CN102586144A - 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: M2011411. 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 active bacillus pumilus of vibrios, and utilize its probiotics preparation as active ingredient.
Background technology:
Along with developing rapidly of mass-producing, intensive aquaculture industry; Aquiculture disease frequently takes place, and for control disease, some antibiotic medicines are widely used in aquaculture and abuse; Cause not only that the environment Chinese traditional medicine is residual, bacterial drug resistance increases; And because the flowability of water body environment, drug resistant gene horizontal transfer more easily causes the appearance of more persisters.In order to tackle resistance, have to strengthen consumption in the breed and constantly change drug kinds, and then cause 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 focus of the current aquaculture of alternate bio-control method becoming; Wherein being applied in the aquaculture of probiotic bacterium more and more comes into one's own; They not only have the effect that suppresses bacterial pathogen, also have the aquatic animal of promotion and grow, improve effects such as immunity.
Aquatic products is used probiotic products on the market, a multitude of names, and layer goes out infinite, gets up to exist following problem but summarize: the probiotic bacterium of (1) most products is directed to the probiotic bacterium kind of human and terrestrial animal.Whether these Lu Yuan probiotic bacteriums can then almost not have relevant report in the enteron aisle of water body environment or hydrocoles by long-term surviving.Panigrahi etc. (2004) think directly that then those use for reference the deficiency that there is difficult field planting in aquatic products probiotic bacterium that livestock and poultry thinkings are developed, can not in digestive canal of aquatic animal, retain and breeding in a large number.(2) the aquatic products probiotic products is main with water conditioner, 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) probiotic products as additive agent for feeding is common with subtilis, Bacillus licheniformis, bacillus cereus, the rare genus bacillus of other type, and the common flora that above-mentioned three kinds of genus bacillus are not ocean environment.(4) the enzyme active of bacterial strain fruit is indeterminate in the aquatic products probiotic products, fungistatic effect is indeterminate, and this binomial index is to estimate the core index of the effect of feeding probiotic bacterium.Use maximum genus bacillus to be example with current aquatic products probiotic bacterium, the genus bacillus of having reported at present is antibiotic to have 169 kinds, and these microbiotic mainly are the peptide classes, and multiple acting is in gram positive bacterium.The enteron aisle The main pathogenic fungi of people and terrestrial animal is a 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 a Gram-negative bacteria, like vibrios.Genus bacillus is also rarely reported for these inhibition effects from the Gram-negative bacteria of aquatic animal.Therefore can not indiscriminately imitate peculation, the genus bacillus of inhibition effect etc. need be arranged Gram-negative bacterias such as vibrios to the characteristics screening of aquatic animal.In conjunction with the problems referred to above, we press for from the indigenous environment of aquatic animal to grow and enteron aisle and separate probiotic strain, and with enzymic activity and the bacteriostatic activity leading indicator as screening and evaluation effect.
Summary of the invention:
First purpose of the present invention provides a kind of separation from healthy Environment of Litopenaeus vannamei Low intestines wall mucous membrane and have enzymatic activity high and press down the active bacillus pumilus of vibrios (Bacillus pumilus) LV149; This bacterium is preserved in Chinese typical 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 intestines wall mucous membrane.
The biological property of bacillus pumilus (Bacillus pumilus) LV149 is following:
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: 5-35 ‰, 7% above NaCl does not grow, and growth appropriate pH scope is 5-9.
Gram-positive, catalase (-), oxydase (+), V-P tests (-); Nitrate reduction (-), starch hydrolysis (+), gelatine liquefication (+), the D-glucose that can ferment, L-arabinose, D-N.F,USP MANNITOL, D-glucose; Glucose fermentation is aerogenesis not, PD (-), Citrate trianion utilization (+), 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 shown in SEQ ID NO.1.Through BLAST comparison, itself and the consistent degree 100% of 16S rRNA gene of many bacillus pumilus (Bacillus pumilus) of announcement are below 99% with the consistent degree of other genus bacillus.Therefore, this bacterium of Molecular Identification is a 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 biochemical identification to bacterium.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, and this bacterium is preserved in Chinese typical 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 the difference of the two finger printing.Visible by Fig. 2; 7 random primers that adopt all can provide the finger printing of two bacillus pumilus; And every primer all can demonstrate the two tangible difference; Though this result shows bacillus pumilus LV149 and bacillus pumilus LV479 and belongs to same kind that hereditary difference is bigger, causes it that different phenotypic characteristics is arranged.
