CN102586144B - Bacillus pumilus, probiotics preparation and preparation method and application thereof - Google Patents
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Abstract
The invention discloses bacillus pumilus, a probiotics preparation and a preparation method and application thereof. The Bacillus pumilus LV149 is preserved in China center for type culture collection (CCTCC) in Nov. 23th, 2011, and the preservation number is CCTCC NO: M 2011411. The bacillus pumilus LV149 has strong extracellular protease, lipase and amylase activities, has wide rejection capability to vibrio and has no hemolytic activity. The Bacillus pumilus LV149 serves as fermenting bacterial strains and is performed with solid fermentation, drying and smashing so as to prepare bacillus pumilus probiotics preparation which uses Bacillus pumilus LV149 as the active ingredients. The bacillus pumilus probiotics preparation can be added into prawn feeds for feeding prawns, so that growth of prawn intestinal pathogenic vibrio can be restrained, prawn vibriosis can be reduced, simultaneously prawn growth is promoted, fish bait coefficient is reduced, quality of commodity is improved, culture cycle is shortened, accordingly culture risk and culture cost are reduced, biological safety is high and the bacillus pumilus, the probiotics preparation and the preparation method and application thereof have wide application prospect in aquaculture.
Description
Technical field
The invention belongs to microorganism field, be specifically related to a kind of tool enzymatic activity high and press down the bacillus pumilus of vibrios activity, and utilize it as probiotics preparation of active ingredient and its preparation method and application.
Background technology:
Along with developing rapidly of mass-producing, intensive aquaculture industry; aquiculture disease frequently occurs; in order to control disease; some antibiotic medicines are widely used in aquaculture and abuse; not only cause environment drug residue, bacterial drug resistance to increase; and due to the mobility of water body environment, the easier horizontal transfer of drug resistant gene causes the appearance of more persisters.In order to tackle resistance, have to strengthen consumption in cultivation and constantly change medicament categories, and then causing vicious cycle.A large amount of uses of medicine on the other hand, the medicine residual food-safety problem that causes in the aquatic animal body also causes concern day by day.In order to tackle these problems, adopt the bio-control method that substitutes to become the focus of current aquaculture, wherein being applied in aquaculture of probiotic bacterium more and more comes into one's own, they not only have the former effect of anti-bacteria venereal disease, also have the growth of the aquatic animal of promotion, improve the effects such as immune.
Aquatic products probiotic products on market, a multitude of names, layer goes out infinite, but sums up the following problem that exists: the probiotic bacterium of (1) most products is directed to the probiotic bacterium kind of the mankind and terrestrial animal.These Lu Yuan probiotic bacteriums whether can long-term surviving in the enteron aisle of water body environment or hydrocoles almost without relevant report.Panigrahi etc. (2004) think directly that those use for reference the deficiency that there is difficult field planting in aquatic products probiotic bacterium that livestock and poultry thinkings develop, can not retain in digestive canal of aquatic animal and amount reproduction.(2) the aquatic products probiotic products is take water conditioner as main, and additive agent for feeding is less important.Some probiotic products indicate it and both can make water conditioner and also can be used as additive agent for feeding, have obviously run counter to the rule that the microorganism growth environment there are differences.(3) as the probiotic products of additive agent for feeding take subtilis, Bacillus licheniformis, bacillus cereus as common, the genus bacillus of rare other type, and the common flora that above-mentioned three kinds of genus bacillus are not ocean environment.(4) in the aquatic products probiotic products, the enzyme active of bacterial strain fruit is indefinite, fungistatic effect is indefinite, and this binomial index is to estimate the core index of the effect of feeding probiotic bacterium.Use maximum genus bacillus as example with current aquatic products probiotic bacterium, the genus bacillus of having reported at present is antibiotic have 169 kinds, and these antibiotic are mainly the peptide classes, and multiaction is in gram positive bacterium.The enteron aisle the main pathogenic fungi of people and terrestrial animal is gram positive bacterium, and therefore for people and terrestrial animal, genus bacillus is proper probiotic bacterium.But for the marine fishery animal, main pathogenic bacteria is Gram-negative bacteria, as vibrios.Genus bacillus for these from the inhibition of the Gram-negative bacteria of aquatic animal rare report also.Therefore can not indiscriminately imitate peculation, the genus bacillus of inhibition etc. need to be arranged Gram-negative bacterias such as vibrios for the characteristics screening of aquatic animal.In conjunction with the problems referred to above, we are in the urgent need to separating probiotic strain from the indigenous environment of aquatic animal to grow and enteron aisle, and with enzymic activity and the bacteriostatic activity leading indicator as the screening and evaluation effect.
