CN104397358A - Application of bacillus pumilus and immunopotentiator powdery preparation - Google Patents
Application of bacillus pumilus and immunopotentiator powdery preparation Download PDFInfo
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- CN104397358A CN104397358A CN201410752560.2A CN201410752560A CN104397358A CN 104397358 A CN104397358 A CN 104397358A CN 201410752560 A CN201410752560 A CN 201410752560A CN 104397358 A CN104397358 A CN 104397358A
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Abstract
The invention relates to application of a bacillus pumilus and immunopotentiator powdery preparation to preparation of biological agents for preventing and treating aquatic bacterial diseases. The bacillus pumilus and immunopotentiator powdery preparation comprises effective components of bacillus pumilus and an immunopotentiator, wherein the bacillus pumilus is a powdery bacterial agent prepared through solid fermentation or liquid fermentation of bacillus pumilus with the collection number of CCTCC M 2013240; the viable count of the bacillus pumilus is 109-1011cfu/g; the immunopotentiator comprises vitamin C, astragalus membranaceus, eucommia, codonopsis pilosula, Chinese angelica, honeysuckle, radix bupleuri, black plum, pomegranate peel and fructus schisandrae. The bacillus pumilus and immunopotentiator powdery preparation has the advantages as follows: the bacillus pumilus and immunopotentiator powdery preparation can significantly improve the resistance of fishes to infection of aeromonas hydrophila, enhance the body immunity of cultured fishes, significantly inhibit the growth of intestinal pathogenic bacteria of the fishes, promote the rapid growth of the fishes and improve the weight gain rate and the disease resistance of the cultured fishes.
Description
Technical field
The present invention relates to microorganism field, specifically, is the application of a kind of bacillus pumilus and immunopotentiator powderous preparations.
Background technology
Bacteriosis is one of major reason of puzzlement culture fishery development always.In recent years, the intensive production model of high density is while promotion aquatic products industry improves output, and the chance also making bacterial disease occur increases, frequency increases, harm increasing; In addition, antibiotic usage causes drug resistance, medicament residue and the destruction to ecological environment also become the focus of social concerns.Therefore, find antibiotic alternative medicine, explore new controlling mode and become aquatic products disease control research one of important directions.
Quorum sensing (Quorum sensing is called for short QS) refers to the bacterium spontaneous generation of energy and discharges some specific signaling molecules, the change of perception self concentration, and then the expression of regulation and control related gene.At present, regulating the signaling molecule of bacterial community induction to mainly contain three kinds, (a) N-acyl homoserine lactones (AHLs), is the signaling molecule of the quorum sensing be extensively present in gramnegative bacterium; (b) autologous induction peptide (Autoinducing peptides, AIP), AIP is a class short peptide molecules, is signaling molecule common in gram-positive bacterium; C () auto-inducer class Ⅱmolecule (Auto inducing II, AI2) is representative by the signaling molecule of luxS enzymatic AI 2 system with Fermi operator (Vibrio fischeri).Research shows, in gramnegative bacterium, as pseudomonas aeruginosa (Pseudomonas aeruginosa), Aeromonas hydrophila (Aeromonas hydrophila) etc., the behaviors such as the formation of its biofilm, harmful toxins generation, virulence factor release are all subject to the adjustment of AHLs class signaling molecule.The people such as Dong (2000) have found aiiA gene in bacillus cereus (Bacillus cereus), can synthesize AHLs signaling molecule digestive enzyme, block the transmission of QS signaling molecule between bacterium; Yin Shouliang etc. (2009) filter out a strain from Jiaozhou Bay and can to degrade the fungi of AHLs signaling molecule, show that marine microorganism also can as the potential research object of QS inhibitor; Bhavanath etc. (2013) find that the crude extract of Japanese yew shape Asparagus toxiformis (Delile) Coll et Harv (Asparagopsis taxiformis) can be degraded AHLs signaling molecule, and have certain inhibitory action to aeruginosa biofilm.Defoirdt etc. (2011) have isolated the bacillus that vibrio harveyi (Vibrio harveyi) QS signaling molecule can be suppressed to transmit first from the enteron aisle of the aquatic livestock such as Penaeus Vannmei and marine seabass, Natrah etc. (2011) isolate micro-algae that a strain can suppress AHLs signaling molecule, this micro-algae can block vibrio harveyi QS system, reduces its pathogenicity.These researchs are inquire into the support providing theoretical foundation and science data from quorum sensing angle prevention and control aquatic products bacteriosis.Bacillus (Bcaillus sp) has the features such as easy production, just transport, storage endurance, aquaculture can be played purify water, the effect such as disease preventing and treating and growth promoting effects, be conventional strains of probiotic microorganism.
