN 201510891728 A CN201510891728 A CN 201510891728A CN 105368749 B CN105368749 B CN 105368749B
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Abstract
The present invention relates to functional microorganism screening technique fields, specifically provide one plant of novel bacillus subtilis SY12 (Bacillus subtillis SY12), and deposit number is CCTCC NO：M2015483.The bacillus subtilis can effectively inhibit the pathogens such as Vibrio harveyi and Vibrio anguillarum, improve the immunity of cultivated animals, reduce the generation of disease, the ability of bacterial strain production protease and amylase is relatively strong simultaneously, it can be used as feed addictive, the digestion power of cultivated animals and the utilization rate to feed are significantly improved, growth of animal is promoted.
Description
Technical field
The present invention relates to functional microorganism screening technique fields, and in particular to a bacillus subtilis and includes the bacterial strain
Feed addictive.
Background technology
With the increase of aquatic products demand and the international development of aquatic products trade, culture fishery develops prominent in recent years
Fly to push ahead vigorously.But the intensive and high density scale of aquaculture is growing, the deterioration of breeding environment and the harm of disease are given
Culture fishery has brought tremendous economic losses.The method of traditional disease preventing and treating mainly uses antibiotic, antibiotic usage
It is also easy to produce endurance strain problem, and there are problems that residual in aquatic livestock body, therefore finds Substitutes For Antibiotic to become
It is inevitable.
Probiotics can participate in microbial balance in animal body, directly by enhancing animal to intestinal toxic microbiologic population
Inhibiting effect, or prevent disease by enhancing non-specific immune function, and then play the work for promoting growth of animal indirectly
With improving the conversion ratio of animal feed.Therefore, research hotspot in the art is become for the screening of probiotics in recent years,
Have great importance to the improvement of aquatic feeds formula.
Invention content
The object of the present invention is to provide one plant of novel bacillus subtilis and include the feeding additive aquatic animal of the bacterial strain.Institute
Bacillus subtilis (Bacillus subtillis) is stated screened from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds, can effectively inhibit pathogen,
The immunity and premunition of aquatic livestock are improved, promotes aquatic livestock growth, application prospect extensive.
One aspect of the present invention provides a bacillus subtilis SY12 (Bacillus subtillis SY12), in
The China typical culture collection center for being preserved in Wuhan, China Wuhan University on the 10th of August in 2015, deposit number CCTCC
NO:M2015483。
Another aspect of the present invention provides the feed addictive for including above-mentioned bacillus subtilis.
The feed addictive further includes one or more groups in polysaccharide, vitamin, Chinese herbal medicine, enzyme or emulsifier
It closes.
The present invention also provides application of the above-mentioned bacillus subtilis in aquaculture.
Beneficial effects of the present invention：
The bacillus subtilis SY12 that the present invention screens can effectively inhibit the pathogens such as Vibrio harveyi and Vibrio anguillarum, carry
The immunity of high cultivated animals reduces the generation of disease, while the ability of bacterial strain production protease and amylase is relatively strong, can make
For feed addictive, the digestion power of cultivated animals and the utilization rate to feed are significantly improved, promotes growth of animal.
The bacillus subtilis SY12 can be used as probiotics applied in the breeding production of grass carp and Penaeus Vannmei.With
Control group is compared, and the rate of body weight gain for feeding the experimental group grass carp of bacillus subtilis SY12 of the present invention improves 26.8%；And
Lysozyme in experimental group Grass Carp Serum, alkaline phosphatase, glutamic-oxalacetic transaminease, glutamic-pyruvic transaminase enzyme activity be respectively increased 4.1%,
10.5%, 3.9%, 80.4%；The activity of protease, amylase and lipase is respectively increased in experimental group grass carp alimentary canal
23.2%, 70.1%, 6.2%.；Penaeus Vannmei in the experimental group of compound additive of the feeding comprising bacillus subtilis SY12
Rate of body weight gain and specific growth rate 17.3% and 9.4% has been respectively increased；And blood cell count and breathing in experimental group prawn blood
38.4% and 14.7% has been respectively increased in outburst vigor；Phenol oxidase, superoxide dismutase and one in experimental group prawn serum
The enzyme activity of nitric oxide synthase has been respectively increased 42.9%, 3.8% and 21.4%；Protease and shallow lake in experimental group prawn digestive system
62.5% and 71.8% has been respectively increased in the enzyme activity of powder enzyme, achieves significant technique effect.
Specific implementation mode
Separation, screening and the identification of 1 bacterial strain of embodiment
1, sample
It is collected in Qingdao City Jiangzhou culture of Penaeus vannamei field aquaculture pond water sample.