Summary of the invention:
First purpose of the present invention is to provide a kind of separation from healthy Environment of Litopenaeus vannamei Low intestinal mucosa and has enzymatic activity high and bacillus pumilus (Bacillus pumilus) LV149 that presses down the vibrios activity, this bacterium is preserved in Chinese Typical Representative culture collection center (CCTCC), deposit number: CCTCC NO:M2011411 on November 23rd, 2011.
Bacillus pumilus of the present invention (Bacillus pumilus) LV149 separates from healthy Environment of Litopenaeus vannamei Low intestinal mucosa.
The biological property of bacillus pumilus (Bacillus pumilus) LV149 is as follows:
1, colonial morphology and cell morphological characteristic: the colonial morphology of bacterial strain on LB nutrient agar and 2216E substratum is: oyster white, flat, the surface is wet, opaque, fold, edge are irregular, cultivates 24 hours colony diameter 2-4mm.Shaft-like, gemma is oval, and neutral or inclined to one side end is given birth to.
2, physiological and biochemical property:
Strain growth suitable salinity scope: the above NaCl of 5-35 ‰, 7% does not grow, and growth appropriate pH scope is 5-9.
Gram-positive, catalase (-), oxydase (+), V-P tests (-), nitrate reduction (-), Starch Hydrolysis (+), gelatine liquefication (+), can ferment D-Glucose, L-arabinose, PEARLITOL 25C, D-Glucose, glucose fermentation is aerogenesis not, phenylalanine deaminase (-), Citrate trianion utilizes (+), lecithinase (-), indole test (-), casein hydrolysis (+), tyrosine hydrolysis (-).
Ordinary method is extracted bacillus pumilus (Bacillus pumilus) LV149 genomic dna and is done 16S rRNA gene sequencing, and its sequence is as shown in SEQ ID NO.1.Through BLAST comparison, itself and the 16S rRNA gene identity 100% of many bacillus pumilus (Bacillus pumilus) of announcing, degree consistent with other genus bacillus is below 99%.Therefore, this bacterium of Molecular Identification is bacillus pumilus.With reference to " the listed method of uncle's Jie Shi Bacteria Identification handbook (the 9th edition) is carried out conventional physiology and chemistry to bacterium and identified.The similarity of itself and type strain is 100%, belongs to bacillus pumilus, and this result is consistent with the result of Molecular Identification.Therefore this Pseudomonas is in bacillus pumilus, called after bacillus pumilus (Bacillus pumilus) LV149, this bacterium is preserved in Chinese Typical Representative culture collection center (CCTCC), deposit number: CCTCC NO:M2011411 on November 23rd, 2011.Bacillus pumilus LV149 and LV479 have been carried out randomly amplified polymorphic DNA (RAPD) amplification, to detect both difference of finger printing.As seen from Figure 2,7 random primers that adopt all can provide the finger printing of two bacillus pumilus, and every primer all can demonstrate both significantly difference, although this result shows bacillus pumilus LV149 and bacillus pumilus LV479 and belongs to same kind, but hereditary difference is larger, causes it that different phenotypic characteristics is arranged.