Chinese patent literature CN103525723A discloses one and has the inhibiting bacillus pumilus of intervention school-based, has the effect of anti-bacteria quorum sensing, can be used for the microorganism formulation preparing control aquatic products bacterial disease.But about a kind of have significantly control aquatic products bacterial disease, improve the bacillus pumilus of survival rate and the application of immunopotentiator powderous preparations yet there are no report.
Summary of the invention
The object of the invention is, for deficiency of the prior art, to provide the application of a kind of bacillus pumilus and immunopotentiator powderous preparations.
For achieving the above object, the technical scheme that the present invention takes is: the application in the biologic product of preparation control aquatic products bacterial disease of bacillus pumilus and immunopotentiator powderous preparations, the active ingredient of described powderous preparations is made up of bacillus pumilus and immunopotentiator, the powdery microbial inoculum that the bacillus pumilus of described bacillus pumilus to be deposit number be CCTCC M 2013240 is made through solid state fermentation or liquid fermentation, wherein bacillus pumilus viable count is 10
9-10
11cfu/g, described immunopotentiator is vitamin C, the Radix Astragali, the bark of eucommia, Radix Codonopsis, Radix Angelicae Sinensis, honeysuckle, radix bupleuri, dark plum, granatum and the fruit of Chinese magnoliavine, and in described powderous preparations, the weight portion of bacillus pumilus and immunopotentiator is respectively: bacillus pumilus 14-16 part, vitamin C 10-12 part, Radix Astragali 7-8 part, bark of eucommia 4-5 part, Radix Codonopsis 2-4 part, Radix Angelicae Sinensis 3-5 part, honeysuckle 6-8 part, radix bupleuri 9-10 part, dark plum 3-5 part, granatum 4-6 part, fruit of Chinese magnoliavine 9-10 part.
Preferably, in described powderous preparations, the weight portion of bacillus pumilus and immunopotentiator is respectively: bacillus pumilus 16 parts, vitamin C 12 parts, the Radix Astragali 8 parts, the bark of eucommia 5 parts, Radix Codonopsis 4 parts, Radix Angelicae Sinensis 5 parts, honeysuckle 8 parts, radix bupleuri 10 parts, dark plum 5 parts, granatum 6 parts, 10 parts, the fruit of Chinese magnoliavine.
Preferably, described solid-state fermentation culture medium is by expecting to form with water, and described material, by gross mass mark 100%, comprises rice bran 10%, wheat bran 25%, dregs of beans 32%, corn flour 30%, yeast extract 2%, NaCl 1%, and material-water ratio is 1:1.
Preferably, described bacillus pumilus is made powdery microbial inoculum through solid state fermentation or liquid fermentation by the preparation method of described powderous preparations, and in described powdery microbial inoculum, bacillus pumilus viable count is 10
9-10
11cfu/g, after described powdery microbial inoculum, vitamin C, the Radix Astragali, the bark of eucommia, Radix Codonopsis, Radix Angelicae Sinensis, honeysuckle, radix bupleuri, dark plum, granatum and the fruit of Chinese magnoliavine were pulverized 100 mesh sieves respectively, according to weight mixing, obtains described powderous preparations.
Preferably, described powderous preparations uses as feeding additive aquatic animal or aquaculture water body additives.
Preferably, described aquatic products is freshwater fish.
Preferably, the addition of described powderous preparations in feed is 1%-3%.
The invention has the advantages that:
Powderous preparations of the present invention is for preventing and treating aquatic products bacterial disease; to the immune protective rate of fish up to 73-90%; the infection ability of fish body opposing Aeromonas hydrophila can be significantly improved; strengthen the immunity of organisms of cultured fishes; the growth of remarkable suppression fish intestines pathogenic bacteria; promote that fish body grows fast, improve rate of body weight gain and the premunition of cultured fishes.
Detailed description of the invention
Below in conjunction with embodiment, detailed description of the invention provided by the invention is elaborated.
The invention provides bacillus pumilus and the application of immunopotentiator powderous preparations in the biologic product of preparation control aquatic products bacterial disease, the active ingredient of described powderous preparations is made up of bacillus pumilus and immunopotentiator, the powdery microbial inoculum that the bacillus pumilus of described bacillus pumilus to be deposit number be CCTCC M 2013240 is made through solid state fermentation or liquid fermentation, wherein bacillus pumilus viable count is 10
9-10
11cfu/g, described immunopotentiator is vitamin C, the Radix Astragali, the bark of eucommia, Radix Codonopsis, Radix Angelicae Sinensis, honeysuckle, radix bupleuri, dark plum, granatum and the fruit of Chinese magnoliavine, and in described powderous preparations, the weight portion of bacillus pumilus and immunopotentiator is respectively: bacillus pumilus 14-16 part, vitamin C 10-12 part, Radix Astragali 7-8 part, bark of eucommia 4-5 part, Radix Codonopsis 2-4 part, Radix Angelicae Sinensis 3-5 part, honeysuckle 6-8 part, radix bupleuri 9-10 part, dark plum 3-5 part, granatum 4-6 part, fruit of Chinese magnoliavine 9-10 part.
