CN102559533B - Bacillus atrophaeus, and preparation and application of bacillus atrophaeus - Google Patents
Bacillus atrophaeus, and preparation and application of bacillus atrophaeus Download PDFInfo
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Abstract
The invention discloses a bacillus atrophaeus strain Aj080319IA-12. The bacillus atrophaeus strain is characterized by being separated from an intestinal tract of a wild stichopus japonicus selenka and collected in China Center for Type Culture Collection (CCTCC) along with a collection number of CCTCC M 2010316. The screened bacillus atrophaeus has remarkable antagonistic action on pathogenic bacteria of a sea cucumber, and has the effects of increasing the growth speed, immunity and intestinal digestive enzyme activity of the stichopus japonicus selenka. The screened bacillus atrophaeus is indigenous bacterium screened from the intestinal tract of the sea cucumber, which is safe and non-toxic for the sea cucumber, fast in breeding speed, easy to culture and strong in adaptability on environment, and is capable of being bred quickly to form predominant bacteria. A microbiological preparation, as a feed additive, can increase the specific growth rate of the sea cucumber, reduce the death rate of the sea cucumber, improve the immunity of the sea cucumber and the digestive enzyme activity of the intestinal tract, and improve the disease resistance of the sea cucumber at the same time, so that the preparation achieves the effects of resisting bacteria, immunizing and promoting growth, and is capable of reducing and replacing the use of antibiotics.
Description
Technical field
The present invention relates to aquatic products using microbe technical field, particularly a kind of atrophy genus bacillus and utilize this atrophy genus bacillus to produce microbial preparation and its preparation method and application.
Background technology
Beneficial bacteria of intestinal tract is that a kind of digestive tube that mostly derives from acts on again gastral active bacteria formulation, can effectively supplement the beneficial microorganism in animal digestive tract, suppress the growth of pathogenic bacteria, improve archenteric flora balance, improve immunity function or the speed of growth of aquatic animal.
Stichopus japonicus (Apostichopus japonicus Liao claims again imitative stichopus japonicus), belongs to invertebrates, Echinodermata, and Holothuroidea, imitative stichopus japonicus belongs to.Because it is of high nutritive value, cultivation scale constantly expands, and has developed into one of sea farming kind of China coast maximum at present.Relatively lagging behind due to stichopus japonicus high-density breeding and irregularoperation and disease control technology in recent years, disease problem is increasingly outstanding, especially breaking out of culturing stichopus japonicus " skin ulcer syndrome ", has caused serious financial loss, has restricted the healthy and sustainable development of this industry.Due to the special life habit of stichopus japonicus and cultivation feature, and there are again some shortcomings and limitations in the prophylaxis such as antibiotic medicine itself, therefore adopt probiotics to become, prevents one of pathogenetic effective measure of apostichopus japonicus culture disease.
Genus bacillus is current the most frequently used probiotic bacterium kind.The present invention is according to deriving from nature, the principle of back to nature, from sea cucumber enteron aisle, filter out a strain atrophy genus bacillus with antagonism stichopus japonicus pathogenic bacteria and high inulinase-producing activity, and be developed to probiotics, effectively avoid the difference between different sources probiotic strain, the security and the high efficiency that guarantee probiotics, have realistic meaning to sea cucumber healthy aquaculture.
According to deriving from nature, the principle of back to nature, screening is the preferred approach of exploitation probiotics with the bacterial classification that former host, former habitat, former Micro Ecosystem, pathogenic microorganism groupy phase adapt to.In the face of the situation of the special-purpose probiotics scarcity of current sea cucumber, from sea cucumber enteron aisle, screen probiotic bacterium and be developed to probiotics, avoided the difference between different strains, guarantee security and the high efficiency of probiotics.Therefore, separated indigenous probiotic bacterium from sea cucumber enteron aisle, by the compatibility of relevant bacterial strain is reached to antibacterial, immune, somatotrophic multiple effect, has realistic meaning to sea cucumber healthy aquaculture.
Summary of the invention
Technical problem to be solved by this invention is, the atrophy Bacillus strain that is adapted to sea cucumber enteron aisle is provided, and provides production method and the using method of said preparation in holothruian cultures of preparing microbial preparation with this bacterial strain.Microbial preparation of the present invention is as fodder additives, sea cucumber specific growth rate be can improve, reduce sea cucumber mortality ratio, the immunological competence of sea cucumber and the digestive enzyme activity of enteron aisle strengthened, improve the disease resistance of sea cucumber simultaneously, reached antibacterial, immune, growth promotion multiple effect, can reduce and replace antibiotic use, to improving cultivation surviving rate and quality product, ensure that the stable and high yields tool of this industry is of great significance.
For solving the problems of the technologies described above, the invention provides a kind of atrophy Bacillus strain Aj080319IA-12, described strains separation, in wild stichopus japonicus enteron aisle, is preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCC M 2010316.
For solving the problems of the technologies described above, the present invention also provides a kind of fodder additives, comprising: atrophy Bacillus strain Aj080319IA-12, and described strains separation is in wild stichopus japonicus enteron aisle, be preserved in Chinese Typical Representative culture collection center, preserving number is CCTCC M 2010316.
