CN105368749A - Bacillus subtilis and feed additive containing same - Google Patents
Bacillus subtilis and feed additive containing same Download PDFInfo
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- CN105368749A CN105368749A CN201510891728.2A CN201510891728A CN105368749A CN 105368749 A CN105368749 A CN 105368749A CN 201510891728 A CN201510891728 A CN 201510891728A CN 105368749 A CN105368749 A CN 105368749A
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- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
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- Enzymes And Modification Thereof (AREA)
- Fodder In General (AREA)
- Feed For Specific Animals (AREA)
Abstract
The invention relates to the technical field of function microorganism screening and particularly provides novel bacillus subtilis SY12 with the preservation number being CCTCC No: M2015483. The bacillus subtilis SY12 has the advantages that the bacillus subtilis SY12 is capable of effectively inhibiting pathogenic bacteria such as vibrio harveyi and vibrio anguillarum, improving immunity of aquaculture animals and reducing diseases; the bacillus subtilis SY12 with high proteinase-producing and amylase producing capabilities can serve as a feed additive, so that digestive ability of the aquaculture animals is improved remarkably, feed use ratio is increased remarkably, and animal growth is promoted.
Description
Technical field
The present invention relates to functional microorganism triage techniques field, be specifically related to a bacillus subtilis and comprise the fodder additives of this bacterial strain.
Background technology
Along with the increase of fishery products demand and the international development of aquatic products trade, culture fishery development in recent years is advanced by leaps and bounds.But the intensive and high-density scale expanding day of aquaculture, the deterioration of breeding environment and the harm of disease bring huge financial loss to culture fishery.The method of tradition disease preventing and treating mainly uses microbiotic, and antibiotic usage easily produces endurance strain problem, and there is problem residual in aquatic animal body, and therefore finding Substitutes For Antibiotic becomes inevitable.
Probiotic bacterium can participate in microbial balance in animal body, directly by strengthening animal to the restraining effect of intestinal toxic microflora, or carry out preventing disease by strengthening non-specific immune function, and then indirectly play the effect promoting growth of animal, improve the transformation efficiency of animal-feed.Therefore, in recent years the screening of probiotic bacterium was become to the study hotspot in this area, also the improvement of aquatic feeds formula was had great importance.
Summary of the invention
The object of this invention is to provide the novel subtilis of a strain and comprise the feeding additive aquatic animal of this bacterial strain.Described subtilis (Bacillussubtillis) screening, from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds, effectively can suppress pathogenic bacteria, and improve immunizing power and the disease resistance of aquatic animal, promote aquatic animal to grow, application prospect is extensive.
One aspect of the present invention provides a bacillus subtilis SY12 (BacillussubtillisSY12), be preserved in the China typical culture collection center of Wuhan, China Wuhan University on August 10th, 2015, its deposit number is CCTCCNO:M2015483.
The present invention provides the fodder additives comprising above-mentioned subtilis on the other hand.
Described fodder additives also comprises one or more the combination in polysaccharide, VITAMIN, herbal medicine, enzyme or emulsifying agent.
Present invention also offers the application of above-mentioned subtilis in aquaculture.
Beneficial effect of the present invention:
The subtilis SY12 that the present invention screens, can effectively suppress the pathogenic bacteria such as Vibrio harveyi and Vibrio anguillarum, improve the immunizing power of cultivated animals, reduce the generation of disease, simultaneously this bacterial strain produce proteolytic enzyme and diastatic ability all stronger, can be used as fodder additives, significantly improve the digestion ability of cultivated animals and the utilization ratio to feed, promote growth of animal.
