CN108865953A - One plant of wide spectrum inhibits bacillus and its composite bacteria preparation of aquatic products vibrio pathogen - Google Patents
One plant of wide spectrum inhibits bacillus and its composite bacteria preparation of aquatic products vibrio pathogen Download PDFInfo
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Abstract
The present invention relates to the bacillus subtilis BSXE-1601 that one plant of wide spectrum inhibits aquatic products vibrio pathogen；The invention further relates to more than one to state bacillus subtilis composite bacteria preparation as main component；Meanwhile the invention further relates to the preparation methods that more than one state bacillus subtilis composite bacteria preparation as main component.Bacillus subtilis (Bacillus subtilis) BSXE-1601 of the invention is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), culture presevation number is CGMCC NO.15949, and the deposit date is on June 19th, 2018.The bacillus subtilis can effectively inhibit the growth of the Vibrios such as vibrio parahaemolytious, vibrio alginolyticus, Vibrio vulnificus and Vibrio harveyi, while can also promote the growth of cultivated animals to a certain extent, improve survival rate and bait utilization.
Description
Technical field
The invention patent belongs to microorganism and its application field more particularly to one plant of wide spectrum inhibits aquatic products vibrio pathogen
Bacillus subtilis (Bacillus subtilis) BSXE-1601；The invention further relates to more than one to state bacillus subtilis
(Bacillus subtilis) BSXE-1601 composite bacteria preparation as main component；Meanwhile the invention further relates to more than one
State the preparation method of bacillus subtilis (Bacillus subtilis) BSXE-1601 composite bacteria preparation as main component.
Background technique
Vibriosis is as caused by vibrio bacteria, is the important disease of one of aquaculture activities.Vibrios disease tool
There is burst strong, the high feature of the death rate can betide in a variety of aquiculture animals, such as fish, shellfish and shell-fish.
In fish culture, leading to the arch-criminal of all village's net cage Larimichthys crocea Large Scale Deaths of Bay, Ningde, Fujian Province Beidou in 1999 is exactly molten algae
Vibrios and vibrio parahaemolytious；In shellfish culture, the main pathogenic bacteria of vibriosis of clam is also vibrio alginolyticus and vibrio parahaemolytious；
The Hepatopancreatic necrosis syndrome occurred in prawn culturing, pathogenic bacteria are mainly exactly vibrio parahaemolytious.The outburst of vibriosis can
The mortality for leading to cultivated animals causes huge economic loss.Vibrios disease can even be made in the sprawling of nursery enterprise
At the total crop failure of nursery unit, consequence is very serious.It therefore, is one of the research field of aquatic products disease to the prevention and control of this kind of disease,
By the concern of domestic and international aquatic products research worker.
For many years, the chemotherapys such as antibiotic and disinfectant are mainly used to the prevention and treatment of vibrios disease, antibiotic
A large amount of endurance strains can occur by natural selection in a large amount of uses, be difficult to control disease more.And chemicals is a large amount of
Use, not only result in pathogenic bacterial strains drug resistance enhancing, while can also polluted-water, reduce the immunity of aquaculture organism,
Aquaculture organism is damaged, vicious circle is formed.
Carrying out prevention and treatment using probiotics is a kind of novel biological control method gradually developed in recent years, and principle is
Using the antagonism between microorganism, the probiotics that can effectively inhibit vibrios to grow, fungus treatment, to reach raw are filtered out
The purpose of state diseases prevention.Meanwhile in the research to probiotics, it was found that probiotics may be implemented in addition to having to disease microorganism
Outside the advantages of effectively controlling, also having improves cultivation water, enhances biological immune power and improves many beneficial effects such as yield.
Therefore, method aquaculture vibrios disease prevented and treated using probiotics will be expected to as following the main direction of development it
One.
Summary of the invention
An object of the present invention is to provide a bacillus subtilis (Bacillus subtilis) BSXE-1601,
The growth that the Vibrios such as vibrio parahaemolytious, vibrio alginolyticus, Vibrio vulnificus and Vibrio harveyi can effectively be inhibited, to by these types
A variety of aquiculture diseases caused by vibrios have effectively preventing effect, while can also promote cultivated animals to a certain extent
Growth, improve survival rate and bait utilization.
Bacillus subtilis (Bacillus subtilis) BSXE-1601 of the invention is preserved in Chinese microorganism strain
Preservation administration committee common micro-organisms center (CGMCC), culture presevation number are CGMCC NO. 15949, the deposit date is
On June 19th, 2018, preservation address are Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
The main feature of bacillus subtilis (Bacillus subtilis) BSXE-1601 of the invention is as follows：
1. morphological feature
Bacillus subtilis (Bacillus subtilis) BSXE-1601 shape on nutrient agar of the invention
At single bacterium colony be round or irregular shape, colony edge is irregular, and lawn is opaque, canescence, and surface wettability is easily chosen
It takes.Gram's staining is the positive, and thallus is rod-shaped, production gemma, and life in gemma, shape is ellipse.