The preparation method of described fodder additives can comprise the following steps:
A. dull and stereotyped cultivation rejuvenation: the atrophy Bacillus strain Aj080319IA-12 of cryopreservation is inoculated on TSB solid plate substratum, in 20～35 ℃ of cultivation 12～36h, makes rejuvenation of spawn and form single bacterium colony; Then picking list colony inoculation, on TSB slant medium, is cultivated 12～36h in 20～35 ℃;
B. the preparation of seed liquor: the bacterial classification that steps A is cultivated makes 1 * 10 with the physiological saline of 5～30 ‰ sterilizings
5the bacteria suspension of CFU/mL, the inoculum size with 2～10% is inoculated in TSB liquid nutrient medium, and 20～35 ℃, 100～200rpm shaking culture, 18～20h, obtains seed liquor;
C. fermentation culture: the seed liquor in step B is inoculated in fermention medium with 2～10% inoculum size, 20～35 ℃, 100～200rpm shaking culture, 50～150h, to viable count be 0.2～8 * 10
9cFU/mL, stuck fermentation;
Component and the weight percent thereof of described fermention medium are respectively: glucose 1～5%, and soybean muffin 1～5%, sodium-chlor 1～3%, all the other are water;
D. the preparation of microbial preparation: add zeolite powder in the fermented liquid of step C, in every 100mL fermented liquid, add 5～30g zeolite powder, static 20～60min after 100～200rpm vibration, 20～60min, by above-mentioned fermented liquid in 4 ℃ of centrifugal 20min of 6000rpm, abandon supernatant, after adding 20～40g zeolite powder to mix in every 100g precipitation, in 30～60 ℃, dry 2～5h.
Described fodder additives is preferably the fodder additives for sea cucumber, and additive capacity can be 0.5 of holothurian feed gross weight～7 ‰.
Described fodder additives can be solid-state powderous preparations.
For solving the problems of the technologies described above, the present invention provides again a kind of microbial preparation, and the preparation method of described microbial preparation comprises the following steps:
A. dull and stereotyped cultivation rejuvenation: the inoculation of cryopreservation, on TSB solid plate substratum, in 22～33 ℃ of cultivation 14～38h, is made to rejuvenation of spawn and forms single bacterium colony; Then picking list colony inoculation, on TSB slant medium, is cultivated 14～36h in 22～33 ℃;
B. the preparation of seed liquor: the bacterial classification that steps A is cultivated makes 1 * 10 with the physiological saline of 5～30 ‰ sterilizings
5the bacteria suspension of CFU/mL, the inoculum size with 3～10% is inoculated in TSB liquid nutrient medium, and 20～35 ℃, 100～200rpm shaking culture, 18～22h, obtains seed liquor;
C. fermentation culture: the seed liquor in step B is inoculated in fermention medium with 3～10% inoculum size, 23～35 ℃, 100～200rpm shaking culture, 60～150h, to viable count be 0.2～8 * 10
9cFU/mL, stuck fermentation;
Component and the weight percent thereof of described fermention medium are respectively: glucose 1～5%, and soybean muffin 1～5%, sodium-chlor 1～3%, all the other are water;
D. the preparation of microbial preparation: add zeolite powder in the fermented liquid of step C, in every 100mL fermented liquid, add 6～30g zeolite powder, static 20～60min after 100～200rpm vibration, 20～60min, by above-mentioned fermented liquid in 4 ℃ of centrifugal 20min of 6000rpm, abandon supernatant, after adding 20～40g zeolite powder to mix in every 100g precipitation, in 30～60 ℃, dry 2～5h.
For solving the problems of the technologies described above, the present invention provides a kind of preparation method of microbial preparation again, comprises the following steps:
A. dull and stereotyped cultivation rejuvenation: the inoculation of cryopreservation, on TSB solid plate substratum, in 18～37 ℃ of cultivation 14～36h, is made to rejuvenation of spawn and forms single bacterium colony; Then picking list colony inoculation, on TSB slant medium, is cultivated 12～36h in 24～38 ℃;
B. the preparation of seed liquor: the bacterial classification that steps A is cultivated makes 1 * 10 with the physiological saline of 5～30 ‰ sterilizings
5the bacteria suspension of CFU/mL, the inoculum size with 3～9% is inoculated in TSB liquid nutrient medium, and 20～35 ℃, 100～200rpm shaking culture, 18～20h, obtains seed liquor;
C. fermentation culture: the seed liquor in step B is inoculated in fermention medium with 2～10% inoculum size, 20～35 ℃, 100～200rpm shaking culture, 50～150h, to viable count be 0.2～8 * 109CFU/mL, stuck fermentation;
Component and the weight percent thereof of described fermention medium are respectively: glucose 1～5%, and soybean muffin 1～4%, sodium-chlor 1～3%, all the other are water;
D. the preparation of microbial preparation: add zeolite powder in the fermented liquid of step C, in every 100mL fermented liquid, add 7～30g zeolite powder, static 20～60min after 100～200rpm vibration, 20～60min, by above-mentioned fermented liquid in 4 ℃ of centrifugal 20min of 6000rpm, abandon supernatant, after adding 20～40g zeolite powder to mix in every 100g precipitation, in 32～60 ℃, dry 2～5h.
For solving the problems of the technologies described above, the present invention also provides the application of a kind of described atrophy Bacillus strain Aj080319IA-12 as fodder additives and/or microbial preparation.