Described bacillus pumilus (Baillus pumilus), depositary institution: China typical culture collection center (CCTCC), address: Wuhan University's school of life and health sciences preservation center, preservation date on June 2nd, 2013, deposit number is CCTCC NO:M 2013240.Confirm that it has intervention school-based inhibitory action.
Described bacillus pumilus is made powdery microbial inoculum through solid state fermentation or liquid fermentation by the preparation method of described powderous preparations, and in described powdery microbial inoculum, bacillus pumilus viable count is 10
9-10
11cfu/g, after described powdery microbial inoculum, vitamin C, the Radix Astragali, the bark of eucommia, Radix Codonopsis, Radix Angelicae Sinensis, honeysuckle, radix bupleuri, dark plum, granatum and the fruit of Chinese magnoliavine were pulverized 100 mesh sieves respectively, according to weight mixing, obtains described powderous preparations.
Embodiment 1 bacillus pumilus liquid fermentation
Short and small bud pole bacteria strain is pressed the order cultivation of inclined-plane, liquid seeds, liquid fermentation, slant medium is conventional nutrient agar medium, and liquid seed culture medium is conventional nutrient meat soup fluid nutrient medium; Liquid fermentation medium formula (W/V): glucose 2%, beancake powder 8%, corn steep liquor 1%, MgSO
47H
2o0.02%, NaCl 0.4%, KH
2pO
40.1%, MnCl
20.04%, fermentation temperature 30 DEG C, aerlbic culture 24-48h.Obtain the zymocyte liquid of high concentration, wherein viable count 10
9-10
11cfu/g.By zymocyte liquid centrifugal segregation zymotic fluid, obtain wet bacterium mud, after adding hat resistant pretectants, spraying dry, obtains solid, powdery microbial inoculum, viable count 10
9-10
11cfu/g.
Embodiment 2 bacillus pumilus solid state fermentation
Short and small bud pole bacteria strain is pressed the order cultivation of inclined-plane, liquid seeds, solid state fermentation, slant medium is conventional nutrient agar medium, and liquid seed culture medium is conventional nutrient meat soup fluid nutrient medium.Be inoculated in solid-state fermentation culture medium with the inoculum concentration of 5-10% by seed culture fluid and carry out solid state fermentation, fermentation time is 24-48h, fermentation temperature 30 DEG C, and tunning obtains bacillus pumilus powdery microbial inoculum after drying and pulverizing.Wherein viable count 10
9-10
11cfu/g.
Wherein solid-state fermentation culture medium is by expecting to form with water, and described material, by gross mass mark 100%, comprises rice bran 10%, wheat bran 25%, dregs of beans 32%, corn flour 30%, yeast extract 2%, NaCl 1%, and material-water ratio is 1:1.
The preparation of embodiment 3 powderous preparations
Described bacillus pumilus is made powdery microbial inoculum through solid state fermentation, and in described powdery microbial inoculum, bacillus pumilus viable count is 10
9-10
11cfu/g, by described powdery microbial inoculum 16 parts, vitamin C 12 parts, the Radix Astragali 8 parts, the bark of eucommia 5 parts, Radix Codonopsis 4 parts, Radix Angelicae Sinensis 5 parts, honeysuckle 8 parts, radix bupleuri 10 parts, dark plum 5 parts, granatum 6 parts, 10 parts, the fruit of Chinese magnoliavine.After pulverizing 100 mesh sieves respectively, according to weight mixing, obtain described powderous preparations.
The preparation of embodiment 4 powderous preparations
Described bacillus pumilus is made powdery microbial inoculum through liquid fermentation, and in described powdery microbial inoculum, bacillus pumilus viable count is 10
9-10
11cfu/g, by described powdery microbial inoculum 14 parts, vitamin C 10 parts, the Radix Astragali 7 parts, the bark of eucommia 4 parts, Radix Codonopsis 2 parts, Radix Angelicae Sinensis 3 parts, honeysuckle 6 parts, radix bupleuri 9 parts, dark plum 3 parts, granatum 4 parts, 9 parts, the fruit of Chinese magnoliavine.After pulverizing 100 mesh sieves respectively, according to weight mixing, obtain described powderous preparations.