2. physiological and biochemical property
Bacillus subtilis (Bacillus subtilis) BSXE-1601 of the invention has motility, Gram-positive
Bacterium produces gemma, cannot utilize propionate, and hydrolyzable starch can restore nitrate, and V-P measurement, catalase, PEARLITOL 25C produce acid
The measurement result that acid is produced with L-arabinose is the positive.
3. bacterial strain 16SrDNA is identified
Bacillus subtilis (Bacillus subtilis) BSXE-1601, is identified through 16S rDNA, find its with
The sequence similarity of bacillus subtilis determines that it belongs to bacillus subtilis through phylogenetic tree analysis up to 99% in Genebank
Pseudomonas (Bacillus subtilis) bacterium.
The present invention also provides a kind of above-mentioned bacillus subtilis (Bacillus subtilis) BSXE-1601 to prevent and treat
The application of the aquiculture disease as caused by the Vibrios such as vibrio parahaemolytious, vibrio alginolyticus, Vibrio vulnificus and Vibrio harveyi.
The present invention also provides one kind with bacillus subtilis (Bacillus subtilis) BSXE-1601 be mainly at
Point composite bacteria preparation, the composite bacteria preparation be by bacillus subtilis (Bacillus subtilis) BSXE-1601 and
Bacillus cereus (Bacillus cereus) BCXE-01 is with 5~8:1 bacterial number ratio is combined.Described is waxy
The bacterial strain that bacillus (Bacillus cereus) BCXE-01 goes out for my laboratory screening, is now preserved in Chinese microorganism strain
Preservation administration committee common micro-organisms center (CGMCC), culture presevation number are CGMCC NO.6940, and the deposit date is 2012
In on December 7, in, preservation address is Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.The composite bacteria preparation can
To generate broad-spectrum antibacterial effect to the vibrio disease in aquaculture process, while it can effectively improve aquiculture animal
Specific growth rate and bait utilization promote the healthy growth of aquiculture animal and volume increase, increase income.
It is described with bacillus subtilis (Bacillus subtilis) BSXE-1601 composite bacteria agent as main component
Specific preparation process is as follows：
(1) seed liquor culture
Bacillus subtilis (Bacillus subtilis) BSXE-1601 and waxy bud for taking separation, purifying to obtain respectively
The conservation liquid of spore bacillus (Bacillus cereus) BCXE-01 is crossed on nutrient agar and 2216E solid plate, and
It is respectively placed under the conditions of 37 DEG C and 28 DEG C, activation is for 24 hours；Then bacillus subtilis (Bacillus subtilis) is picked them separately
BSXE-1601 and bacillus cereus (Bacillus cereus) BCXE-01 single colonie are inoculated in the nutrient agar after improvement
In culture medium, the seed liquor for obtaining two kinds of bacteriums for 24 hours is cultivated under the conditions of 28 DEG C, 190rpm；
The formula of the nutrient agar is as follows：Peptone 10g, powdered beef 3g, sodium chloride 5g, agar 20g, distillation
Water 1000ml, pH 7.3 uses 121 DEG C of prepared culture medium after 101KPa sterilizing 30min；
The formula of the 2216E solid medium is as follows：Peptone 10g, yeast powder 5g, agar 20g, Chen Haishui
1000mL, pH 7.6 uses 121 DEG C of prepared culture medium after 101KPa sterilizing 30min；
Nutrient agar formula after the improvement is as follows：Peptone 10g, powdered beef 3g, sodium chloride 5g,
KH2PO41.5g, MgSO4·7H2O 0.5g, distilled water 1000ml, pH 7.3, by 121 DEG C of prepared culture medium, 101KPa
It is used after sterilizing 30min；
(2) expand culture
The seed liquor of two kinds of bacterium presses bacillus subtilis (Bacillus subtilis) BSXE-1601：Bacillus cereus
BCXE-01=5~8 (Bacillus cereus):After 1 bacterial number ratio is mixed, control seed mixture liquid at this time
Bacterial content is 1*106~1*107Cfu/ml, according to seed mixture liquid：The volume ratio of nutrient agar after improvement is 1:
10 ratio, by seed mixture liquid be inoculated into equipped with improvement after nutrient agar fermentor in, in temperature be 28 DEG C,
Mixing speed is 190rpm, ventilatory capacity 4V/V.min, and culture is for 24 hours；
(3) preparation of composite bacteria agent
According to seed mixture liquid：The volume ratio of liquid fermentation medium is 1:Seed mixture liquid is inoculated into dress by 10 ratio
Have in the fermentor of liquid fermentation medium, the bacterial content for controlling seed mixture liquid at this time is 1*106~1*107Cfu/ml,
It is 28 DEG C, mixing speed 190rpm, ventilatory capacity 4V/V.min in temperature, after cultivating 48h, is concentrated to give high concentration bacterium solution；Institute
The formula for stating liquid fermentation medium is as follows：Peptone 10g, corn pulp 10g, glucose 10g, sodium chloride 5g, KH2PO41.5g
MgSO4·7H2O 0.5g, distilled water 1000ml, pH are adjusted to 7.0 for 121 DEG C of prepared culture medium, and 101KPa sterilizes after 30min
It uses；
By peanut meal, corn flour, diatomite, bentonite according to 2:2:1:1 weight ratio is uniformly mixed and crushes, and is made
Auxiliary material carrier；By auxiliary material carrier and high concentration bacterium solution according to 1:2 weight ratio is uniformly mixed, and adds polysorbate and xanthan
Glue, wherein the additional amount of polysorbate is the 2-3% of high concentration bacterium solution quality, and the additional amount of xanthan gum is high concentration bacterium solution quality
1-3%；It is uniformly mixed, drying is crushed, remixed uniformly to get composite bacteria preparation.