For solving the problems of the technologies described above, the present invention also provides the application of a kind of described atrophy Bacillus strain Aj080319IA-12 as enteron aisle prebiotics.
For solving the problems of the technologies described above, the present invention separately provides the application of a kind of described atrophy Bacillus strain Aj080319IA-12 in the preparation of fodder additives, microbial preparation and/or enteron aisle prebiotics.
The present invention has following useful technique effect:
1) the atrophy bacillus of screening has significant antagonistic action to sea cucumber pathogenic bacteria, and has the function that improves the stichopus japonicus speed of growth, immunizing power and enteron aisle digestive enzyme activity.
2) screening atrophy bacillus is the indigenous bacterium of screening from sea cucumber enteron aisle, fast to sea cucumber safety non-toxic, reproduction speed, be easy to cultivate, strong to adaptive capacity to environment, can breed faster formation dominant bacteria.
3) microbial preparation developed is used working method easy, only said preparation need be added in feed and throws something and feeds, and is applicable to the disease control in the apostichopus japonicus culture process of children's ginseng cultivating and various patterns.
4) use the present invention to reduce or the use of substitute antibiotics, to reduce medicine residual, improve sea cucumber commercial quality, ensuring food safety has vital role.
Accompanying drawing explanation
Fig. 1 is the evolutionary tree result figure obtaining with PHYLIP software according to 16S rDNA sequence described in the embodiment of the present invention;
Fig. 2 is that atrophy Bacillus strain Aj080319IA-12 preparation affects result figure to sea cucumber coelomic fluid enzyme activity described in the embodiment of the present invention;
Fig. 3 is that Aj080319IA-12 atrophy bacillus preparation affects result figure to sea cucumber enteron aisle enzyme activity described in the embodiment of the present invention.
Embodiment
Below with reference to drawings and Examples, describe embodiments of the present invention in detail, to the present invention, how utilisation technology means solve technical problem whereby, and the implementation procedure of reaching technique effect can fully understand and implement according to this.
Embodiment 1:
The screening and separating process of Aj080319IA-12 bacterial strain:
Aj080319IA-12 bacterial strain is Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science's separated obtaining from wild stichopus japonicus enteron aisle.Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science wild stichopus japonicus that arrest in marine site, peaceful angle from Qingdao in 2008, under aseptic condition, stichopus japonicus is carried out to vivisection, get its enteron aisle, extrude intestinal contents, with 15 ‰ normal saline flushing enteron aisle of sterilizing three times, by the enteron aisle homogenate after rinsing, after proportional diluted, be coated on TSB substratum and 2216E seawater nutrient agar, select suitable dilution flat board carry out the separated of bacterium and pass through repeatedly streak culture purification of bacterial, record morphological structure, the quantity of bacterium and take pictures; Take stichopus japonicus encountered pathogenic bacteria-Vibrio splindidus (Vibrio splendidus), Pseudoalteromonas (Pseudoalteromonas nigrifaciens) and Aeromonas hydrophila (Aeromonas hydrophlia) is indicator, utilize dibbling method, right-angled intersection method and Oxford agar diffusion method to detect the bacteriostatic action of institute's separation of bacterial to stichopus japonicus pathogenic bacteria, by its fungistatic effect and the contrast of antibiotic fungistatic effect, filter out the significant bacterial strain of fungistatic effect; Utilize dibbling method and Oxford agar diffusion method to measure the enzymatic productivity with antagonistic action bacterial strain simultaneously; Result therefrom filters out 1 bacteriostatic action and is greater than or approaches the antibacterial effect of microbiotic and the strong bacterial strain of enzymatic productivity, is numbered Aj080319IA-12.
Embodiment 2:
The morphological specificity of Aj080319IA-12 bacterial strain
The cell gramstaining of strains A j080319IA-12 is positive, and thalline is shaft-like, and spore staining result shows gemma, and capsule stain shows that thalline is without pod membrane.On TSB solid plate substratum, bacterium colony is yellow, and there is fold on surface, wire drawing shape, and opaque, edge is irregular, projection, periphery of bacterial colonies blackout look after cultivation 48h, has mycoderm and has precipitation to generate during liquid culture.
Embodiment 3:
16S rDNA sequencing
As shown in Figure 1, the evolutionary tree result figure for obtaining with PHYLIP software according to 16S rDNA sequence described in the embodiment of the present invention.
Universal primer for the bacterial 16 S rDNA that increases is 27F:5 '-AGAGTTTGATC (C/A) TGGCTCAG-3 ' and reverse primer 1492R:5 '-TACGG (C/T) TACCTTGTTACGACTT-3 '.Take the genomic dna of Aj080319IA-12 bacterial strain as template amplification 16S rDNA and transform order-checking, the sequence of gained is shown in sequence table.The 16SrDNA sequence of the Aj080319IA-12 bacterial strain of gained is carried out to homology comparison in Genbank, adopting Clustalw software to carry out multisequencing coupling arranges, by system, occur to infer that software package PHYLIP4.0 carries out statistical study and cluster analysis, adopt ortho position phase connection to obtain development system tree (Fig. 1), by bootstrapping, analyze the assessment of carrying out systematic evolution tree, the data set of bootstrapping is 1000 times.Homology comparative result shows, the homology of Aj080319IA-12 bacterial strain and atrophy genus bacillus (Bacillus atrophaeus) reaches 99.93%.