Find through experiment, bacillus pumilus of the present invention (Bacillus pumilus) LV149 can produce proteolytic enzyme, lipase and amylase simultaneously, and these three kinds of enzymes have high activity.Bacillus pumilus (Bacillus pumilus) LV149 has inhibition widely to vibrios such as Vibrio harveyi E385, Vibrio parahaemolyticus E154, Vibrio parahaemolyticus E347, Vibrio parahaemolyticus E346, wheel animalcule vibrios E231, Vibrio harveyi E379, Vibrio harveyi E345, Vibrio furnissii E341, Vibrio vulnificus 11758, vibrio alginolyticus E333, vibrio alginolyticus A056 and vibrio alginolyticus E066, and does not have hemolytic activity.Further bacillus pumilus (Bacillus pumilus) LV149 is carried out solid state fermentation, drying and crushing prepares the bacillus pumilus probiotics preparation that contains bacillus pumilus (Bacillus pumilus) LV149, add with it Environment of Litopenaeus vannamei Low of feeding in feed to, found that along with the culture-cycle increases, the experimental group of the feed that added the bacillus pumilus probiotics preparation of throwing something and feeding is compared with the control group that does not add probiotics preparation, the body weight increase is obvious gradually, and liver body index reduces obvious gradually.When finishing to experiment, the mean body weight of experimental group prawn is compared obvious difference with control group, has increased by 11.7%; The liver body index of experimental group prawn is compared obvious difference with control group, has reduced by 10.2%; After calculating, feed coefficient has reduced by 12.1%.Explanation thus uses the bacillus pumilus probiotics preparation can reduce significantly feed coefficient in the cultivation of prawn, promote the prawn growth, thereby Shortening culturing period reduces aquaculture cost and risk.The use of bacillus pumilus probiotics preparation has also obviously improved the commercial quality of prawn, has increased dressing percentage.In addition, this result also shows in conjunction with hemolytic experiment: bacillus pumilus (Bacillus pumilus) LV149 has biological safety.The present invention is divided into experimental group and control group with prawn, the commodity prawn feed that the experimental group prawn is thrown something and fed and is added with the bacillus pumilus probiotics preparation, the control group commodity prawn feed of only throwing something and feeding.Prawn is soaked 24 hours in containing the water of Vibrio harveyi after, normally change water, throw something and feed, observe ingesting of prawn and show and add up mortality ratio, result shows: adopt the forage feed prawn 5 days be added with the bacillus pumilus probiotics preparation, prawn namely shows has certain resistibility to Vibrio harveyi, and mortality ratio is lower than control group; After throwing something and feeding 10 days, 15 days, the prawn mortality ratio is starkly lower than control group, after throwing something and feeding 20 days, the mortality ratio of the prawn of experimental group only has 5%, show thus: in cultivation was used, bacillus pumilus (Bacillus pumilus) LV149 can suppress Vibrio harveyi, adheres to using the bacillus pumilus probiotics preparation more than 10 days, can greatly reduce the morbidity of vibriosis penaeus, life-time service can be stopped the generation of vibriosis substantially.
Therefore, second purpose of the present invention is to provide the application of bacillus pumilus (Bacillus pumilus) LV149 in preparation inhibition vibrios probiotics.
Described vibrios cause of disease is Vibrio harveyi E385, Vibrio parahaemolyticus E154, Vibrio parahaemolyticus E347, Vibrio parahaemolyticus E346, wheel animalcule vibrios E231, Vibrio harveyi E379, Vibrio harveyi E345, Vibrio furnissii E341, Vibrio vulnificus 11758, vibrio alginolyticus E333, vibrio alginolyticus A056 and vibrio alginolyticus E066.
The 3rd purpose of the present invention is to provide a kind of bacillus pumilus probiotics preparation, it is characterized in that, with bacillus pumilus (Bacillus pumilus) LV149 as active ingredient.
The 4th purpose of the present invention is to provide a kind of preparation method of bacillus pumilus probiotics preparation, it is characterized in that, as fermentation strain, through solid state fermentation, oven dry makes after pulverizing with bacillus pumilus (Bacillus pumilus) LV149.
the preparation method of described bacillus pumilus probiotics preparation, its preferred concrete steps are: bacillus pumilus (Bacillus pumilus) LV149 is inoculated into to cultivate in the LB substratum makes seed culture fluid, be inoculated in solid-state fermentation culture medium with 5%～10% inoculum size again, fermentation time is 20～48 hours, leavening temperature is 30 ℃, fermented product is 50 ℃ of oven dry in baking oven, the oven dry thing was pulverized 60 mesh sieves, be the bacillus pumilus probiotics preparation, described solid-state fermentation culture medium is comprised of material and water, described material is by total mass mark 100%, comprise rice bran 5～20%, wheat bran 10～30%, dregs of beans 20～40%, Semen Maydis powder 20～40%, skim-milk 1～5%, sucrose 1～5%, yeast extract 0.1～2%, NaCl 0.1～1% and feeding many ore deposits 0.1～0.5% (Guangzhou feed technology company limited of China Telecom, name of product is: Jiang Feng many ore deposits, article No. is: JC1000), material-water ratio is 1: 1～1.3.