Find that through experiment bacillus pumilus of the present invention (Bacillus pumilus) LV149 can produce proteolytic enzyme, lypase and glycase 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 the Environment of Litopenaeus vannamei Low of feeding in the feed to it; The result finds 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, and 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%; Calculate the back feed coefficient and reduced by 12.1%.Explanation thus uses the bacillus pumilus probiotics preparation can reduce feed coefficient significantly in the breed of prawn, promotes the prawn growth, thereby shortens the culture-cycle, 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 combines hemolytic experiment also to show: 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 then only throwing something and feeding.Prawn soaked 24 hours in containing the water of Vibrio harveyi after; Normally change water, throw something and feed; Observe ingest performance and the statistics mortality ratio of prawn; The result shows: adopt the rat chow prawn 5 days be added with the bacillus pumilus probiotics preparation, prawn promptly 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 has only 5%; Show thus: in culturing application, bacillus pumilus (Bacillus pumilus) LV149 can suppress Vibrio harveyi, adheres to using the bacillus pumilus probiotics preparation more than 10 days; Can reduce the morbidity of vibriosis penaeus greatly, life-time service can be stopped the generation of vibriosis basically.
Therefore, second purpose of the present invention provides 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 provides 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 provides a kind of preparation method of bacillus pumilus probiotics preparation, it is characterized in that, as fermentation strain, through solid state fermentation, oven dry is pulverized the back and made 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 in the LB substratum to cultivate processes seed culture fluid, be inoculated in the solid-state fermentation culture medium with 5%～10% inoculum size, fermentation time is 20～48 hours; Leavening temperature is 30 ℃; Fermented product is 50 ℃ of oven dry in baking oven, and the oven dry thing was pulverized 60 mesh sieves, was the bacillus pumilus probiotics preparation; Described solid-state fermentation culture medium is made up 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% (feed technology ltd of Guangzhou China Telecom, name of product is: the many ore deposits of Jiang Feng; Article No. is: JC1000), material-water ratio is 1: 1～1.3.
The 5th purpose of the present invention provides the application of bacillus pumilus probiotics preparation as fodder additives.Bacillus pumilus probiotics preparation of the present invention and the prawn finished product feedstuff mixed by weight 0.1～1% is even; Adsorbed 10～60 minutes; The prawn of can throwing something and feeding not only can suppress the growth of prawn enteron aisle pathogenic vibrio, reduce the generation of vibriosis penaeus, promotes the prawn growth simultaneously, reduces feed coefficient, improves commercial quality, shortens the culture-cycle; Culture risk, reduction aquaculture cost thereby reduce, 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; Make the bacillus pumilus probiotics preparation as activeconstituents through solid state fermentation, drying and crushing with bacillus pumilus (Bacillus pumilus) LV149; It is added in the prawn feed, and the prawn of feeding not only can play the generation that suppresses the growth of prawn enteron aisle pathogenic vibrio, reduces vibriosis penaeus; Promote the prawn growth simultaneously, reduce feed coefficient, improve commercial quality, shorten the culture-cycle; Culture risk, reduction aquaculture cost thereby reduce, and biological safety is high, in aquaculture, is with a wide range of applications.
Bacillus pumilus of the present invention (Bacillus pumilus) LV149 is preserved in Chinese typical culture collection center (CCTCC), address on November 23rd, 2011: Chinese Wuhan Wuhan University, deposit number: CCTCC NO:M2011411.