Embodiment 4:
Physiological and biochemical property
Adopt the French Mei Liai API ID 32E of company indentifying substance bar to analyze the physio-biochemical characteristics of bacterial strain, for uncertain reaction, adopt the strain identification pipe of Beijing Luqiao Technology Co., Ltd. to carry out measurement result respectively in Table 1.According to the feature of < < Bergey ' s Manual of Determinative Bacteriology > > (the 9th edition) and Gordon < < genus bacillus > > genus, bacterial strain is carried out to classification position evaluation, this bacterial strain has atrophy genus bacillus physiological and biochemical property, in conjunction with 16S rDNA sequence homology analysis result, illustrate that Aj080319IA-12 bacterial strain belongs to a kind of atrophy genus bacillus simultaneously.
Table 1:Aj080319IA-12 strains A PI ID 32E biochemical reactions result
Note :+: reacting positive represented;-: represent reaction negative;
Embodiment 5:
The preparation method of Aj080319IA-12 microbial preparation
The microbial preparation of producing with separated atrophy genus bacillus Aj080319IA-12 bacterial strain, is solid-state powderous preparations, and its preparation method comprises the steps:
A. dull and stereotyped cultivation rejuvenation: the atrophy Bacillus strain Aj080319IA-12 of cryopreservation is inoculated on TSB solid plate substratum, in 28 ℃ of cultivation 24h, makes rejuvenation of spawn and form single bacterium colony; Then picking list colony inoculation, on TSB slant medium, is cultivated 24h in 28 ℃;
B. the preparation of seed liquor: the bacterial classification that steps A is cultivated makes 1 * 10 with the physiological saline of 15 ‰ sterilizings
5the bacteria suspension of CFU/mL, the inoculum size with 5% is inoculated in TSB liquid nutrient medium, and 28 ℃, 170rpm shaking culture 18～20h, obtains seed liquor;
C. fermentation culture: the seed liquor in step B is inoculated in fermention medium with 5% inoculum size, 28 ℃, 170rpm shaking culture 96h, to viable count be 2～4 * 10
9cFU/mL, stuck fermentation;
Component and the weight percent thereof of described fermention medium are respectively: glucose 1～2%, and soybean muffin 1～2%, sodium-chlor 1%, all the other are water;
D. the preparation of microbial preparation: add zeolite powder in the fermented liquid of step C, in every 100mL fermented liquid, add 10g zeolite powder, static 40min after 170rpm vibration 40min, by above-mentioned fermented liquid in 4 ℃ of centrifugal 20min of 6000rpm, abandon supernatant, every 100g adds the zeolite powder of 20～40g to mix in precipitating after, after 45～50 ℃ of oven dry 3～4h, obtain the microbial preparation of this atrophy genus bacillus;
The viable bacteria content that utilizes dilution plate coating method to detect in preparation reaches 1 * 10
9more than CFU/g.
Embodiment 6:
The using method of microbial preparation of the present invention
Atrophy bacillus preparation of the present invention can be used as holothurian feed additive and uses, and using method, for according to the addition of weight ratio 3 ‰, this preparation and sea cucumber compound feed being mixed, carries out sea cucumber to throw something and feed, to reach antibacterial, immune, somatotrophic effect.
Embodiment 7:
The enzymatic productivity of Aj080319IA-12 bacterial strain and antagonism sea cucumber pathogenic bacteria ability
Take that stichopus japonicus encountered pathogenic bacteria---Vibrio splindidus (V.splendidus), Pseudoalteromonas (P.nigrifaciens) and Aeromonas hydrophila (A.hydrophlia) are indicator, utilize dibbling method, right-angled intersection method and Oxford agar diffusion method to detect the bacteriostatic action of this bacterial strain to stichopus japonicus pathogenic bacteria, by its fungistatic effect and the contrast of antibiotic fungistatic effect.Adopt dibbling method, Oxford agar diffusion method by casein hydrolyzing culture medium and Starch Hydrolysis culture medium culturing, to measure its product enzyme activity of this bacterial strain.Bacterium colony antagonistic effect result and product enzyme result (table 2) show, this atrophy genus bacillus is the growth of these three kinds of stichopus japonicus pathogenic bacterias of antagonism significantly, inhibition zone size is almost suitable with effects of antibiotics, and this bacterial strain has good product proteolytic enzyme and amylase activity.Therefore, this bacterial strain can be used as and has antibacterial, somatotrophic probiotic bacterium and be applied to apostichopus japonicus Selenka feed additive.