2, culture medium：
1) 2216E sea water mediums：Peptone 5g, yeast extract 1g, ferric phosphate 0.01g, seawater 1000ml, pH7.6-
7.8, prepare the agar that 15-20g is added in solid medium, 121 DEG C of sterilizing 20min.
2) TSB culture mediums：Pancreas peptone soybean broth 30g, sodium chloride 15g, distilled water 1000ml, pH7.4-7.6 are prepared
The agar of 15-20g, 121 DEG C of sterilizing 20min are added in solid medium.
3) MRS culture mediums：Peptone 10g, beef extract 10g, glucose 20g, yeast powder 5g, Tween-80 lml, di(2-ethylhexyl)phosphate
Hydrogen potassium 2g, dibasic ammonium citrate 2g, sodium acetate 5g, magnesium sulfate 0.58g, manganese sulfate 0.15g, distilled water 1000ml, pH6.2-6.4,
The agar of 15-20g is added to prepare solid medium, 115 DEG C of sterilizing 20min.
4) Starch Hydrolysis culture medium：Peptone 10g, beef extract 5g, sodium chloride 5g, soluble starch 2g, distilled water
1000ml, pH7.4-7.6,115 DEG C of sterilizing 20min.
5) casein hydrolyzing culture medium：Solution A：Yeast extract 3g, peptone 10g, agar 10g, seawater 1000ml；Solution B：Junket
Protein 10 g, distilled water 250ml；Solution C：Agar 10g, distilled water 250ml；It is cold respectively by 121 DEG C of three kinds of solution sterilizing 20min
But first B liquid and C liquid are mixed after, tablet is poured into and forms a thin layer, A liquid is poured into after thin layer solidification, double-layer plate is made.
3, screening technique
1ml water samples are taken, with 10 times of gradient dilutions of sterile saline, are inoculated in 2216E, TSB and MRS solid culture respectively
On base, 28 DEG C are cultivated -48h for 24 hours.Uniform clearly single bacterium colony is chosen, purification of bacterial of crossing.Single bacterium colony be named as according to this SY1,
SY2、SY3……、SY16。
Using pathogenic bacteria --- Vibrio harveyi and Vibrio anguillarum screen probiotics as indicator bacteria, using tablet antagonism method.It is being coated with
Whether dibbling bacterial solution to be screened on the tablet of indicator bacteria, 28 DEG C of constant temperature incubations, 48h press down around interior observation dibbling area
Bacterium clear area or the area of coverage.The ratio of antibacterial circle diameter and colony diameter is expressed as to the inhibiting effect of pathogen, 1 is not pressed down
It making and uses, 1-2 indicates there is certain inhibiting effect,>2 indicate that bacteriostasis is stronger.Concrete outcome is shown in Table 1
1 potential probiotic strain antibacterial situation of table
It is equal to Vibrio harveyi and Vibrio anguillarum that only 5 plants of bacterium in the bacterial strain that the present invention screens are can be seen that from the data of table 1
With certain inhibiting effect, wherein most strong with the bacteriostasis of bacterial strain SY12.
SY1, SY2, SY5, SY8 and SY12 for filtering out this 5 pathogen strain bacterium Antagonistic Fungi difference dibbling are trained in Starch Hydrolysis
It supports on base and ferment hydrolyzing culture medium, whether there is or not hydrolysis for observation after 28 DEG C of constant temperature incubation 48h form apparent bacterium colony.Producing enzyme energy
Power is expressed as the ratio of transparent loop diameter and colony diameter, and 1 indicates not generate enzyme, and 1-2 indicates that enzymatic productivity is general,>2 indicate production
Enzyme ability is stronger.Concrete outcome is shown in Table 2
2 potential probiotic strain enzymatic productivity of table
Only SY8 and SY12 in the 5 pathogen strain bacterium Antagonistic Fungis that the present invention screens is can be seen that from the data of table 2 to lay eggs
The ability of white enzyme and amylase is relatively strong, and wherein most strong with the synthesis enzymatic productivity of SY12 bacterial strains.
4, bacterial strain is identified
1) colony morphology characteristic：Above-mentioned bacterial strains SY12 to be crossed culture again, bacterium colony is rendered as flat, canescence,
It is opaque, edge sawtooth shape.
2) the genome SYA of extraction above-mentioned bacterial strains SY12 expands 16SrRNA sequences using round pcr, passes through sequencing
BLAST compares analysis, is up to 99% with the 16S rRNA sequence similarities for the more bacillus subtilis announced, identification card
Real bacterial strain SY12 is bacillus subtilis (Bacillus subtillis), consistent with biochemical identification result.