The 5th purpose of the present invention is to provide the bacillus pumilus probiotics preparation as the application of fodder additives.Ratio with bacillus pumilus probiotics preparation of the present invention and prawn finished product feedstuff by weight 0.1～1% mixes, adsorbed 10～60 minutes, the prawn of can throwing something and feeding, not only can suppress the generation of the growth of gut of shrimp pathogenic vibrio, minimizing vibriosis penaeus, promote simultaneously the prawn growth, reduce feed coefficient, improve commercial quality, Shortening culturing period, thereby reduce the cultivation risk, reduce aquaculture cost, and biological safety is high.
Described feed is preferably prawn feed, and further preferred bacillus pumilus probiotics preparation is pressed 0.1～1% of prawn feed weight and added.
In sum, bacillus pumilus of the present invention (Bacillus pumilus) LV149 has inhibition widely to vibrios, and does not have hemolytic activity.with this bacterium as fermentation strain, through solid state fermentation, drying and crushing makes the bacillus pumilus probiotics preparation as activeconstituents with bacillus pumilus (Bacillus pumilus) LV149, it is added in prawn feed, the prawn of feeding, not only can play and suppress the growth of gut of shrimp pathogenic vibrio, reduce the generation of vibriosis penaeus, promote simultaneously the prawn growth, reduce feed coefficient, improve commercial quality, Shortening culturing period, thereby reduce the cultivation risk, reduce aquaculture cost, and biological safety is high, be with a wide range of applications in aquaculture.
Bacillus pumilus of the present invention (Bacillus pumilus) LV149 is preserved in Chinese Typical Representative culture collection center (CCTCC), address on November 23rd, 2011: Wuhan, China Wuhan University, deposit number: CCTCC NO:M2011411.
Description of drawings:
Fig. 1 is three kinds of enzymic activity detection figure of bacillus pumilus LV149, wherein 1: amylase activity; 2: lipase activity; 3: protease activity;
Fig. 2 is the RAPD molecular fingerprint trace analysis of bacillus pumilus LV149 and bacillus pumilus LV479.Wherein: 1,3,5,7,9,11,13 are respectively Shanghai gives birth to work random primer S9, S10, S11, S12, S13, S14, the RAPD amplification of S15 to bacillus pumilus LV149; 2,4,6,8,10,12,14 are respectively Shanghai gives birth to work random primer S9, S10, S11, S12, S13, S14, the RAPD amplification of S15 to bacillus pumilus LV479; M, DNA molecular Marker DL 15000;
Fig. 3 is that bacillus pumilus presses down the active typical detection figure of vibrios.Wherein (1): bacillus pumilus LV448 (indicator); (2): bacillus pumilus LV149;
Fig. 4 is bacillus pumilus LV149 and the hemolytic detection figure that contrasts bacterium vibrio alginolyticus A056, wherein (1): bacillus pumilus LV149; (2): vibrio alginolyticus A056 (indicator).
Embodiment:
Following examples are to further illustrate of the present invention, rather than limitation of the present invention.Method therefor and technology if no special instructions, are ordinary method and technology.
Embodiment 1: the screening of bacillus pumilus (Bacillus pumilus) LV149 with separate
(1) enteron aisle adheres to the bacterium separation
Buy 5 batches of healthy Environment of Litopenaeus vannamei Low respectively at different time and different market, every batch of 30 tails, therefrom picking is individual large, body colour is bright, smooth, individuality 5 tails of not damaged, liver brown, the long 12cm of average body.With the alcohol disinfecting prawn body surface of volume fraction 75%, then use aseptic seawater flushing, get its enteron aisle after dissection on aseptic masking foil, draw the sterilization seawater with syringe, rinse out rapidly the ight soil in enteron aisle, every enteral irrigation 2-3 time is with in the visual inspection enteron aisle not till residual content.Add stroke-physiological saline solution with enteron aisle homogenate in aseptic homogenizer.Get the 0.5mL homogenate and be enteron aisle adhesion bacterial suspension with stroke-physiological saline solution (1%NaCl) by 10 times of serial dilutions.