Specifically, this technology can produce following good effect compared with prior art：
It, can be high simultaneously 1. the antimicrobial spectrum of bacillus subtilis (Bacillus subtilis) BSXE-1601 of the invention is wide
Effect inhibits the multiple diseases caused by a variety of pathogenic vibrios such as vibrio parahaemolytious, vibrio alginolyticus, Vibrio vulnificus and Vibrio harveyi,
To the inhibiting rate of vibrios up to 80% or more.And the growth of bacillus of the invention is fast, and in light, seawater, temperature 60-100
DEG C, pH can effectively play bacteriostatic activity in the range of being 3-10, a variety of aquaculture kind vibriosises evil can be widely used in
Prevention and treatment.In addition, bacillus of the invention can produce a variety of secondary metabolites, the growth of cultivated animals can be effectively facilitated,
Immunity is improved, survival rate is increased, promotes absorption and utilization of the cultivated animals to bait.
2. of the invention by bacillus subtilis (Bacillus subtilis) BSXE-1601 and bacillus cereus
(Bacillus cereus) BCXE-01 is with 5~8:1 volume ratio cooperates obtained composite bacteria agent that antimicrobial spectrum is made to expand as brilliance
Vibrios, vibrio parahaemolytious, vibrio alginolyticus, Vibrio vulnificus and Vibrio harveyi increase to 91.28% and simultaneous to the inhibiting rate of vibrios
Tool improves the effect of breeding water body environment, and composite bacteria agent is substantially better than single strain to the control efficiency of vibrio pathogen, it is seen that this
There is synergistic effect between two kinds of bacteriums.
Detailed description of the invention
The phylogenetic tree that Fig. 1 is bacillus subtilis BSXE-1601 is analyzed；
Fig. 2 is the growing state of bacillus subtilis BSXE-1601 at different temperatures；
Fig. 3 is growing state of the bacillus subtilis BSXE-1601 at different pH；
Fig. 4 is growing state of the bacillus subtilis BSXE-1601 under different rotating speeds.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.
Reagent and instrument
Culture medium：
2216E fluid nutrient medium：Tryptone 10g, yeast powder 5g, Chen Haishui 1000mL, pH7.6,121 DEG C, 101KPa
Sterilize 30min.
2216E solid medium：Tryptone 10g, yeast powder 5g, agar 20g, Chen Haishui 1000mL, pH7.6,121
DEG C, 101KPa sterilizing 30min.
Nutrient agar：Peptone 10g, powdered beef 3g, sodium chloride 5g, agar 20g, distilled water 1000ml, pH are
7.3,121 DEG C, 101KPa sterilizing 30min.
Nutrient broth medium：Peptone 10g, powdered beef 3g, sodium chloride 5g, distilled water 1000ml, pH 7.3,121
DEG C, 101KPa sterilizing 30min.
Nutrient agar after improvement：Peptone 10g, powdered beef 3g, sodium chloride 5g, KH2PO41.5g, MgSO4·
7H2O 0.5g, distilled water 1000ml, pH 7.3 use 121 DEG C of prepared culture medium after 101KPa sterilizing 30min.
Liquid fermentation medium：Peptone 10g, corn pulp 10g, glucose 10g, sodium chloride 5g, KH2PO41.5g
MgSO4·7H2O 0.5g, distilled water 1000ml, pH are adjusted to 7.0, by 121 DEG C of prepared culture medium, 101KPa sterilizing 30min
After use.
TCBS agar (the rich biology in Qingdao sea)：89.1g is weighed, is dissolved in 1000ml distilled water, boils, be cooled to 45-
At 50 DEG C, it is poured into sterilized petri dishes.
Instrument：Optical microscopy, temperature control shaking table, superclean bench, high-pressure sterilizing pot, pH meter, supercentrifuge, constant temperature training
Support case etc..
The screening and separation of 1 bacillus subtilis BSXE-1601 of embodiment
From water body, bed mud and the enteron stool of prawn of prawn culturing field acquisition cultivating pool, nutrient broth medium is used
After carrying out Enrichment of bacteria, pass through the isolated pure culture of plate streaking.
Isolated single strain is inoculated into and is coated with vibrio parahaemolytious, vibrio alginolyticus, Vibrio vulnificus and Vibrio harveyi
Nutrient agar panel on, by observation whether there is or not inhibition zone appearances, finally screen and obtain one plant and have preferable suppression to this 4 kinds of vibrios
The bacterial strain of bacterium effect, is named as BSXE-1601.