The preparation of embodiment 5 powderous preparations
Described bacillus pumilus is made powdery microbial inoculum through solid state fermentation, and in described powdery microbial inoculum, bacillus pumilus viable count is 10
9-10
11cfu/g, by described powdery microbial inoculum 15 parts, vitamin C 11 parts, the Radix Astragali 7 parts, the bark of eucommia 4 parts, Radix Codonopsis 3 parts, Radix Angelicae Sinensis 4 parts, honeysuckle 7 parts, radix bupleuri 10 parts, dark plum 4 parts, granatum 5 parts, 10 parts, the fruit of Chinese magnoliavine.After pulverizing 100 mesh sieves respectively, according to weight mixing, obtain described powderous preparations.
The water splashing effect experimental of embodiment 6 powderous preparations
Crucian, buys from freshwater fish culturing center, Xuzhou, body weight 100 ± 0.5g, feeds in advance 10 days, without any Aeromonas hydrophila symptom.Feed is bought from Ningbo Tianbang Co., Ltd, feeds according to 3% of body weight.
First raise and train before the test and within one week, determine to close Aeromonas hydrophila symptom without appointing, then get the crucian 90 of body weight (100 ± 2.3) g, be divided into three groups often to organize three repetitions at random, often organize 10, cultivation, at 700 × 500 × 50mm, in volume 90L aquarium, keeps water temperature 16 ~ 20 DEG C.
Experimental design three groups, totally 90, often organize 10, three repetition, control group: in water body, adding the Aeromonas hydrophila bacterium liquid 100mL of cultivation, (finally bacterial concentration reaches 10 after dilution
8cfu/mL, formerly experiment determines that the absolute lethal concentration of Aeromonas hydrophila is 1 × 10
8cfu/mL), test group adds the powderous preparations 10g of the present invention that Aeromonas hydrophila bacterium liquid 100mL and embodiment 3 prepare, and blank group does not add anything in blank water body.Change water every three days, add the running water of the gas that exposed to the sun, and the bacterium liquid again cultivated, the upgrowth situation of record crucian and death toll.
In the breeding cycle of two weeks, blank group without any the phenomena of mortality; The all samples of control group are all dead in 9 days, and add the test group of Aeromonas hydrophila and powderous preparations of the present invention in water body, and after seven days, reach stable, final survival rate is 90%.
Embodiment 7 powderous preparations growth promotion and challenge viral dosage
Growth experiment: the crucian that test selects health, specification, weight basically identical, individual just counterpoise is about 50g.Random packet, be provided with control group, test one group, test two groups, test three groups and test four groups, often group establishes 3 parallel tests, every parallel group 50 tail test fish, totally 6 net cages (specification is 250cm × 150cm × 100cm).After crucian domestication 30d, control group is thrown something and fed normal diet, test the feed of one group of interpolation 3.0% percentage by weight powderous preparations of the present invention (embodiment 3) of throwing something and feeding, test the feed of two groups of interpolation 1.0% percentage by weight powderous preparations of the present invention of throwing something and feeding, test the feed of three groups of interpolation 3.0% percentage by weight bacillus pumilus of the present invention (not comprising the powderous preparations of immunopotentiator) of throwing something and feeding, test the feed of four groups of interpolation 3.0% percentage by weight immunopotentiators (not comprising the immunopotentiator of bacillus pumilus) of throwing something and feeding, raise 56d, measure the impact of its growth.
Challenge viral dosage: control group, test one group, test two groups, test three groups and test four groups often group randomly draw 15 tail crucians.1 × 10
8cfu/mL Aeromonas hydrophila, by every 100g fish heavy lumbar injection bacterium liquid 1.0ml, after put in the cement pit net cage of same specification volume (specification is 100cm × 100cm × 100cm) in a suitable place to breed, and ensure sufficient oxygen, observe fish body death condition every day, observe 10d, statistics survival rate.
Concrete result of the test sees the following form 1.