3) above-mentioned bacterial strains SY12 is named as bacillus subtilis SY12 (Bacillus subtillis SY12), in
The China typical culture collection center for being preserved in Wuhan, China Wuhan University on the 10th of August in 2015, deposit number CCTCC
NO:M2015483。
Embodiment 2：Bacillus subtilis SY12 is to the influence that grass carp is immune and grows
Healthy grass carp is chosen, is randomly divided into 2 groups --- control group and experimental group, every group of 3 repetitions are each to repeat 30 tails grass
Fish (original body mass 50.86 ± 2.35).Control group fed basal feed, the feed of experimental group are added in basal feed
107CFU/g bacillus subtilises SY12.Daily bait is fed by the 3% of grass carp weight, daily 08:00、12:00 and 19:00 point
Bait throwing in three times, period 45d.It tests and is carried out in circulation indoors, during which water temperature is 23-25 DEG C, pH7.4-7.8, molten
It solves oxygen and is not less than 7.5mg/L.
Stop to raise after raising experiment for 24 hours, each repetition is randomly selected 10 tail fishes and is detected.Tail bone venous blood collection, blood
4000r/min centrifuges 15min after 4 DEG C of sample is stood for 24 hours, and supernatant turns ammonia for detecting serum lysozyme, alkaline phosphatase, millet straw
Enzyme and gpt activity.Anatomy experiment fish takes its enteron aisle, fat and alimentary canal content is rejected, with glass homogenizer in ice
It is homogenized in bath, subsequent 4 DEG C of 10000r/min centrifuge 10min, and the supernatant of acquisition measures digestive enzyme activity.Concrete outcome is shown in Table 3.
Influences of the 3 bacillus subtilis SY12 of table to Growth of Grass Carps Ctenopharyngodon Idellus, immunity and digestive enzyme
| Project | Control group | Experimental group |
| Rate of body weight gain % | 17.42±0..68a | 22.09+1.36b |
| Lysozyme U/L | 972.12±14.25b | 1012.32±12.36b |
| Alkaline phosphatase U/L | 61.21±3.62a | 67.65±4.23b |
| Glutamic-oxalacetic transaminease U/L | 69.26±5.34 | 71.98±5.46 |
| Glutamic-pyruvic transaminase U/L | 9.23±1.46a | 16.65±2.13b |
| Proteinase activity U/mg Protein | 23.996±1.124a | 29.569±1.147b |
| Amylase activity U/mg Protein | 58.455±2.192a | 99.432±2.420b |
| Lipase active U/mg Protein | 25.537±1.595 | 27.115±0.570 |
Note：Different letters indicate significant difference (P ＜ 0.05).
It can be seen that the experiment for feeding bacillus subtilis SY12 of the present invention compared with the control group from the data of table 3
The rate of body weight gain of group grass carp improves 26.8%；And lysozyme, alkaline phosphatase, glutamic-oxalacetic transaminease, paddy in experimental group Grass Carp Serum
The enzyme activity of pyruvic transaminase has been respectively increased 4.1%, 10.5%, 3.9%, 80.4%；Protease, shallow lake in experimental group grass carp alimentary canal
The activity of powder enzyme and lipase has been respectively increased 23.2%, 70.1%, 6.2%.To illustrate, by adding withered grass in feed
Bacillus SY12 is remarkably improved the immune and digestive function of grass carp, and then Growth of Grass Carps Ctenopharyngodon Idellus is greatly facilitated.
Embodiment 3：Including the compound additive of bacillus subtilis SY12 grows Penaeus Vannmei, is immune and digestion
It influences
Experiment sets control group and experimental group, and every group of three repetitions are each to repeat 30 tail Penaeus Vannmeis.With fish meal, dregs of beans
It is main protein sources with peanut meal, fish oil and soybean lecithin are that main fat source prepares basal feed, which can meet
The nutritional need of prawn growth.Control group fed basal feed, the feed of experimental group are that 10 are added in basal feed9CFU/g
Bacillus subtilis SY12,500mg/kg glucan and 5000U/kg protease.
Experiment carries out in circulation, continues 8 weeks.Bait is fed by the 5%-10% of prawn weight daily, daily
It feeds 3 times, Feeding time is respectively 6:00,11:00,18:00, then suitably adjusted according to the situation of ingesting of daily prawn
It is whole.Continuous charge during raising, 24-28 DEG C of water temperature, salinity 30-32, pH7.8-8.2, dissolved oxygen is not less than 6.5mg/L.