Description of drawings:
Fig. 1 is three kinds of enzymic activity detections of bacillus pumilus LV149 figure, wherein 1: amylase activity; 2: lipase activity; 3: protease activity;
Fig. 2 is that the RAPD molecular fingerprint collection of illustrative plates of bacillus pumilus LV149 and bacillus pumilus LV479 compares.Wherein: 1,3,5,7,9,11,13 are respectively Shanghai gives birth to worker's random primer S9, S10, and S11, S12, S13, S14, S15 is to the RAPD amplification of bacillus pumilus LV149; 2,4,6,8,10,12,14 are respectively Shanghai gives birth to worker's random primer S9, S10, and S11, S12, S13, S14, S15 is to the RAPD amplification of 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 specify 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 markets, every batch 30 tail, therefrom picking is individual big, 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(tric)fraction 75%, use aseptic seawater flushing again, get its enteron aisle after the dissection on aseptic masking foil; Draw the sterilization seawater with syringe; Rinse out the ight soil in the enteron aisle rapidly, every enteral irrigation 2-3 time is with in the visual inspection enteron aisle not till the residual content.In aseptic homogenizer, add SPSS with enteron aisle homogenate.Get the 0.5mL homogenate and be enteron aisle adhesion bacterial suspension by 10 times of serial dilutions with SPSS (1%NaCl).
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 flat board is fallen in sterilization.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 prawn enteron aisle adhesion bacterium.
(2) three kinds of enzymic activity enteron aisles adhere to bacteria screening
Detect protease activity, lipase activity, the amylase activity that enteron aisle adheres to bacterium by ordinary method.The result finds that the bacterial strain that can produce three kinds of extracellular enzymes in the 500 strain bacteriums simultaneously has 90 strains.
(3) the enzymatic activity high enteron aisle adheres to screening bacteriostatic activity strain in the bacterium
Adopt dull and stereotyped filter paper diffusion process 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 substratum is the LB substratum, and indicator is vibrio alginolyticus A056, Vibrio harveyi E385, Vibrio parahaemolyticus E154.Tried bacterium and indicator and all used LB substratum incubated overnight in advance.It is dull and stereotyped to adopt sterilization 1%NaCl suitably to dilute back coating LB indicator liquid, obedient filter paper on the LB flat board, and diameter 5mm, each filter paper drip and are tried bacterium liquid 20 μ L, cultivate 12-24 hour for 30 ℃, observe the appearance of inhibition zone, and measure the diameter of inhibition zone.The result shows: 90 strains have in the enzymatic activity high enteron aisle adhesion bacterium has only 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
From the bacterium of 90 strain tool enzymic activitys, select growth 69 strain bacteriums very fast, that be easy to cultivate and carried out 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.The result shows: they belong to acinetobacter (Acinetobacter), bacillus (Bacillus), Staphylococcus (Staphylococcus), Pseudoalteromonas genus (Pseudoalteromonas), Aeromonas (Aeromonas) respectively, have a liking for salt zygosaccharomyces (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 shown in SEQ ID NO.1.Through BLAST comparison, itself and the consistent degree 100% of 16S rRNA gene of many bacillus pumilus (Bacillus pumilus) of announcement are below 99% with the consistent degree of other genus bacillus.Therefore, this bacterium of Molecular Identification is a 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 biochemical identification to bacterium.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 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 the bacterium,, has only a bacillus pumilus LV149 that three kinds of vibrios are had obvious restraining effect comprising other bacillus pumilus strain.This is illustrated in the 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 the worker has carried out the RAPD amplification to bacillus pumilus LV149 and LV479 respectively, to detect the difference of the two finger printing.RAPD amplification program: 93 ℃ of preparatory sex change 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, ultraviolet imagery, and the result sees Fig. 2.Visible by Fig. 2; 7 random primers that adopt all can provide the finger printing of two bacillus pumilus, and every primer all can demonstrate the two tangible difference, belong to same kind though this result shows LV149 and LV479; But hereditary difference is bigger, 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 filter paper diffusion process of same employing.Judge the power of bacteriostasis according to the size of inhibition zone, the antimicrobial spectrum analytical results is seen 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.