Described subtilis SY12 can be used as probiotic bacterium and is applied in the breeding production of grass carp and Penaeus vannamei.Compared with control group, the rate of body weight gain of the experimental group grass carp of the subtilis SY12 of the present invention that feeds improves 26.8%; And the enzyme work of N,O-Diacetylmuramidase, alkaline phosphatase, glutamic-oxal(o)acetic transaminase, gpt improves 4.1%, 10.5%, 3.9%, 80.4% respectively in experimental group Grass Carp Serum; In experimental group grass carp digestive tube, the activity of proteolytic enzyme, amylase and lipase improves 23.2%, 70.1%, 6.2% respectively.; Feed the composite additive comprising subtilis SY12 experimental group in the rate of body weight gain of Penaeus vannamei and specific growth rate improve 17.3% and 9.4% respectively; And blood cell count and respiratory burst vigor improve 38.4% and 14.7% respectively in experimental group prawn blood; In experimental group prawn serum, the enzyme activity of phenol oxidase, superoxide-dismutase and nitricoxide synthase improves 42.9%, 3.8% and 21.4% respectively; In experimental group prawn digestive system, proteolytic enzyme and diastatic enzyme activity improve 62.5% and 71.8% respectively, all achieve significant technique effect.
Embodiment
The separation of embodiment 1 bacterial strain, selection systems
1, sample
Be collected in culture of Penaeus vannamei field, Jiangzhou, Qingdao City culturing pool water sample.
2, substratum:
1) 2216E sea water medium: peptone 5g, yeast extract 1g, tertiary iron phosphate 0.01g, seawater 1000ml, pH7.6-7.8, preparation solid medium adds the agar of 15-20g, 121 DEG C of sterilizing 20min.
2) TSB substratum: pancreas peptone soybean broth 30g, sodium-chlor 15g, distilled water 1000ml, pH7.4-7.6, preparation solid medium adds the agar of 15-20g, 121 DEG C of sterilizing 20min.
3) MRS substratum: peptone 10g, extractum carnis 10g, glucose 20g, yeast powder 5g, tween-80 lml, potassium primary phosphate 2g, dibasic ammonium citrate 2g, sodium acetate 5g, magnesium sulfate 0.58g, manganous sulfate 0.15g, distilled water 1000ml, pH6.2-6.4, adds the agar of 15-20g to prepare solid medium, 115 DEG C of sterilizing 20min.
4) Starch Hydrolysis substratum: peptone 10g, extractum carnis 5g, sodium-chlor 5g, Zulkovsky starch 2g, distilled water 1000ml, pH7.4-7.6,115 DEG C of sterilizing 20min.
5) casein hydrolyzing culture medium: solution A: yeast extract paste 3g, peptone 10g, agar 10g, seawater 1000ml; Solution B: casein 10g, distilled water 250ml; Solution C: agar 10g, distilled water 250ml; Respectively by three kinds of solution, 121 DEG C of sterilizing 20min, first by B liquid and the mixing of C liquid after cooling, pour dull and stereotyped formation skim into, after thin layer solidifies, pour A liquid into, make double-layer plate.
3, screening method
Get 1ml water sample, with stroke-physiological saline solution 10 times of gradient dilutions, be inoculated on 2216E, TSB and MRS solid medium respectively, cultivate 24h-48h for 28 DEG C.Choose even single bacterium colony clearly, line purification of bacterial.Single bacterium colony called after SY1, SY2, SY3 according to this ..., SY16.
With pathogenic bacterium---Vibrio harveyi and Vibrio anguillarum are indicator, adopt dull and stereotyped antagonism method to screen probiotic bacterium.The Bacteria liquid that dibbling is to be screened on the flat board scribbling indicator, 28 DEG C of constant temperature culture, observe around dibbling district whether occur antibacterial clear area or coverage area in 48h.The restraining effect of pathogenic bacteria is expressed as to the ratio of antibacterial circle diameter and colony diameter, 1 does not have restraining effect, and 1-2 represents to have certain restraining effect, and >2 represents that bacteriostatic action is stronger.Concrete outcome is in table 1
The antibacterial situation of the potential probiotic strain of table 1
As can be seen from the data of table 1,5 strain bacterium in the bacterial strain that the present invention screens, are only had all to have certain restraining effect to Vibrio harveyi and Vibrio anguillarum, wherein the strongest with the bacteriostasis of bacterial strain SY12.