Enteron aisle is adhered to bacterial suspension coating prawn feed substratum.The prawn feed medium component: by total mass mark 100%, comprise that prawn feed smashes powder (cross 100 mesh sieves) 10%, NaCl 1%, agar 2%, and all the other be water, and then sterilize and be down flat plate.Cultivate after 24～48 hours for 30 ℃, picking colony is further rule at the LB substratum and is separated and purifying.Amount to and obtain 500 strains of gut of shrimp adhesion bacterium.
(2) three kinds of enzymic activity enteron aisles adhere to bacteria screening
Detect according to a conventional method protease activity, lipase activity, amylase activity that enteron aisle adheres to bacterium.Found that the bacterial strain that can produce simultaneously three kinds of extracellular enzymes in 500 strain bacteriums has 90 strains.
(3) the enzymatic activity high enteron aisle adheres to screening bacteriostatic activity strain in bacterium
Adopt dull and stereotyped disc diffusion method that the enzymatic activity high enteron aisle adhesion bacterium of above-mentioned acquisition is screened, purpose is to obtain to have the bacterial strain that suppresses aquatic animal pathogenic vibrio ability and enzymatic activity high.Used medium is the LB substratum, and indicator is vibrio alginolyticus A056, Vibrio harveyi E385, Vibrio parahaemolyticus E154.Tested bacterium and indicator are all used LB substratum incubated overnight in advance.After indicator liquid is adopted the suitable dilution of sterilization 1%NaCl, coating LB is dull and stereotyped, note filter paper on the LB flat board, and diameter 5mm, the tested bacterium liquid 20 μ L of each filter paper dropping cultivated 12-24 hour for 30 ℃, observed the appearance of inhibition zone, and measured the diameter of inhibition zone.Result shows: 90 strains have in enzymatic activity high enteron aisle adhesion bacterium only has a strain bacterium LV149 inhibited to three kinds of vibrios simultaneously.
(4) the enzymatic activity high enteron aisle adheres to bacteria molecule evaluation and biochemical identification
Select from the bacterium of 90 strain tool enzymic activitys that growth is very fast, 69 strain bacteriums that be easy to cultivate have carried out the Molecular Identification based on 16S rRNA gene.Pcr amplification primer: 27F:5 '-AGAGTT TGATC (C/A) TGG CTCAG-3 '; 1492R:5 '-TACGG (C/T) TAC CTT GTT ACG ACT T-3 '.Positive amplified fragments is directly submitted Invitrogen company purifying, order-checking.Result shows: they belong to respectively acinetobacter (Acinetobacter), bacillus (Bacillus), Staphylococcus (Staphylococcus), Pseudoalteromonas (Pseudoalteromonas), Aeromonas (Aeromonas), have a liking for Halomonas (Halomonas), Li Sidun Bordetella (Listonella) and moraxella (Moraxella).Wherein quantity is at most Bacillus, accounts for to identify 53.62% of total plate count.
The 16S rRNA gene order of bacterium LV149, its sequence is as shown in SEQ ID NO.1.Through BLAST comparison, itself and the 16S rRNA gene identity 100% of many bacillus pumilus (Bacillus pumilus) of announcing, degree consistent with other genus bacillus is below 99%.Therefore, this bacterium of Molecular Identification is bacillus pumilus.With reference to " the listed method of uncle's Jie Shi Bacteria Identification handbook (the 9th edition) is carried out conventional physiology and chemistry to bacterium and identified.The similarity of itself and type strain is 100%, belongs to bacillus pumilus, and this result is consistent with the result of Molecular Identification.Therefore this Pseudomonas was in bacillus pumilus, and called after bacillus pumilus (Bacillus pumilus) LV149 is preserved in Chinese Typical Representative culture collection center (CCTCC), deposit number: CCTCC NO:M2011411 on November 23rd, 2011.
There is genus bacillus to occupy the majority although 69 plant height enzymic activity enteron aisles adhere in bacterium, comprising other bacillus pumilus strain, only has a bacillus pumilus LV149 to three kinds of vibrios, obvious restraining effect to be arranged.This shows in to enteron aisle, has enzymatic activity high and have a ratio that the genus bacillus strain that suppresses vibrios occurs very low.