The contrast of table 1 three groups of rates of body weight gain and the death rate
| Control group | Test one group | Test two groups | Test three groups | Test four groups | |
| Rate of body weight gain | 5.02％ | 15.48％ | 10.25％ | 8.34％ | 9.15％ |
| Survival rate | 0 | 86％ | 73％ | 61％ | 53％ |
Result of the test shows, in feed, add the powderous preparations of the present invention of 3.0%, 1.0% test one group and test two groups of rates of body weight gain reach 15.48% and 10.25%, compared with control group, and P < 0.05.After lumbar injection Aeromonas hydrophila 10d, control group crucian is all dead, and the test one group of powderous preparations of the present invention of feeding and test two groups of final survival rates are respectively 86% and 73%, compared with control group, and P < 0.01.Simultaneously, the immune indexes such as superoxide dismutase activity, lysozyme content, antibacterial activity tested in one group and test two groups of crucian serum is all significantly higher than control group (P < 0.05), and the quantity of the pathogenic bacteria such as the total number of bacteria in enteron aisle, Escherichia coli, Aeromonas hydrophila is significantly lower than control group (P < 0.05).Rate of body weight gain and the survival rate of testing one group are also significantly higher than the test three groups and test four groups that are used alone bacillus pumilus or immunopotentiator, show powderous preparations of the present invention to use simultaneously there is the inhibiting bacillus pumilus of intervention school-based and immunopotentiator, fish body anti-infection ability can be significantly improved, strengthen the immunity of organisms of cultured fishes, promote that fish body grows fast simultaneously.
Above experimental result display; powderous preparations of the present invention to the immune protective rate of fish up to 73-90%; the infection ability of fish body opposing Aeromonas hydrophila can be significantly improved; strengthen the immunity of organisms of cultured fishes; the growth of remarkable suppression fish intestines pathogenic bacteria; promote that fish body grows fast, improve rate of body weight gain and the premunition of cultured fishes.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (7)
1. bacillus pumilus and the immunopotentiator powderous preparations application in the biologic product of preparation control aquatic products bacterial disease, the active ingredient of described powderous preparations is made up of bacillus pumilus and immunopotentiator, the powdery microbial inoculum that the bacillus pumilus of described bacillus pumilus to be deposit number be CCTCC M 2013240 is made through solid state fermentation or liquid fermentation, wherein bacillus pumilus viable count is 10
9-10
11cfu/g, described immunopotentiator is vitamin C, the Radix Astragali, the bark of eucommia, Radix Codonopsis, Radix Angelicae Sinensis, honeysuckle, radix bupleuri, dark plum, granatum and the fruit of Chinese magnoliavine, and in described powderous preparations, the weight portion of bacillus pumilus and immunopotentiator is respectively: bacillus pumilus 14-16 part, vitamin C 10-12 part, Radix Astragali 7-8 part, bark of eucommia 4-5 part, Radix Codonopsis 2-4 part, Radix Angelicae Sinensis 3-5 part, honeysuckle 6-8 part, radix bupleuri 9-10 part, dark plum 3-5 part, granatum 4-6 part, fruit of Chinese magnoliavine 9-10 part.
2. application according to claim 1, it is characterized in that, in described powderous preparations, the weight portion of bacillus pumilus and immunopotentiator is respectively: bacillus pumilus 16 parts, vitamin C 12 parts, the Radix Astragali 8 parts, the bark of eucommia 5 parts, Radix Codonopsis 4 parts, Radix Angelicae Sinensis 5 parts, honeysuckle 8 parts, radix bupleuri 10 parts, dark plum 5 parts, granatum 6 parts, 10 parts, the fruit of Chinese magnoliavine.
3. application according to claim 1, is characterized in that, described solid-state fermentation culture medium is by expecting to form with water, and described material is by gross mass mark 100%, comprise rice bran 10%, wheat bran 25%, dregs of beans 32%, corn flour 30%, yeast extract 2%, NaCl 1%, material-water ratio is 1:1.
4. application according to claim 1, is characterized in that, described bacillus pumilus is made powdery microbial inoculum through solid state fermentation or liquid fermentation by the preparation method of described powderous preparations, and in described powdery microbial inoculum, bacillus pumilus viable count is 10
9-10
11cfu/g, after described powdery microbial inoculum, vitamin C, the Radix Astragali, the bark of eucommia, Radix Codonopsis, Radix Angelicae Sinensis, honeysuckle, radix bupleuri, dark plum, granatum and the fruit of Chinese magnoliavine were pulverized 100 mesh sieves respectively, according to weight mixing, obtains described powderous preparations.
5. according to the arbitrary described application of claim 1-4, it is characterized in that, described powderous preparations uses as feeding additive aquatic animal or aquaculture water body additives.
6. application according to claim 5, is characterized in that, described aquatic products is freshwater fish.
7. application according to claim 5, is characterized in that, the addition of described powderous preparations in feed is 1%-3%.
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Cited By (1)
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| CN107223776A (en) * | 2017-07-07 | 2017-10-03 | 芜湖市木子生态园有限公司 | A kind of green and healthy trionyx sinensis feed additive |
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Application publication date: 20150311 |