The taxonomic identification of 2 bacillus subtilis BSXE-1601 of embodiment
1. morphological feature
It is in the single colonie feature of nutrient agar by bacillus subtilis BSXE-1601：Round or irregular shape
Shape, colony edge is irregular, and lawn is opaque, canescence, surface wettability, easy picking.Gram's staining and microscopy are shown：The bacterium
It is rod-shaped for Gram-positive, gemma is produced, life in gemma is oval.
2. molecular genetics feature
Extract bacillus subtilis BSXE-1601 DNA, to the region 16SrDNA amplification after, sequencing, as the result is shown its with
Bacillus subtilis sequence height similar (being detailed in Figure of description 1), finally determines that it belongs to bacillus subtilis Pseudomonas
(Bacillus subtilis)。
3. physiological and biochemical property
The physiological and biochemical property of bacillus subtilis BSXE-1601 is as shown in table 1：
1 bacillus subtilis BSXE-1601 physiological and biochemical property of table
The measurement of 3 bacillus subtilis BSXE-1601 growth conditions of embodiment
1. taking bacillus subtilis BSXE-1601 bacterium solution, it is inoculated in nutrient broth medium, is respectively placed in 22 DEG C, 24
DEG C, 26 DEG C, its growing state of culture assay under the conditions of 28 DEG C and 30 DEG C.
2. taking bacillus subtilis BSXE-1601 bacterium solution to be inoculated in pH value respectively is 6.5,7.0,7.5,8.0 and 8.5
In nutrient broth, its growing state of culture assay under the conditions of being placed in 28 DEG C.
3. taking bacillus subtilis BSXE-1601 bacterium solution, it is inoculated in nutrient broth medium, is 70r/ in revolving speed
It is cultivated under conditions of min, 100r/min, 130r/min, 160r/min and 190r/min, measures its growing state respectively.
The result shows that：As shown in Figure of description 2-4, bacillus subtilis BSXE-1601 is at 28 DEG C, pH7.0, revolving speed
It is grown under the conditions of 190r/min best.
The measurement of 4 bacillus subtilis BSXE-1601 fungistatic effect of embodiment
Bacillus subtilis BSXE-1601 bacterium solution is taken, is inoculated into respectively is coated with Vibrio harveyi, false alternately list after cultivation
Born of the same parents bacterium, Vibrio splindidus, Edwardsiella tarda, vibrio parahaemolytious, Huanghai Sea Shewanella, Aeromonas hydrophila, vibrio alginolyticus, wound
On the nutrient agar panel for hurting vibrios and Streptococcus iniae, then the inhibition zone of formation is measured, is calculated.
The result shows that：As shown in table 2, bacillus subtilis BSXE-1601 is in addition to Edwardsiella tarda, thermophilic aqueous vapor list
Born of the same parents bacterium and Streptococcus iniae without obvious inhibiting effect outside, have inhibitory effect to other kinds pathogen.Especially to secondary haemolysis arc
Bacterium, vibrio alginolyticus, the inhibitory effect of Vibrio vulnificus and Vibrio harveyi are significant, and the antibacterial circle diameter formed is respectively
19.31mm, 19.67mm, 25.14mm and 15.31mm.
Inhibitory effect of the 2 bacillus subtilis BSXE-1601 of table to pathogen
Note："-" is for muting sensitive or in vain
Embodiment 5 cultivates influence of the duration to bacillus subtilis BSXE-1601 fungistatic effect
Bacillus subtilis BSXE-1601 bacterium solution is taken to carry out inoculated and cultured in nutrient broth medium, in the of culture
18h, for 24 hours, 48h, 72h and 96h take bacterium solution to be added on the nutrient agar panel for filling Vibrio vulnificus, then to the antibacterial of formation
Circle is measured, is calculated.
The result shows that：As shown in table 3, fungistatic effect of the bacillus subtilis BSXE-1601 after culture for 24 hours is best, with
The extension of incubation time, fungistatic effect gradually decrease.
Influence of the different incubation times of table 3 to bacillus subtilis BSXE-1601 fungistatic effect
Influence of 6 high temperature of embodiment to bacillus subtilis BSXE-1601 fungistatic effect
Bacillus subtilis BSXE-1601 bacterium solution is taken persistently to cultivate 60min, phase under the conditions of 50 DEG C, 75 DEG C and 100 DEG C
Between, bacterium solution, which is drawn, respectively at 10min, 20min, 40min and 60min is added on the plate for filling Vibrio vulnificus, it is right afterwards for 24 hours
The inhibition zone of formation is measured, is calculated.
The result shows that：As shown in table 4, the fungistatic effect of bacillus subtilis BSXE-1601 is relatively more steady under the high temperature conditions
Fixed, bacteriostatic activity only reduces by 4.7% and 7.2% after heating 1h under the conditions of 50 DEG C and 75 DEG C, heats 1h under the conditions of 100 DEG C
Bacteriostatic activity also only reduces by 12.8% afterwards.Illustrate that bacillus subtilis BSXE-1601 can be protected under conditions of within 100 DEG C
Hold good bacteriostatic activity.