Three kinds of extracellular enzymes of bacillus pumilus (Bacillus pumilus) LV149 all have high activity, and three kinds of enzyme biopsy mappings are seen Fig. 1.
(5) the molecular fingerprint figure spectrum signature of bacillus pumilus LV149
Randomly amplified polymorphic DNA (RAPD) the random primer S9-S15 that adopts Shanghai to give birth to work has carried out the RAPD amplification to bacillus pumilus LV149 and LV479 respectively, to detect both difference of finger printing.RAPD amplification program: 93 ℃ of denaturation 2min; 93 ℃ of 1min, 36 ℃ of 1min, 72 ℃ of 7min carry out 35 circulations altogether; Last 72 ℃ are extended 7min.The RAPD product is with 0.8% agarose electrophoresis 30min, and ultraviolet imagery the results are shown in Figure 2.As seen from Figure 2,7 random primers that adopt all can provide the finger printing of two bacillus pumilus, and every primer all can demonstrate both significantly difference, belong to same kind although this result shows LV149 and LV479, but hereditary difference is larger, causes it that different phenotypic characteristics is arranged.The RAPD finger printing of bacillus pumilus can be used for distinguishing congener not homophyletic, therefore to a certain extent can be false proof, and prevent illegal product plagiarization.
Embodiment 2: the inhibition vibrios spectrum analysis of bacillus pumilus LV149 and hemolytic analysis
Employing is analyzed the inhibition vibrios spectrum of bacillus pumilus LV149 from 9 13 strain vibrios not of the same race.The above-mentioned dull and stereotyped disc diffusion method of same employing.Judge the power of bacteriostasis according to the size of inhibition zone, the antimicrobial spectrum analytical results sees Table 1:
Table 1: the inhibition vibrios activation analysis of bacillus pumilus LV149
Annotate: +++: bacteriostatic activity is strong; ++: bacteriostatic activity is medium; +: bacteriostatic activity is arranged.
By as seen from Table 1, from tens kinds of encountered pathogenic vibrios analyzing, bacillus pumilus LV149 has the bacteriostatic activity of wide spectrum to vibrios.Bacillus pumilus LV149 sees Fig. 3 to the typical inhibition zone that vibrios forms.
Adopt common LB blood agar plate that the hemolytic activity of bacillus pumilus LV149 is detected, bacillus pumilus LV149 bacterium liquid dibbling 5 μ L cultivated 24 hours for 30 ℃.Found that bacillus pumilus LV149 does not have hemolytic activity, and contrast bacterium vibrio alginolyticus A056 hemolytic activity is obvious.The hemolytic activity detection figure of bacillus pumilus LV149 sees Fig. 4.
Embodiment 3: the preparation of bacillus pumilus probiotics preparation
Primary seed solution: take LB as substratum, the bacillus pumilus LV149 that very low temperature is preserved joins in the LB liquid nutrient medium with the ratio of 1% (volume percent), and 30 ℃ of concussions were cultivated 24 hours, obtained primary seed solution.
Secondary seed solution: take LB as substratum, the ratio of primary seed solution with 2% (volume percent) joined in the LB liquid nutrient medium, 30 ℃ of concussions were cultivated 24 hours, obtained secondary seed solution.
Solid-state fermentation culture medium: with rice bran 1000g, wheat bran 2500g, dregs of beans 3000g, Semen Maydis powder 3000g, skim-milk 200g, sucrose 200g, yeast extract 50g, NaCl 40g, feeding many ore deposits 10g (Guangzhou feed technology company limited of China Telecom, name of product is: Jiang Feng many ore deposits, article No. is: JC1000), amount to 10kg, be dissolved in the 11kg tap water, material-water ratio is 1: 11, mixes.
121 ℃ of autoclaving solid-state fermentation culture medium 20 minutes naturally cool to below 40 ℃, and the ratio take inoculum size as 5% adds secondary seed solution 500ml, mixes.Cultivated 40 hours for 30 ℃ in the 10L beaker of air-permeable envelope sealing, therebetween shaken several times firmly.Fermentation ends, microscopy gemma yield is 90%.With fermented product 50 ℃ of oven dry in baking oven, the oven dry thing was pulverized 60 mesh sieves, was the bacillus pumilus probiotics preparation.