This 5 pathogen strain bacterium Antagonistic Fungi of SY1, SY2, SY5, SY8 and SY12 filtered out is distinguished dibbling on Starch Hydrolysis substratum and ferment hydrolyzing culture medium, observes with or without hydrolysis after 28 DEG C of constant temperature culture 48h form obvious bacterium colony.Enzymatic productivity is expressed as the ratio of transparent circle diameter and colony diameter, and 1 expression does not produce enzyme, and 1-2 represents that enzymatic productivity is general, and >2 represents that enzymatic productivity is stronger.Concrete outcome is in table 2
The potential probiotic strain enzymatic productivity of table 2
As can be seen from the data of table 2, in the 5 pathogen strain bacterium Antagonistic Fungis that the present invention screens, only have SY8 and SY12 product proteolytic enzyme and diastatic ability all comparatively strong, and wherein the strongest with the comprehensive enzymatic productivity of SY12 bacterial strain.
4, identification of strains
1) colony morphology characteristic: above-mentioned bacterial strains SY12 is again streak culture, its bacterium colony is rendered as flats, and canescence is opaque, edge sawtooth shape.
2) the genome SYA of above-mentioned bacterial strains SY12 is extracted, utilize round pcr amplification 16SrRNA sequence, by order-checking BLAST compare of analysis, its 16SrRNA sequence similarity with the many bacillus subtilis to have announced is up to 99%, identify and confirm that bacterial strain SY12 is subtilis (Bacillussubtillis), consistent with biochemical identification result.
3) by above-mentioned bacterial strains SY12 called after subtilis SY12 (BacillussubtillisSY12), be preserved in the China typical culture collection center of Wuhan, China Wuhan University on August 10th, 2015, its deposit number is CCTCCNO:M2015483.
Embodiment 2: subtilis SY12 on grass carp immunity and growth impact
Choose healthy grass carp, be divided into 2 groups at random---control group and experimental group, often organize 3 repetitions, each repetition 30 tail grass carp (original body mass 50.86 ± 2.35).Control group fed basal feed, the feed of experimental group adds 10 in basal feed
7cFU/g subtilis SY12.Every day to throw something and feed bait by 3% of grass carp body weight, and every day, 08:00,12:00 and 19:00 divided three bait throwing in, and the cycle is 45d.Experiment carry out in indoor circulation water system, period water temperature be 23-25 DEG C, pH7.4-7.8, dissolved oxygen is not less than 7.5mg/L.
Stop raising 24h after raising experiment terminates, eachly repeat to randomly draw 10 tail fishes and detect.Tail bone venous blood collection, the centrifugal 15min of 4000r/min after blood sample 4 DEG C of standing 24h, supernatant liquor is for detecting serum lysozyme, alkaline phosphatase, glutamic-oxal(o)acetic transaminase and gpt activity.Anatomy experiment fish gets its enteron aisle, rejects fat and alimentary canal content, and with glass homogenizer homogenate in ice bath, 4 DEG C of centrifugal 10min of 10000r/min subsequently, the supernatant liquor of acquisition measures digestive enzyme activity.Concrete outcome is in table 3.
Table 3 subtilis SY12 is on the impact of Growth of Grass Carps Ctenopharyngodon Idellus, immunizing power and digestive enzyme
| Project | Control group | Experimental group |
| Rate of body weight gain % | 17.42±0..68 a | 22.09+1.36 b |
| N,O-Diacetylmuramidase U/L | 972.12±14.25 b | 1012.32±12.36 b |
| Alkaline phosphatase U/L | 61.21±3.62 a | 67.65±4.23 b |
| Glutamic-oxal(o)acetic transaminase U/L | 69.26±5.34 | 71.98±5.46 |
| Gpt U/L | 9.23±1.46 a | 16.65±2.13 b |
| Protease activity U/mg Protein | 23.996±1.124 a | 29.569±1.147 b |
| Amylase activity U/mg Protein | 58.455±2.192 a | 99.432±2.420 b |
| Lipase activity U/mg Protein | 25.537±1.595 | 27.115±0.570 |
Note: different letter representation significant differences (P < 0.05).