Influence of 4 high-temperature process of table to bacillus subtilis BSXE-1601 fungistatic effect
Influence of the 7 difference pH of embodiment to bacillus subtilis BSXE-1601 fungistatic effect
Take bacillus subtilis BSXE-1601 bacterium solution, be inoculated in respectively pH value be 3.0,4.0,5.0,6.0,7.0,8.0,
9.0, in 10.0,11.0 and 12.0 culture medium, after culture for 24 hours, take bacterium solution be added to fill Vibrio vulnificus nutrient agar it is flat
On plate, after culture for 24 hours, the inhibition zone of formation is measured, is calculated.
The result shows that：As shown in table 5, bacillus subtilis BSXE-1601 can be kept preferable under strongly acidic conditions
Fungistatic effect, but under strongly alkaline conditions, fungistatic effect can be increased with pH and be gradually decreased, until pH can completely lose when being 12
Bacteriostatic activity.
Influence of the 5 difference pH of table to bacillus subtilis BSXE-1601 fungistatic effect
Influence of 8 protease of embodiment to bacillus subtilis BSXE-1601 bacteriostatic activity
Bacillus subtilis BSXE-1601 bacterium solution is taken to be separately added into 2 sterile triangular flasks.An addition albumen wherein
Enzyme, another is without any processing, then by two triangular flask heating water bath 120min.Period, in 20min, 40min,
80min and 120min draws bacterium solution respectively from two triangular flasks and is added on the plate for filling Vibrio vulnificus, after culture for 24 hours,
The inhibition zone of formation is measured, is calculated.
The result shows that：As shown in table 6, after Protease Treatment 2h, bacillus subtilis BSXE-1601 still keeps higher antibacterial
Activity illustrates that the bacteriostatic activity of bacillus subtilis BSXE-1601 is not influenced by protease.
Influence of 6 Protease Treatment of table to bacillus subtilis BSXE-1601 bacteriostatic activity
The bacillus subtilis BSXE-1601 that splashes of embodiment 9 imitates the inhibition of litopenaeus vannamei culture water vibrios quantity
Fruit and the influence that prawn is grown
Sample plot point is selected in Chinese Marine University's aquaculture base, 1 test group of experimental setup and 1 control group, right
Test group is splashed bacillus subtilis BSXE-1601, and with no treatment, other cultivating conditions are consistent control group.
It first takes breeding water body to be applied on TCBS culture medium flat plate before experiment, carries out bacterium colony counting afterwards for 24 hours, be calculated as initial value.
After experiment starts, takes the water body of each group to detect amount of vibrio daily, persistently detect 5d.The specific life of prawn is calculated after cultivation 42d
Long rate, rate of body weight gain, survival rate and bait utilization.
The result shows that：As shown in table 7, in the inhibitory effect to prawn culturing water vibrios, bacillus subtilis is carried out
The preceding 2d of the processing of splashing of BSXE-1601, compared to control group, test group is significant to the inhibitory effect of vibrios, test group after 5d
Pond also remains preferable inhibitory effect to the average inhibition of vibrios up to 83.23% to vibrios.
As shown in table 8, in the influence grown to prawn, compared with the control group, the specific growth rate of prawn in test group,
Body weight increase rate, survival rate and bait utilization significantly increase.Illustrate to splash bacillus subtilis BSXE-1601 to prawn
Growth also has good facilitation.
Table 7 splash various concentration bacillus subtilis BSXE-1601 to litopenaeus vannamei culture water vibrios quantity
It influences
Embodiment 10 feeds bacillus subtilis BSXE-1601 and imitates to the inhibition of litopenaeus vannamei culture water vibrios quantity
Fruit and the influence that prawn is grown
Sample plot point is selected in Chinese Marine University's aquaculture base, tests 1 test group and 1 control group, test group
Bacillus subtilis BSXE-1601 bacterium solution is added in the feed of prawn, control group feeds basal feed always, other cultivation items
Part is consistent.
It first takes each breeding water body to be applied on TCBS culture medium flat plate respectively before experiment, carries out bacterium colony counting afterwards for 24 hours, be calculated as just
Initial value.After experiment starts, periodically takes the water body of each sink to detect its amount of vibrio daily, persistently detect 5d.After cultivating 42d, meter
Calculate specific growth rate, rate of body weight gain, survival rate and the bait utilization of prawn.
The result shows that：As shown in table 9, the prawn of test group feeds in the 5d after bacillus subtilis BSXE-1601, right
Vibrios average inhibition reaches 80.47% or more, illustrates that feeding bacillus subtilis BSXE-1601 has good suppression to vibrios
Production is used.
As shown in table 10, compared with the control group, withered feed to test group prawn in the influence grown to prawn
After the processing of careless bacillus BSXE-1601, specific growth rate, body weight increase rate, survival rate and the bait benefit of test group prawn
Risen with rate, illustrates that feeding bacillus subtilis BSXE-1601 has significant facilitation to the growth of prawn.