Get bacillus pumilus probiotics preparation 1g, after the NaCl solution gradient dilution through adding massfraction 0.9%, get 100 μ L coating LB dull and stereotyped, cultivated 24 hours for 30 ℃, calculate viable count.The bacillus pumilus probiotics preparation viable count that obtains as calculated reaches 1.2 * 10
9Cfu/g.
Embodiment 4: promoter action and the Evaluation of Biocompatibility of bacillus pumilus probiotics preparation to the prawn growth
Get Environment of Litopenaeus vannamei Low a collection of, the long 8.1cm of average body, mean body weight 7.3g.Support after 3 days temporarily, be divided into control group (C) and experimental group (E), every group of each 150 tail prawns.The experimental group prawn commodity prawn feed that is added with the bacillus pumilus probiotics preparation (No. 2 material of " permanent emerging board " penaeus vannamei boone feed) of throwing something and feeding, the control group above-mentioned commodity prawn feed of only throwing something and feeding.Bacillus pumilus probiotics preparation addition means: the bacillus pumilus probiotics preparation adds 5g by the per kilogram feed and takes, join in a certain amount of seawater (15% feed is heavy), the genus bacillus on carrier fully stirs evenly, so that can be shed in water.This seawater is evenly joined in feed, stir evenly with hand, adsorb and to throw something and feed after 20 minutes.Control group and experimental group are thrown something and fed 3 times every day.The feed consumption of each group of record.30 ℃ of temperature of cultivation change the water secondary every day, change water 50% at every turn.Get 20 tail shrimp statistical weights in average every 15 days, and then took out liver and weigh, calculate liver body index.45 days culture-cycles, add up altogether 3 times, add up feed coefficient after 45 days.The results are shown in Table 2.
Table 2: the growth-promoting effect of bacillus pumilus probiotics preparation to Environment of Litopenaeus vannamei Low
By as seen from Table 2: along with the culture-cycle increases, the experimental group of having added bacillus pumilus probiotics preparation feed of throwing something and feeding is compared with control group, and the body weight increase is obvious gradually, and liver body index reduces obvious gradually.When finishing to experiment, the mean body weight of experimental group prawn is compared obvious difference with control group, has increased by 11.7%; The liver body index of experimental group prawn is compared obvious difference with control group, has reduced by 10.2%; After calculating, feed coefficient has reduced by 12.1%.Explanation thus uses the bacillus pumilus probiotics preparation can reduce significantly feed coefficient in the cultivation of prawn, promote the prawn growth, thereby Shortening culturing period reduces aquaculture cost and risk.The use of bacillus pumilus probiotics preparation has also obviously improved the commercial quality of prawn, has increased dressing percentage.In addition, this result also shows in conjunction with hemolytic experiment: bacillus pumilus LV149 has biological safety.
Embodiment 5: the restraining effect of bacillus pumilus probiotics preparation to the prawn vibrio infection
Get Environment of Litopenaeus vannamei Low a collection of, the long 8.1cm of average body, mean body weight 7.5g.Support after 3 days temporarily, be divided into control group C1, C2, C3, C4 and experimental group E1, E2, E3, E4, every group of each 40 tail prawns.The experimental group prawn commodity prawn feed that is added with the bacillus pumilus probiotics preparation (No. 2 material of " permanent emerging board " penaeus vannamei boone feed) of throwing something and feeding, the control group above-mentioned commodity prawn feed of only throwing something and feeding.Bacillus pumilus probiotics preparation addition means and prawn feeding method and daily administration method are with embodiment 4.To not adding Vibrio harveyi E385 suspension on the same group prawn culturing cylinder, making Vibrio harveyi E385 is 2 * 10 at the final concentration of raising water body when raising 5 days, 10 days, 15 days, 20 days
6Cfu/ml, prawn is soaked after 24 hours in the water that contains Vibrio harveyi E385, normally changes water, throws something and feeds, and observes ingesting of prawn and shows and add up mortality ratio, the results are shown in Table 3.