After culture experiment, 6 tail shrimps are taken out at random from each repetition, according to hemolymph and anti-coagulants 1:2 ratio,
It carries out taking blood from abdomen blood sinus；An anticoagulation part is directly used in count blood cells sum and measures respiratory burst activity；Another portion
Divide 4 DEG C, 700 × g centrifuges 10min, and gained supernatant is used for serum phenol oxidase, superoxide dismutase and nitricoxide synthase
Enzyme activity determination.Meanwhile 10 tail prawns are taken at random from each experimental group, its enteron aisle is taken, enteron stool, deionized water punching are removed
It washes, filter paper blots, and weighs, and is homogenized at 0 DEG C, and then 4 DEG C of centrifugation 30min (10000r/min), take supernatant to measure albumen respectively
Enzyme and amylase activity.Concrete outcome is shown in Table 4
Table 4 includes the compound additive of bacillus subtilis SY12 to Penaeus Vannmei growth and the influence of immunity
| Project | Control group | Experimental group |
| Rate of body weight gain % | 116.89±1.99a | 137.08±6.73b |
| Specific growth rate % | 1.17±0.01a | 1.28±0.02b |
| Blood cell count 107cell/ml | 1.74±0.072a | 2.408±0.095b |
| Respiratory burst vigor OD630/106cells | 1.415±0.038a | 1.623±0.047b |
| Phenoloxidase Activities U/ml | 50.783±2.219a | 72.594±3.978b |
| Superoxide dismutase activity U/ml | 41.805±0.643 | 43.395±0.469 |
| Nitric oxide synthase U/ml | 4.843±0.088a | 5.878±0.136b |
| Prolease activity U/mg Protein | 0.08±0.0012a | 0.13±0.0043b |
| Amylase activity U/mg Protein | 0.71±0.014a | 1.22±0.057b |
It can be seen that compared with the control group from the data of table 4, feeding includes the compound additive of bacillus subtilis SY12
Experimental group in Penaeus Vannmei rate of body weight gain and specific growth rate 17.3% and 9.4% has been respectively increased；And experimental group prawn
38.4% and 14.7% has been respectively increased in blood cell count and respiratory burst vigor in blood；Phenol aoxidizes in experimental group prawn serum
The enzyme activity of enzyme, superoxide dismutase and nitricoxide synthase has been respectively increased 42.9%, 3.8% and 21.4%；Experimental group
62.5% and 71.8% has been respectively increased in the enzyme activity of protease and amylase in prawn digestive system.To illustrate, packet of the present invention
The compound additive of the SY12 containing bacillus subtilis can effectively facilitate the growth of Penaeus Vannmei, significantly improve Penaeus Vannmei body
The immunocompetence and digestion power of chamber cell.
To sum up, bacillus subtilis SY12 provided by the invention can effectively inhibit pathogen, improve animal immunizing power, subtract
Few cultivated animals disease odds, while it can also be used as feed addictive, improve cultivated animals digestion power and to feed
Utilization rate, promote growth of animal, help to improve the economic benefit of raiser.In addition, bacillus subtilis SY12 can be with
Other substances, such as polysaccharide immunopotentiator, vitamins, Chinese herbal medicine etc. are compounded, and the aquatic products such as fish, shrimp crab are applied to
In the feed of animal, cultivated animals principal immune index value 7-12% can be improved, complete period survival rate 15-20% is improved, carries
Seedling height rate 13-17% reduces feed coefficient 5-10%, has a extensive future.
Claims (3)
1. a kind of bacillus subtilis (Bacillus subtilis), deposit number is CCTCC NO:M 2015483.
2. a kind of feed addictive including bacillus subtilis described in claim 1.
3. feed addictive as claimed in claim 2, which is characterized in that the feed addictive further includes polysaccharide, dimension life
One or more combinations in element, Chinese herbal medicine, enzyme or emulsifier.
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| CN110029074B (en) * | 2019-03-15 | 2020-12-29 | 河南科技大学 | Bacillus subtilis and application thereof in prevention and treatment of fish and shrimp culture diseases |
| CN111690558B (en) * | 2020-05-31 | 2022-04-08 | 青岛玛斯特生物技术有限公司 | Bacillus subtilis strain and application thereof in aquaculture |
| CN112239735B (en) * | 2020-09-18 | 2022-05-24 | 华中农业大学 | Bacillus subtilis, microbial inoculum, screening method and application |
| CN112715749A (en) * | 2020-11-11 | 2021-04-30 | 广西海洋研究所有限责任公司 | Trachinotus ovatus feed additive capable of improving immunity |
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