Table 9 feeds the bacillus subtilis BSXE-1601 of various concentration to litopenaeus vannamei culture water vibrios quantity
It influences
Table 10 feeds influence of the bacillus subtilis BSXE-1601 of various concentration to Growth of Litopenaeus vannamei
Embodiment 11 splash and feed bacillus cereus (Bacillus cereus) BCXE-01 to litopenaeus vannamei support
Grow the inhibitory effect of water vibrios quantity and the influence to prawn growth
Sample plot point is selected in Chinese Marine University's aquaculture base, 2 test groups of experimental setup and 1 control group, sprinkles
It spills test group bacillus cereus BCXE-01 bacterium solution is splashed into the sink of cultured prawn；Test group is fed by waxy gemma
Bacillus BCXE-01 bacterium solution is added in feed.Control group is without any processing, other cultivating conditions are consistent.
It first takes the water body of each sink to be applied on TCBS culture medium flat plate respectively before experiment, carries out bacterium colony counting afterwards for 24 hours, be calculated as
Initial value.After experiment starts, periodically takes the water body of each sink to detect its amount of vibrio daily, persistently detect 5d.After cultivating 42d,
Calculate specific growth rate, rate of body weight gain, survival rate and the bait utilization of prawn.
The result shows that：As shown in table 11, compared with the control group, splashing and feed bacillus cereus BCEX-01 can be effective
Inhibit the growth of prawn culturing water vibrios, the average inhibition for test group of splashing in 5d is 59.57%, feeds the suppression of test group
Rate processed is 52.15%.
As shown in table 12, compared with the control group, splash and feed the specific growth rate of test group prawn, body weight increase rate,
Survival rate and bait utilization significantly increase, but the effect for feeding test group shows waxy gemma bar better than test group of splashing
Bacterium BCXE-01, which equally has, promotes prawn growth, improves the effect of bait utilization.
Table 11 is splashed and feeds influence of the bacillus cereus BCXE-01 to litopenaeus vannamei culture water vibrios quantity
Table 12 is splashed and feeds influence of the bacillus cereus BCXE-01 to Growth of Litopenaeus vannamei
The antagonistic experiment of embodiment 12 bacillus subtilis BSXE-1601 and bacillus cereus BCXE-01
The single colonie of bacillus subtilis BSXE-1601 and bacillus cereus BCXE-01 are picked them separately in nutrient agar
Cross is crossed on culture medium, under the conditions of the plate of scribing line is placed in 28 DEG C, after culture for 24 hours, observes the growth feelings of two plants of bacterium
Condition.
The result shows that：Antagonism is not present between two plants of bacterium.
The fungistatic effect of embodiment 13 bacillus subtilis BSXE-1601 and bacillus cereus BCXE-01 mixed bacteria liquid
Measurement
The bacterium solution for taking bacillus subtilis BSXE-1601 and bacillus cereus BCXE-01 respectively, respectively according to 5:1,6:
1,7:1 and 8:1 bacterial number ratio mixes the bacterium solution of two kinds of bacteriums, is then inoculated in nutrient agar respectively, and 28
DEG C, after culture for 24 hours, 4 kinds of mixed bacteria liquids are inoculated into respectively and are coated with Pseudoalteromonas, Vibrio splindidus, vibrio parahaemolytious, thermophilic water
Aeromonas, Huanghai Sea Shewanella, Edwardsiella tarda, vibrio alginolyticus, Vibrio harveyi, Vibrio vulnificus and Streptococcus iniae
Nutrient agar panel on, culture for 24 hours after, the inhibition zone of formation is measured, is calculated.
The result shows that：As shown in table 13, the bacillus subtilis BSXE-1601 of different proportion and bacillus cereus
BCXE-01 mixed bacteria liquid is to Pseudoalteromonas, Vibrio splindidus, vibrio parahaemolytious, Huanghai Sea Shewanella, vibrio alginolyticus, Ha Wei
Family name vibrios and Vibrio vulnificus have inhibitory effect.Wherein, to Vibrio splindidus, vibrio parahaemolytious, vibrio alginolyticus, Vibrio harveyi and
The inhibitory effect of Vibrio vulnificus is more significant, illustrates that the mixing of two kinds of bacteriums is effectively expanded to the antimicrobial spectrum of pathogen and antibacterial
Effect.
13 different proportion bacillus subtilis BSXE-1601 of table and bacillus cereus BCXE-01 mixed bacteria liquid it is antibacterial
Effect
Note：" ﹣ " is for muting sensitive or in vain
The preparation method of 14 composite bacteria preparation of embodiment
The preparation step of the composite bacteria preparation of the present embodiment is as follows：
1. seed liquor culture
Bacillus subtilis (Bacillus subtilis) BSXE-1601 and waxy bud for taking separation, purifying to obtain respectively
The conservation liquid of spore bacillus (Bacillus cereus) BCXE-01 is crossed on nutrient agar and 2216E solid plate, and
It is respectively placed under the conditions of 37 DEG C and 28 DEG C, activation is for 24 hours.Then picking bacillus subtilis (Bacillus subtilis) BSXE-
1601 and bacillus cereus (Bacillus cereus) BCXE-01 single colonie be inoculated in improvement after nutrient agar
In, the seed liquor for obtaining two kinds of bacteriums for 24 hours is cultivated under the conditions of 28 DEG C, 190rpm.