Table 3: the retarding effect that bacillus pumilus infects the Environment of Litopenaeus vannamei Low Vibrio harveyi
By as seen from Table 3: adopt the forage feed prawn 5 days that is added with the bacillus pumilus probiotics preparation, prawn namely shows has certain resistibility to Vibrio harveyi, and mortality ratio is lower than control group; After throwing something and feeding 10 days, 15 days, the prawn mortality ratio is starkly lower than control group, and after throwing something and feeding 20 days, the mortality ratio of the prawn of experimental group only has 5%.Result shows: in the cultivation of reality is used, bacillus pumilus can suppress Vibrio harveyi E385, adhere to using the bacillus pumilus probiotics preparation more than 10 days, can greatly reduce the morbidity of vibriosis penaeus, life-time service can be stopped the generation of vibriosis substantially.
Claims (9)
1. bacillus pumilus (Bacillus pumilus) LV149, deposit number: CCTCC NO:M2011411.
2. the application of bacillus pumilus claimed in claim 1 (Bacillus pumilus) LV149 in preparation inhibition vibrios probiotics.
3. application according to claim 2, it is characterized in that, described vibrios is Vibrio harveyi E385, Vibrio parahaemolyticus E154, Vibrio parahaemolyticus E347, Vibrio parahaemolyticus E346, wheel animalcule vibrios E231, Vibrio harveyi E379, Vibrio harveyi E345, Vibrio furnissii E341, Vibrio vulnificus 1.1758, vibrio alginolyticus E333, vibrio alginolyticus A056 or vibrio alginolyticus E066.
4. a bacillus pumilus probiotics preparation, is characterized in that, with bacillus pumilus claimed in claim 1 (Bacillus pumilus) LV149 as active ingredient.
5. the preparation method of a bacillus pumilus probiotics preparation claimed in claim 4, is characterized in that, as fermentation strain, through solid state fermentation, oven dry makes after pulverizing with bacillus pumilus (Bacillus pumilus) LV149.
6. preparation method according to claim 5, it is characterized in that, concrete steps are: bacillus pumilus (Bacillus pu milus) LV149 is inoculated into to cultivate in the LB substratum makes seed culture fluid, be inoculated in solid-state fermentation culture medium with 5%～10% inoculum size again, fermentation time is 20～48 hours, leavening temperature is 30 ℃, fermented product is 50 ℃ of oven dry in baking oven, the oven dry thing was pulverized 60 mesh sieves, be the bacillus pumilus probiotics preparation, described solid-state fermentation culture medium is comprised of material and water, described material is by total mass mark 100%, comprise rice bran 5～20%, wheat bran 10～30%, dregs of beans 20～40%, Semen Maydis powder 20～40%, skim-milk 1～5%, sucrose 1～5%, yeast extract 0.1～2%, NaCl0.1～1% and feeding many ore deposits 0.1～0.5%, material-water ratio is 1:1～1.3.
7. bacillus pumilus probiotics preparation claimed in claim 4 is as the application of fodder additives.
8. application according to claim 7, is characterized in that, described feed is prawn feed.
9. application according to claim 8, is characterized in that, the bacillus pumilus probiotics preparation is pressed 0.1～1% of prawn feed weight and added.
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| CN103602608B (en) * | 2013-08-02 | 2015-07-01 | 国家海洋局第三海洋研究所 | Application of bacillus pumilus in prawn culture |
| CN104087529B (en) * | 2014-06-29 | 2016-08-31 | 新疆农业科学院微生物应用研究所 | bacillus pumilus (Bacillus pumilus) JY2 and application thereof |
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| CN104371957A (en) * | 2014-11-17 | 2015-02-25 | 青岛根源生物技术集团有限公司 | Bacillus pumilus viable preparation and solid fermentation method and applications thereof |
| CN104593299A (en) * | 2015-01-08 | 2015-05-06 | 国家海洋局第三海洋研究所 | Strain for improving intestinal tracts of aquatic animals and application thereof |
| CN106676029B (en) * | 2016-05-03 | 2021-06-11 | 天津昌农科技有限责任公司 | Bacillus pumilus strain, and microecological preparation and feed thereof |
| CN108060096A (en) * | 2017-12-12 | 2018-05-22 | 中国水产科学研究院黄海水产研究所 | A kind of probiotic combinations and its application in litopenaeus vannamei seed rearing |
| CN108740289A (en) * | 2018-08-08 | 2018-11-06 | 广东绿百多生物开发有限公司 | A kind of fermented feed applied to prawn culturing |
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