As can be seen from the data of table 3, compared with control group, the rate of body weight gain of the experimental group grass carp of the subtilis SY12 of the present invention that feeds improves 26.8%; And the enzyme work of N,O-Diacetylmuramidase, alkaline phosphatase, glutamic-oxal(o)acetic transaminase, gpt improves 4.1%, 10.5%, 3.9%, 80.4% respectively in experimental group Grass Carp Serum; In experimental group grass carp digestive tube, the activity of proteolytic enzyme, amylase and lipase improves 23.2%, 70.1%, 6.2% respectively.Thus illustrate, immunity and the digestive function of grass carp can be significantly improved by adding subtilis SY12 in feed, and then greatly facilitate Growth of Grass Carps Ctenopharyngodon Idellus.
Embodiment 3: the composite additive comprising subtilis SY12 is on the impact of Penaeus vannamei growth, immunity and digestion
Control group and experimental group are established in experiment, often organize three repetitions, each repetition 30 tail Penaeus vannamei.With fish meal, dregs of beans and peanut meal for major protein source, fish oil and soybean lecithin are primary fat source preparation basal feed, and this formula can meet the nutritional need of prawn growth.Control group fed basal feed, the feed of experimental group adds 10 in basal feed
9cFU/g subtilis SY12,500mg/kg dextran and 5000U/kg proteolytic enzyme.
Experiment is carried out in circulating water system, continues 8 weeks.Every day to throw something and feed bait by the 5%-10% of prawn body weight, and throw something and feed every day 3 times, Feeding time is respectively 6:00,11:00,18:00, then according to every day prawn situation of ingesting suitably adjust.Continuous charge between feeding period, water temperature 24-28 DEG C, salinity 30-32, pH7.8-8.2, dissolved oxygen is not less than 6.5mg/L.
After culture experiment terminates, take out 6 tail shrimps at random from each repetition, according to the ratio of hemolymph and antithrombotics 1:2, carry out getting blood from abdomen blood sinus; An anticoagulation part is directly used in count blood cells sum and measures respiratory burst activity; Another part 4 DEG C, the centrifugal 10min of 700 × g, gained supernatant liquor is used for the enzyme activity determination of serum phenol oxidase, superoxide-dismutase and nitricoxide synthase.Meanwhile, get 10 tail prawns at random from each experimental group, get its enteron aisle, remove enteron stool, deionized water rinsing, filter paper blots, and weighs, homogenate at 0 DEG C, then 4 DEG C of centrifugal 30min (10000r/min), get supernatant liquor and measure proteolytic enzyme and amylase activity respectively.Concrete outcome is in table 4
The composite additive that table 4 comprises subtilis SY12 is on the impact of Penaeus vannamei growth and immunizing power
| Project | Control group | Experimental group |
| Rate of body weight gain % | 116.89±1.99 a | 137.08±6.73 b |
| Specific growth rate % | 1.17±0.01 a | 1.28±0.02 b |
| Blood cell count 10 7cell/ml | 1.74±0.072 a | 2.408±0.095 b |
| Respiratory burst vigor OD630/10 6cells | 1.415±0.038 a | 1.623±0.047 b |
| Phenoloxidase Activities U/ml | 50.783±2.219 a | 72.594±3.978 b |
| Superoxide dismutase activity U/ml | 41.805±0.643 | 43.395±0.469 |
| Nitric oxide synthase U/ml | 4.843±0.088 a | 5.878±0.136 b |
| Proteinase activity U/mg Protein | 0.08±0.0012 a | 0.13±0.0043 b |
| Amylase activity U/mg Protein | 0.71±0.014 a | 1.22±0.057 b |
As can be seen from the data of table 4, compared with control group, in the experimental group of the composite additive comprising subtilis SY12 of feeding, the rate of body weight gain of Penaeus vannamei and specific growth rate improve 17.3% and 9.4% respectively; And blood cell count and respiratory burst vigor improve 38.4% and 14.7% respectively in experimental group prawn blood; In experimental group prawn serum, the enzyme activity of phenol oxidase, superoxide-dismutase and nitricoxide synthase improves 42.9%, 3.8% and 21.4% respectively; In experimental group prawn digestive system, proteolytic enzyme and diastatic enzyme activity improve 62.5% and 71.8% respectively.Thus illustrate, the composite additive that the present invention comprises subtilis SY12 effectively can promote the growth of Penaeus vannamei, significantly improves immunocompetence and the digestion ability of Penaeus vannamei coelomocyte.