2. expanding culture
The seed liquor of two kinds of bacterium presses bacillus subtilis (Bacillus subtilis) BSXE-1601：Bacillus cereus
BCXE-01=5~8 (Bacillus cereus):After 1 bacterial number ratio is mixed, control seed mixture liquid at this time
Bacterial content is 1*106~1*107Cfu/ml, according to seed mixture liquid：The volume ratio of nutrient agar after improvement is 1:
10 ratio, by seed mixture liquid be inoculated into equipped with improvement after nutrient agar fermentor in, in temperature be 28 DEG C,
Mixing speed is 190rpm, ventilatory capacity 4V/V.min, and culture is for 24 hours.
3. the preparation of composite bacteria agent
According to seed mixture liquid：The volume ratio of liquid fermentation medium is 1:Seed mixture liquid is inoculated into dress by 10 ratio
Have in the fermentor of liquid fermentation medium, the bacterial content for controlling seed mixture liquid at this time is 1*106~1*107Cfu/ml,
It is 28 DEG C, mixing speed 190rpm, ventilatory capacity 4V/V.min in temperature, after cultivating 48h, is concentrated to give high concentration bacterium solution；Institute
The formula for stating liquid fermentation medium is as follows：Peptone 10g, corn pulp 10g, glucose 10g, sodium chloride 5g, KH2PO41.5g
MgSO4·7H2O 0.5g, distilled water 1000ml, pH are adjusted to 7.0 for 121 DEG C of prepared culture medium, and 101KPa sterilizes after 30min
It uses；
By peanut meal, corn flour, diatomite, bentonite according to 2:2:1:1 weight ratio is uniformly mixed, and then uses mesh
The beater disintegrating machine of 0.9mm carries out crushing material, then after mixing with double worm mixer, and auxiliary material carrier is made；It will be auxiliary
Expect carrier and high concentration bacterium solution according to 1:2 weight ratio is uniformly mixed, and polysorbate and xanthan gum is added, wherein poly- sorb
The additional amount of ester is the 2-3% of high concentration bacterium solution quality, and the additional amount of xanthan gum is the 1-3% of high concentration bacterium solution quality；Mixing
Uniformly, after being dried with fluid bed dryer, crushing material is carried out with the beater disintegrating machine of mesh 0.9mm again, then
It is uniformly mixed with double worm mixer to get composite bacteria preparation.
Embodiment 15 splash and feed composite bacteria preparation to the inhibitory effect of litopenaeus vannamei culture water vibrios quantity and
Influence to prawn growth
Sample plot point is selected in Chinese Marine University's aquaculture base, 2 test groups of experimental setup and 1 control group, sprinkles
Test group is spilt according to 1.5g/m3Dosage composite bacteria preparation is splashed into the sink of cultured prawn；Test group is fed according to feeding
Composite bacteria preparation is added in feed by the ratio of material weight 1%.Control group is without any processing, other cultivating conditions are kept
Unanimously.
It first takes the water body of each sink to be applied on TCBS culture medium flat plate respectively before experiment, carries out bacterium colony counting afterwards for 24 hours, be calculated as
Initial value.After experiment starts, periodically takes the water body of each sink to detect its amount of vibrio daily, persistently detect 5d.After cultivating 42d,
Calculate specific growth rate, rate of body weight gain, survival rate and the bait utilization of prawn.
The result shows that：As shown in table 14, compared with the control group, test group of splashing and the vibrios number in test group water body is fed
Amount is effectively suppressed, and average inhibition of the test group of especially splashing in 5 days is up to 91.28%, it was demonstrated that composite bacteria preparation pair
The prevention and treatment of culture water vibrios has significant effect.
As shown in Table 15, compared with the control group, test group of splashing and the prawn of test group is fed in specific growth rate, opposite
It is significantly increased on rate of body weight gain, survival rate and bait utilization, at the end of experiment, the survival rate for feeding test group is reachable
98.47%, bait utilization also reaches 80% or more.Show that composite bacteria preparation is promoting prawn growth, improves immunity and bait
Also there is remarkable effect in terms of material utilization rate.
Table 14 is splashed and feeds influence of the composite bacteria preparation to litopenaeus vannamei culture water vibrios quantity
Table 15 is splashed and feeds influence of the composite bacteria preparation to Growth of Litopenaeus vannamei
The above is only a preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, without departing from the principle of the present invention, several improvement can also be made, these improvement should be regarded as guarantor of the invention
Protect range.
Claims (8)
1. one plant of wide spectrum inhibits the bacillus of aquatic products vibrio pathogen, it is bacillus subtilis (Bacillus subtilis)
BSXE-1601 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), culture presevation number
For CGMCC NO.15949, the deposit date is on June 19th, 2018.