Enzymatic productivity and the antagonistic action to sea cucumber pathogenic bacteria of table 2:Aj080319IA-12 bacterial strain
Embodiment 8:
With Aj080319IA-12 bacterial strain, prepare microbial preparation
With separated atrophy genus bacillus Aj080319IA-12 bacterial strain, preparing microbial preparation, is solid-state powderous preparations, and step is as follows:
A. dull and stereotyped cultivation rejuvenation: the atrophy Bacillus strain Aj080319IA-12 of-80 ℃ of Refrigerator stores is inoculated on TSB solid plate substratum, in 28 ℃ of cultivation 24h, makes rejuvenation of spawn open form become single bacterium colony; Then picking list colony inoculation, on TSB slant medium, is cultivated 24h in 28 ℃;
B. the preparation of seed liquor: the bacterial classification that steps A is cultivated makes 1 * 10 with the physiological saline of 15 ‰ sterilizings
5the bacteria suspension of CFU/mL, the inoculum size with 5% is inoculated in 1000mL TSB liquid nutrient medium, and 28 ℃, 170rpm shaking culture 18～20h, obtains seed liquor;
C. fermentation culture: the seed liquor in step B is inoculated in the fermention medium of 5L with 5% inoculum size, 28 ℃, 170rpm shaking culture, every 12h sampling, amount of bacteria is calculated in dull and stereotyped coating, and observe gemma generation situation by spore staining method, and the demonstration of fermentation culture result, this bacterial strain starts to enter stationary phase at 84h, spore staining shows that major part has formed gemma, but the gemma production rate of 96h is higher than 84h, and nearly all thalline all exists with the form of spore, therefore at 96h stuck fermentation;
Component and the weight percent thereof of described fermention medium are respectively: glucose 1～2%, and soybean muffin 1～2%, sodium-chlor 1%, all the other are water;
D. the preparation of microbial preparation: add zeolite powder in the fermented liquid of step C, in every 100mL fermented liquid, add 10g zeolite powder, static 40min after 170rpm vibration 40min, by above-mentioned fermented liquid in 4 ℃ of centrifugal 20min of 6000rpm, abandon supernatant, every 100g adds the zeolite powder of 20～40g to mix in precipitating after, after 45～50 ℃ of oven dry 3～4h, obtain the microbial preparation of this atrophy genus bacillus.The viable bacteria content that utilizes dilution plate coating method to detect in preparation is 1.56 * 10
9cFU/g, oven drying method detects prepared preparation water content lower than 7%.The preparation pack of preparation is placed on to dry place and preserves, regularly detect the viable bacteria content in preparation, detected result demonstration, said preparation was preserved after half a year, and its viable bacteria content is still more than 70%.
Embodiment 9:
The impact of atrophy bacillus Aj080319IA-12 preparation on the growth of sea cucumber, survival rate, immune indexes and enteron aisle digestive enzyme activity.
Prepared atrophy bacillus Aj080319IA-12 preparation is added in feed to the sea cucumber of throwing something and feeding test according to 3 ‰ of feed weight.
Test is broadcast sea cucumber with sea cucumber at the bottom of from Yantai Tian Yuan aquatic products company limited natural waters, support after 7 days temporarily, select similar, the good sea cucumber of vigor of size and test, sea cucumber body weight is initially 55.01 ± 1.293g, the long 6～8cm of body, duration of test, sea water salinity is that 29～30, pH value is 7.46～8.02, and dissolved oxygen is more than 5.0g/L, water temperature is 16～18 ℃, and feedstuff feeding amount is stichopus japonicus body weight 1%.Test is divided into 3 groups, and test group 1 is added the holothurian feed of 3 ‰ atrophy bacillus preparations for throwing something and feeding, and test group 2 is added 1 ‰ β-1 for throwing something and feeding, the holothurian feed of 3 dextran, control group is thrown something and fed and is not added the holothurian feed of any additive, every group of 3 repetitions, and each repeats 15 sea cucumbers.Be 42d the experimental period of throwing something and feeding, and wherein front 28d throws something and feeds and adds the feed of relevant fodder additives, changes and throw non-additive holothurian feed after 28d.
0d, 4d, 14d, 28d and 42d in test extract stichopus japonicus coelomic fluid, for measuring acid phosphatase (ACP), alkaline phosphatase (AKP), the superoxide-dismutase (SOD) of coelomic fluid, enzymic activity and sea cucumber enteron aisle protease activity and the amylase activity of N,O-Diacetylmuramidase (LZM), at the 42nd day, measure sea cucumber body weight and calculate rate of body weight gain, in addition, companion, raise the 28d that throws something and feeds and from each experimental group, choose at random one of them and repeat manually to attack malicious infection experiment with Pseudoalteromonas, it is 1 * 10 that every sea cucumber is injected 200 μ L concentration
8the Pseudoalteromonas bacterium liquid of CFU/mL, the latter two weeks mortality ratio of interior three groups of poison attacked in record, and then calculate immune protective rate.
Test-results is as follows:
Aspect growth promotion, as can be seen from Table 3, add β-1,3 dextran and atrophy bacillus preparation experimental group of the present invention, all can significantly improve the rate of body weight gain (P < 0.05) of sea cucumber.
Table 3: the impact of atrophy bacillus preparation on sea cucumber growth of throwing something and feeding
As shown in Figure 2, for Aj080319IA-12 atrophy bacillus preparation affects result figure to sea cucumber coelomic fluid enzyme activity.Wherein, contain same letter difference not remarkable (P > 0.05); Contain different alphabetical significant differences (P < 0.05).Aspect raising immunizing power, test-results is as shown in Figure 2: at duration of test, except SOD enzyme activity between 3 groups without significant difference, add β-1, ACP, the AKP of 3 dextran and atrophy bacillus preparation experimental group of the present invention, LZM activity are all higher than control group (P < 0.05), illustrate that atrophy bacillus preparation of the present invention has and β-1, the immunocompetent function of raising sea cucumber body that 3 dextran are identical.