To sum up, subtilis SY12 provided by the invention effectively can suppress pathogenic bacteria, improve animal immunizing power, reduce cultivated animals disease odds, simultaneously can also as fodder additives, improve cultivated animals digestion ability and the utilization ratio to feed, promote growth of animal, contribute to the economic benefit improving raiser.In addition, subtilis SY12 can with other materials, such as polyose immunostimulant, vitamins, herbal medicine etc. carry out composite, be applied in the feed of the aquatic animal such as fish, shrimp crab, cultivated animals principal immune desired value 7-12% can be improved, improve complete period survival rate 15-20%, improve growth rate 13-17%, reduce feed coefficient 5-10%, have a extensive future.
Claims (4)
1. a subtilis, its deposit number is CCTCCNO:M2015483.
2. one kind comprises the fodder additives of subtilis according to claim 1.
3. fodder additives as claimed in claim 2, it is characterized in that, described fodder additives also comprises one or more the combination in polysaccharide, VITAMIN, herbal medicine, enzyme or emulsifying agent.
4. the application of subtilis according to claim 1.
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| CN108977387A (en) * | 2018-08-08 | 2018-12-11 | 广东绿百多生物开发有限公司 | A kind of bacterial strain for fermented feed |
| CN110029074A (en) * | 2019-03-15 | 2019-07-19 | 河南科技大学 | A kind of bacillus subtilis and its application in raising fish and shrimp disease prevention and cure |
| CN111690558A (en) * | 2020-05-31 | 2020-09-22 | 青岛玛斯特生物技术有限公司 | Bacillus subtilis strain and application thereof in aquaculture |
| CN112239735A (en) * | 2020-09-18 | 2021-01-19 | 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 |
| CN116286447A (en) * | 2022-10-09 | 2023-06-23 | 集美大学 | Bacillus subtilis and application thereof |
| CN117487719A (en) * | 2023-11-25 | 2024-02-02 | 山东省海洋科学研究院(青岛国家海洋科学研究中心) | Bacillus with vibrio-resistant activity separated from intestinal tracts of prawns |
| CN117683687A (en) * | 2023-12-19 | 2024-03-12 | 中国热带农业科学院热带生物技术研究所 | Bacillus-W2Z, application thereof and feed |
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| CN110029074A (en) * | 2019-03-15 | 2019-07-19 | 河南科技大学 | A kind of bacillus subtilis and its application in raising fish and shrimp disease prevention and cure |
| 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 |
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| CN111690558A (en) * | 2020-05-31 | 2020-09-22 | 青岛玛斯特生物技术有限公司 | Bacillus subtilis strain and application thereof in aquaculture |
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| 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 |
| CN116286447A (en) * | 2022-10-09 | 2023-06-23 | 集美大学 | Bacillus subtilis and application thereof |
| CN117487719A (en) * | 2023-11-25 | 2024-02-02 | 山东省海洋科学研究院(青岛国家海洋科学研究中心) | Bacillus with vibrio-resistant activity separated from intestinal tracts of prawns |
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