Visible by table 1, see that 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 ℃.The result finds 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: with LB is substratum, and 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: with LB is substratum, and the ratio of primary seed solution with 2% (volume percent) joined in the LB liquid nutrient medium, and 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 (feed technology ltd of Guangzhou China Telecom; Name of product is: the many ore deposits of Jiang Feng; 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 ℃, are that 5% ratio adds secondary seed solution 500ml with inoculum size, mix.In 10L beaker that air-permeable envelope seals, cultivated 40 hours for 30 ℃, 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 that adds massfraction 0.9%, it is dull and stereotyped to get 100 μ L coating LB, cultivates 24 hours for 30 ℃, calculates viable count.Reach 1.2 * 10 through calculating the bacillus pumilus probiotics preparation viable count that is obtained
9Cfu/g.
Embodiment 4: the bacillus pumilus probiotics preparation is to the promoter action and the biological safety evaluation of prawn growth
It is a collection of to get Environment of Litopenaeus vannamei Low, 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 prawn.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 feed) of throwing something and feeding, the control group above-mentioned commodity prawn feed of then 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 by weighing, and joins in a certain amount of seawater (15% feed is heavy), fully stirs, so that the genus bacillus on the carrier can be shed in the water.This seawater is evenly joined in the feed, stir, adsorb and to throw something and feed after 20 minutes with hand.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 per 15 days, and took out liver then and weigh, calculate liver body index.45 days culture-cycles, add up altogether 3 times, add up feed coefficient after 45 days.The result sees table 2.
Table 2: the bacillus pumilus probiotics preparation is to the promotes growth effect of Environment of Litopenaeus vannamei Low
Visible by 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 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%; Calculate the back feed coefficient and reduced by 12.1%.Explanation thus uses the bacillus pumilus probiotics preparation can reduce feed coefficient significantly in the breed of prawn, promotes the prawn growth, thereby shortens the culture-cycle, 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 combines hemolytic experiment also to show: bacillus pumilus LV149 has biological safety.
Embodiment 5: the bacillus pumilus probiotics preparation is to the restraining effect of prawn vibrio infection
It is a collection of to get Environment of Litopenaeus vannamei Low, 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 prawn.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 feed) of throwing something and feeding, the control group above-mentioned commodity prawn feed of then 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 the 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 ingest performance and the statistics mortality ratio of prawn, and the result sees table 3.
Table 3: the retarding effect that bacillus pumilus infects the Environment of Litopenaeus vannamei Low Vibrio harveyi
Visible by table 3: adopt the rat chow prawn 5 days that is added with the bacillus pumilus probiotics preparation, prawn promptly 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 has only 5%.The result shows: in the breed of reality is used; Bacillus pumilus can suppress Vibrio harveyi E385; Adhere to using the bacillus pumilus probiotics preparation more than 10 days, can reduce the morbidity of vibriosis penaeus greatly, life-time service can be stopped the generation of vibriosis basically.
Claims (9)
1. bacillus pumilus (Bacillus pumilus) LV149, deposit number: CCTCC NO:M2011411.
2. the described bacillus pumilus of claim 1 (Bacillus pumilus) LV149 suppresses the application in the vibrios probiotics in preparation.
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 11758, vibrio alginolyticus E333, vibrio alginolyticus A056 or vibrio alginolyticus E066.
4. a bacillus pumilus probiotics preparation is characterized in that, with the described bacillus pumilus of claim 1 (Bacillus pumilus) LV149 as active ingredient.
5. the preparation method of the described bacillus pumilus probiotics preparation of claim 4 is characterized in that, as fermentation strain, through solid state fermentation, oven dry is pulverized the back and made with bacillus pumilus (Bacillus pumilus) LV149.
6. preparation method according to claim 5 is characterized in that, concrete steps are: bacillus pumilus (Bacillus pumilus) LV149 is inoculated into to cultivate in the LB substratum processes seed culture fluid; Be inoculated in the solid-state fermentation culture medium with 5%～10% inoculum size again; Fermentation time is 20～48 hours, and leavening temperature is 30 ℃, and 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 made up of material and water, and 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%, material-water ratio is 1: 1～1.3.
7. the described bacillus pumilus probiotics preparation of claim 4 is used as fodder additives.
8. application according to claim 7 is characterized in that, described feed is a 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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