As shown in Figure 3, for Aj080319IA-12 atrophy bacillus preparation affects result figure to sea cucumber enteron aisle enzyme activity.Wherein, contain same letter difference not remarkable (P > 0.05); Contain different alphabetical significant differences (P < 0.05).Aspect raising Activity of Digestive Enzymes in Intestine, at duration of test, add atrophy bacillus preparation histone enzyme of the present invention and amylase activity and be significantly higher than control group and add β-1,3 dextran test group.Test-results is as shown in Figure 3: in feed, add the digestive enzyme activity that atrophy bacillus preparation can significantly improve stichopus japonicus.
Aspect raising sea cucumber disease resistance; manually attacking after poison; experimental result is as shown in table 4; add β-1; the mortality ratio of 3 dextran and atrophy bacillus preparation experimental group sea cucumber of the present invention is significantly lower than control group (P < 0.05); β-1,3 dextran and atrophy bacillus preparation of the present invention are 85.71% to the immune protective rate of sea cucumber, show that invented atrophy bacillus preparation can significantly improve the immunizing power of stichopus japonicus.
Table 4: the immune protective rate of the mortality ratio of stichopus japonicus and atrophy bacillus preparation on the same group not in artificial challenge experiment
Embodiment result proves: atrophy bacterial strain of bacillus of the present invention has the proteolytic enzyme of producing and amylase activity, has the effect of antagonism sea cucumber pathogenic bacteria simultaneously; The atrophy bacillus preparation viable bacteria content of developing reaches 1.56 * 10
9cFU/g, deposit half a year after viable bacteria content be still greater than 70%; The sea cucumber result of throwing something and feeding shows that this microbial preparation is as fodder additives, sea cucumber specific growth rate be can improve, reduce sea cucumber mortality ratio, the immunological competence of sea cucumber and the digestive enzyme activity of enteron aisle strengthened, improve the disease resistance of sea cucumber simultaneously, reached antibacterial, immune, growth promotion multiple effect, can reduce and replace antibiotic use, to improving cultivation surviving rate and quality product, ensure that the stable and high yields tool of this industry is of great significance.
Embodiment 10:
A kind of atrophy Bacillus strain Aj080319IA-12.
Described strains separation, in wild stichopus japonicus enteron aisle, is preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCC M 2010316.
Embodiment 11:
A kind of fodder additives.
Comprise: atrophy Bacillus strain Aj080319IA-12, described strains separation, in wild stichopus japonicus enteron aisle, is preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCC M 2010316.
The preparation method of described fodder additives can comprise the following steps:
A. dull and stereotyped cultivation rejuvenation: the atrophy Bacillus strain Aj080319IA-12 of cryopreservation is inoculated on TSB solid plate substratum, in 20～35 ℃ of cultivation 12～36h, makes rejuvenation of spawn and form single bacterium colony; Then picking list colony inoculation, on TSB slant medium, is cultivated 12～36h in 20～35 ℃;
B. the preparation of seed liquor: the bacterial classification that steps A is cultivated makes 1 * 10 with the physiological saline of 5～30 ‰ sterilizings
5the bacteria suspension of CFU/mL, the inoculum size with 2～10% is inoculated in TSB liquid nutrient medium, and 20～35 ℃, 100～200rpm shaking culture, 18～20h, obtains seed liquor;
C. fermentation culture: the seed liquor in step B is inoculated in fermention medium with 2～10% inoculum size, 20～35 ℃, 100～200rpm shaking culture, 50～150h, to viable count be 0.2～8 * 10
9cFU/mL, stuck fermentation;
Component and the weight percent thereof of described fermention medium are respectively: glucose 1～5%, and soybean muffin 1～5%, sodium-chlor 1～3%, all the other are water;
D. the preparation of microbial preparation: add zeolite powder in the fermented liquid of step C, in every 100mL fermented liquid, add 5～30g zeolite powder, static 20～60min after 100～200rpm vibration, 20～60min, by above-mentioned fermented liquid in 4 ℃ of centrifugal 20min of 6000rpm, abandon supernatant, after adding 20～40g zeolite powder to mix in every 100g precipitation, in 30～60 ℃, dry 2～5h.
Described fodder additives is preferably the fodder additives for sea cucumber, and additive capacity can be 0.5 of holothurian feed gross weight～7 ‰.
Described fodder additives can be solid-state powderous preparations.
Embodiment 12:
A kind of microbial preparation.
The preparation method of described microbial preparation comprises the following steps:
A. dull and stereotyped cultivation rejuvenation: the inoculation of cryopreservation, on TSB solid plate substratum, in 22～33 ℃ of cultivation 14～38h, is made to rejuvenation of spawn and forms single bacterium colony; Then picking list colony inoculation, on TSB slant medium, is cultivated 14～36h in 22～33 ℃;
B. the preparation of seed liquor: the bacterial classification that steps A is cultivated makes 1 * 10 with the physiological saline of 5～30 ‰ sterilizings
5the bacteria suspension of CFU/mL, the inoculum size with 3～10% is inoculated in TSB liquid nutrient medium, and 20～35 ℃, 100～200rpm shaking culture, 18～22h, obtains seed liquor;
C. fermentation culture: the seed liquor in step B is inoculated in fermention medium with 3～10% inoculum size, 23～35 ℃, 100～200rpm shaking culture, 60～150h, to viable count be 0.2～8 * 10
9cFU/mL, stuck fermentation;
Component and the weight percent thereof of described fermention medium are respectively: glucose 1～5%, and soybean muffin 1～5%, sodium-chlor 1～3%, all the other are water;
D. the preparation of microbial preparation: add zeolite powder in the fermented liquid of step C, in every 100mL fermented liquid, add 6～30g zeolite powder, static 20～60min after 100～200rpm vibration, 20～60min, by above-mentioned fermented liquid in 4 ℃ of centrifugal 20min of 6000rpm, abandon supernatant, after adding 20～40g zeolite powder to mix in every 100g precipitation, in 30～60 ℃, dry 2～5h.