2. wide spectrum inhibits the bacillus of aquatic products vibrio pathogen according to claim 1, which is characterized in that the withered grass
Bacillus BSXE-1601 Gram-positive, the bacterium colony circle or irregular shape formed on nutrient agar panel, edge is not
Neatly, lawn is opaque, canescence, surface wettability, easy picking；Thallus is rod-shaped, production gemma, and life in gemma, shape is ellipse
Shape.
3. a kind of wide spectrum described in claim 1 inhibits the bacillus of aquatic products vibrio pathogen inhibiting the application in vibrios.
4. a kind of wide spectrum described in claim 1 inhibits application of the bacillus of aquatic products vibrio pathogen in aquaculture.
5. wide spectrum according to claim 4 inhibits application of the bacillus of aquatic products vibrio pathogen in aquaculture,
It is characterized in that, the bacillus subtilis full pool spilling head or spice are fed.
6. wide spectrum according to claim 4 inhibits application of the bacillus of aquatic products vibrio pathogen in aquaculture,
It directly splashes it is characterized in that, the bacterium is watered, dosage 108cfu/m3；Or spice is fed, dosage 1011cfu/
kg。
7. a kind of bacillus compound bacteria as main component for inhibiting aquatic products vibrio pathogen with wide spectrum described in claim 1
Preparation, which is characterized in that the composite bacteria preparation is by bacillus subtilis BSXE-1601 and bacillus cereus BCXE-01
With 5~8:1 bacterial population ratio is combined.
8. a kind of preparation method of composite bacteria preparation as claimed in claim 7, which is characterized in that include the following steps：
(1) seed liquor culture
Bacillus subtilis (Bacillus subtilis) BSXE-1601 and waxy gemma bar for taking separation, purifying to obtain respectively
The conservation liquid of bacterium (Bacillus cereus) BCXE-01 is crossed on nutrient agar and 2216E solid plate, and respectively
It is placed under the conditions of 37 DEG C and 28 DEG C, activation is for 24 hours；Then bacillus subtilis (Bacillus subtilis) BSXE- is picked them separately
1601 and bacillus cereus (Bacillus cereus) BCXE-01 single colonie be inoculated in improvement after nutrient agar
In, the seed liquor for obtaining two kinds of bacteriums for 24 hours is cultivated under the conditions of 28 DEG C, 190rpm；
The formula of the nutrient agar is as follows：Peptone 10g, powdered beef 3g, sodium chloride 5g, agar 20g, distilled water
1000ml, pH 7.3 uses 121 DEG C of prepared culture medium after 101KPa sterilizing 30min；
The formula of the 2216E solid medium is as follows：Peptone 10g, yeast powder 5g, agar 20g, Chen Haishui 1000mL, pH
It is 7.6,121 DEG C of prepared culture medium uses after 101KPa sterilizing 30min；
Nutrient agar formula after the improvement is as follows：Peptone 10g, powdered beef 3g, sodium chloride 5g, KH2PO41.5g
MgSO4·7H2O 0.5g, distilled water 1000ml, pH 7.3, by 121 DEG C of prepared culture medium, 101KPa sterilizes after 30min
It uses；
(2) expand culture
The seed liquor of two kinds of bacterium presses bacillus subtilis (Bacillus subtilis) BSXE-1601：Bacillus cereus
BCXE-01=5~8 (Bacillus cereus):After 1 bacterial number ratio is mixed, control seed mixture liquid at this time
Bacterial content is 1*106~1*107Cfu/ml, according to seed mixture liquid：The volume ratio of nutrient agar after improvement is 1:
10 ratio, by seed mixture liquid be inoculated into equipped with improvement after nutrient agar fermentor in, in temperature be 28 DEG C,
Mixing speed is 190rpm, ventilatory capacity 4V/V.min, and culture is for 24 hours；
(3) preparation of composite bacteria agent
According to seed mixture liquid：The volume ratio of liquid fermentation medium is 1:Seed mixture liquid is inoculated into equipped with liquid by 10 ratio
In the fermentor of body fermentation medium, the bacterial content for controlling seed mixture liquid at this time is 1*106~1*107Cfu/ml, in temperature
It is 28 DEG C, mixing speed 190rpm, ventilatory capacity 4V/V.min, after cultivating 48h, is concentrated to give high concentration bacterium solution；The liquid
The formula of fermentation medium is as follows：Peptone 10g, corn pulp 10g, glucose 10g, sodium chloride 5g, KH2PO41.5g, MgSO4·
7H2O 0.5g, distilled water 1000ml, pH are adjusted to 7.0 for 121 DEG C of prepared culture medium, use after 101KPa sterilizing 30min；
By peanut meal, corn flour, diatomite, bentonite according to 2:2:1:1 weight ratio is uniformly mixed and crushes, and auxiliary material is made and carries
Body；By auxiliary material carrier and high concentration bacterium solution according to 1:2 weight ratio is uniformly mixed, and adds polysorbate and xanthan gum,
The additional amount of middle polysorbate is the 2-3% of high concentration bacterium solution quality, and the additional amount of xanthan gum is the 1- of high concentration bacterium solution quality
3%；It is uniformly mixed, drying is crushed, remixed uniformly to get composite bacteria preparation.
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