Embodiment 13:
A kind of preparation method of microbial preparation.
Comprise the following steps:
A. dull and stereotyped cultivation rejuvenation: the inoculation of cryopreservation, on TSB solid plate substratum, in 18～37 ℃ of cultivation 14～36h, is made to rejuvenation of spawn and forms single bacterium colony; Then picking list colony inoculation, on TSB slant medium, is cultivated 12～36h in 24～38 ℃;
B. the preparation of seed liquor: the bacterial classification that steps A is cultivated makes 1 * 10 with the physiological saline of 5～30 ‰ sterilizings
5the bacteria suspension of CFU/mL, the inoculum size with 3～9% is inoculated in TSB liquid nutrient medium, and 20～35 ℃, 100～200rpm shaking culture, 18～20h, obtains seed liquor;
C. fermentation culture: the seed liquor in step B is inoculated in fermention medium with 2～10% inoculum size, 20～35 ℃, 100～200rpm shaking culture, 50～150h, to viable count be 0.2～8 * 10
9cFU/mL, stuck fermentation;
Component and the weight percent thereof of described fermention medium are respectively: glucose 1～5%, and soybean muffin 1～4%, sodium-chlor 1～3%, all the other are water;
D. the preparation of microbial preparation: add zeolite powder in the fermented liquid of step C, in every 100mL fermented liquid, add 7～30g zeolite powder, static 20～60min after 100～200rpm vibration, 20～60min, by above-mentioned fermented liquid in 4 ℃ of centrifugal 20min of 6000rpm, abandon supernatant, after adding 20～40g zeolite powder to mix in every 100g precipitation, in 32～60 ℃, dry 2～5h.
Embodiment 14: a kind of described atrophy Bacillus strain Aj080319IA-12 is as the application of fodder additives and/or microbial preparation.
Embodiment 15: a kind of described atrophy Bacillus strain Aj080319IA-12 is as the application of enteron aisle prebiotics.
Embodiment 16: the application of a kind of described atrophy Bacillus strain Aj080319IA-12 in the preparation of fodder additives, microbial preparation and/or enteron aisle prebiotics.
All above-mentioned these intellecture properties of primary enforcement, do not set restriction this product innovation of other forms of enforcement and/or novel method.Those skilled in the art will utilize this important information, and foregoing is revised, to realize similar implementation status.But all modifications or transformation belong to the right of reservation based on product innovation of the present invention.
The above, be only preferred embodiment of the present invention, is not invention to be done to the restriction of other form, and any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the equivalent embodiment of equivalent variations.But every technical solution of the present invention content that do not depart from, any simple modification, equivalent variations and the remodeling above embodiment done according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.
Wherein, described atrophy genus bacillus Aj080319IA-12 (Bacillus atrophaeus Aj080319IA-12) is preserved in Chinese Typical Representative culture collection center (being called for short CCTCC), depositary institution address: China on November 25th, 2010. Wuhan. and Wuhan University.Deposit number is: CCTCC NO:M 2010316.
Claims (8)
1. an atrophy Bacillus strain Aj080319IA-12, is characterized in that, described strains separation, in wild stichopus japonicus enteron aisle, is preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCC NO:M2010316.
2. a fodder additives, is characterized in that, comprising: atrophy Bacillus strain Aj080319IA-12, and described strains separation, in wild stichopus japonicus enteron aisle, is preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCC NO:M2010316.
3. fodder additives according to claim 2, is characterized in that, the preparation method of described fodder additives comprises the following steps:
A. dull and stereotyped cultivation rejuvenation: the atrophy Bacillus strain Aj080319IA-12 of cryopreservation is inoculated on TSB solid plate substratum, in 20～35 ℃ of cultivation 12～36h, makes rejuvenation of spawn and form single bacterium colony; Then picking list colony inoculation, on TSB slant medium, is cultivated 12～36h in 20～35 ℃;
B. the preparation of seed liquor: the bacterial classification that steps A is cultivated makes 1 * 10 with the physiological saline of 5～30 ‰ sterilizings
5the bacteria suspension of CFU/mL, the inoculum size with 2～10% is inoculated in TSB liquid nutrient medium, and 20～35 ℃, 100～200rpm shaking culture, 18～20h, obtains seed liquor;
C. fermentation culture: the seed liquor in step B is inoculated in fermention medium with 2～10% inoculum size, 20～35 ℃, 100～200rpm shaking culture, 50～150h, to viable count be 0.2～8 * 10
9cFU/mL, stuck fermentation;
Component and the weight percent thereof of described fermention medium are respectively: glucose 1～5%, and soybean muffin 1～5%, sodium-chlor 1～3%, all the other are water;
D. the preparation of microbial preparation: add zeolite powder in the fermented liquid of step C, in every 100mL fermented liquid, add 5～30g zeolite powder, static 20～60min after 100～200rpm vibration, 20～60min, by above-mentioned fermented liquid in 4 ℃ of centrifugal 20min of 6000rpm, abandon supernatant, after adding 20～40g zeolite powder to mix in every 100g precipitation, in 30～60 ℃, dry 2～5h.
4. according to the fodder additives described in claim 2 or 3, it is characterized in that, described fodder additives is the fodder additives for sea cucumber, and additive capacity is 0.5 of holothurian feed gross weight～7 ‰.
5. according to the fodder additives described in claim 2 or 3, it is characterized in that, described fodder additives is solid-state powderous preparations.
6. a microbial preparation, is characterized in that, the preparation method of described microbial preparation comprises the following steps:
A. dull and stereotyped cultivation rejuvenation: the atrophy Bacillus strain Aj080319IA-12 that is CCTCC NO:M2010316 by the preserving number of cryopreservation is inoculated on TSB solid plate substratum, in 22～33 ℃ of cultivation 14～38h, makes rejuvenation of spawn and forms single bacterium colony; Then picking list colony inoculation, on TSB slant medium, is cultivated 14～36h in 22～33 ℃;
B. the preparation of seed liquor: the bacterial classification that steps A is cultivated makes 1 * 10 with the physiological saline of 5～30 ‰ sterilizings
5the bacteria suspension of CFU/mL, the inoculum size with 3～10% is inoculated in TSB liquid nutrient medium, and 20～35 ℃, 100～200rpm shaking culture, 18～22h, obtains seed liquor;
C. fermentation culture: the seed liquor in step B is inoculated in fermention medium with 3～10% inoculum size, 23～35 ℃, 100～200rpm shaking culture, 60～150h, to viable count be 0.2～8 * 10
9cFU/mL, stuck fermentation;
Component and the weight percent thereof of described fermention medium are respectively: glucose 1～5%, and soybean muffin 1～5%, sodium-chlor 1～3%, all the other are water;
D. the preparation of microbial preparation: add zeolite powder in the fermented liquid of step C, in every 100mL fermented liquid, add 6～30g zeolite powder, static 20～60min after 100～200rpm vibration, 20～60min, by above-mentioned fermented liquid in 4 ℃ of centrifugal 20min of 6000rpm, abandon supernatant, after adding 20～40g zeolite powder to mix in every 100g precipitation, in 30～60 ℃, dry 2～5h.
7. a preparation method for microbial preparation, is characterized in that, comprises the following steps:
A. dull and stereotyped cultivation rejuvenation: the atrophy Bacillus strain Aj080319IA-12 that is CCTCC NO:M2010316 by the preserving number of cryopreservation is inoculated on TSB solid plate substratum, in 18～37 ℃ of cultivation 14～36h, makes rejuvenation of spawn and forms single bacterium colony; Then picking list colony inoculation, on TSB slant medium, is cultivated 12～36h in 24～38 ℃;
B. the preparation of seed liquor: the bacterial classification that steps A is cultivated makes 1 * 10 with the physiological saline of 5～30 ‰ sterilizings
5the bacteria suspension of CFU/mL, the inoculum size with 3～9% is inoculated in TSB liquid nutrient medium, and 20～35 ℃, 100～200rpm shaking culture, 18～20h, obtains seed liquor;
C. fermentation culture: the seed liquor in step B is inoculated in fermention medium with 2～10% inoculum size, 20～35 ℃, 100～200rpm shaking culture, 50～150h, to viable count be 0.2～8 * 10
9cFU/mL, stuck fermentation;
Component and the weight percent thereof of described fermention medium are respectively: glucose 1～5%, and soybean muffin 1～4%, sodium-chlor 1～3%, all the other are water;
D. the preparation of microbial preparation: add zeolite powder in the fermented liquid of step C, in every 100mL fermented liquid, add 7～30g zeolite powder, static 20～60min after 100～200rpm vibration, 20～60min, by above-mentioned fermented liquid in 4 ℃ of centrifugal 20min of 6000rpm, abandon supernatant, after adding 20～40g zeolite powder to mix in every 100g precipitation, in 32～60 ℃, dry 2～5h.
8. the application of atrophy Bacillus strain Aj080319IA-12 in the preparation of fodder additives, microbial preparation and/or enteron aisle prebiotics as claimed in claim 1.
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| CN101082026A (en) * | 2006-05-31 | 2007-12-05 | 上海泓宝绿色水产科技发展有限公司 | Preparation of holothurian-nourishing bacterium and method for restoring sea cucumber cultivation pool environment |
| CN101658252A (en) * | 2009-09-17 | 2010-03-03 | 大连水产学院 | Food organism additive for cultivating stichopus japonicus |
| CN101912051A (en) * | 2010-07-13 | 2010-12-15 | 大连海洋大学 | Fermentation process of sea cucumber compound feed |
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| CN101082026A (en) * | 2006-05-31 | 2007-12-05 | 上海泓宝绿色水产科技发展有限公司 | Preparation of holothurian-nourishing bacterium and method for restoring sea cucumber cultivation pool environment |
| CN101658252A (en) * | 2009-09-17 | 2010-03-03 | 大连水产学院 | Food organism additive for cultivating stichopus japonicus |
| CN101912051A (en) * | 2010-07-13 | 2010-12-15 | 大连海洋大学 | Fermentation process of sea cucumber compound feed |
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