CN1321173C - Bacillus subtilis preparation - Google Patents
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Abstract
The present invention relates to a bacillus subtilis preparation capable of effectively improving the water quality for breeding and reducing poisonous and harmful substances in water, and the preparation has no toxicity or pathogenicity to fish and improves the breeding benefit. On the basis of the weight of the preparation, the preparation is prepared from 1.4 to 4.0 % of following fermentation medium solid material, 86 to 90% of following filling material, and water as the rest. The content of bacillus subtilis B115 CGMCC No. 1210 in the preparation is 1 to 100*10<8> units /g; the solid material of the fermentation medium is a composition of two or three of bean cake powder, wheat bran and corn flour; the filling material is mineral powder, straw grain powder of agricultural crops, or a composition of the mineral powder and the straw grain powder of agricultural crops. The preparation of the present invention is suitable for being used as a water quality modifier.
Description
Technical field
The present invention relates to contain the preparation of subtilis, particularly improver of water quality.
Background technology
In recent years, the culture fishery development is extremely rapid, a large amount of microbiotic and other chemicalses of using in the extensive intensification process, though reached volume increase and disease-resistant purpose, but long-term abuse of antibiotics not only can cause the resistance of pathogenic bacteria, and antibiotic chemical residual is directly threatening human beings'health in fishery products and the water surrounding.Residues of antibiotics has become the technology barriers of the fishery products international trade of China, makes the product water outlet of China suffer tremendous loss.The antibiotic use of the strict restriction of country in recent years, and actively seek its substitute.
The existing century-old history of use of probiotics (or claiming probiotics microeclogical modulator), since advocating drinking acid milk and can have good health and a long life in Europe from the earlier 1900s plum Cheney husband of section (Elie Metchnikoff), probiotics is swift and violent in development all over the world, nineteen seventies, Germany Volkor Rusch originally set up microecology institute in the Hull, and was engaged in research and the application of viable bacterias such as bifidus bacillus, Bacterium lacticum, intestinal bacteria being made ecological therapy.Japan has 26 kinds of little ecological goods the beginning of the eighties and is used for medical treatment and health care.Other various countries also have multiple little ecological goods to put on market, and quantity and kind are also in continuous expansion.China uses probiotics lactasinum (Bacterium lacticum) to prevent and treat intestinal tract disease the earliest.Succeeded in developing the beginning of the eighties since the cereobiogen (candlestick genus bacillus), various viable bacteria probioticses are succeeded in developing in succession.Give birth to (Bacillus licheniformis), SANZHU KOUFUYE (bifidus bacillus, milk-acid bacteria, DL bacterium live organism and metabolite thereof), and put on market successively as beautiful pearl intestines happy (bifidus bacillus), whole intestines.The probiotics of probiotics for animals such as bacillus category, lactic acid bacteria class and budding fungus is succeeded in developing and is widely used as fodder additives simultaneously, and the Ministry of Agriculture had stipulated 12 kinds of feed level microbe additives (The Ministry of Agriculture of the People's Republic of China, MOA announces the fodder additives kind catalogue that No. 105 permission used) such as subtilis in 1999.
The water body environment of aquatic living things life is the homestat of a microorganism, profitable strain and the mutual survival of cause of disease flora, but because environmental pollution is serious day by day, the water quality deterioration of aquaculture water, cause pathogenic bacteria to breed in a large number, make that the aquatic animal disease constantly takes place in recent years, and be outbreak of epidemic trend, jeopardize nearly all cultivated animals such as fish shrimp crab shellfish algae soft-shelled turtle.Meanwhile because the residual and drugs used in aquiculture of drugs used in aquiculture in the aquatic food to Effects of water environment, makes the use of drugs used in aquiculture be strictly controlled.Therefore begin with probiotics as the fishing improver of water quality in the eighties.But the kind of using is mainly (microecological carp preparation and preparation method thereof patent publication No. CN1100271 such as photosynthetic bacterium, Bacterium lacticum, bacillus cereus, bacteriophagic Bdellovibrio and compound EM flora; The process patent publication number CN1461734A of microcapsule life tonifying clean water composite bacteria repairing aquaculture environment; The foster water process patent publication number CN1460414A in early stage of shrimp is supported in a kind of pond).But then rarely has report as fishing with improver of water quality with subtilis.The fishing of producing is at present mainly adopted conventional deep fermentation method with probiotics, and this method exists the production cost height, can only solidify shortcomings such as difficulty based on the aqua formulation.
Summary of the invention
The present invention to solve that aquaculture water water quality goes from bad to worse because of pollution and fishing at present with microecological water quality improving agent kind more single or problems such as production cost is high, have secondary pollution, formulation curing difficulty, a kind of bacillus subtilis formulation of the present invention is provided for this reason, this modifying agent can effectively be improved aquaculture water water quality, to fish nontoxicity and pathogenic.
For addressing the above problem, its special character of technical scheme that bacillus subtilis formulation of the present invention adopts is to be that benchmark contains 1.4～4.0% following fermention medium solid substances, 86～90% following stopping composition and the water of surplus with its weight, and subtilis B115 in the described preparation (Bacillus subtilis B115CGMCC No 1210) content is 1～100 * 10
8Individual/gram;
Described fermention medium solid substance can be two kinds or three kinds a composition in soybean cake powder, wheat bran, the Semen Maydis powder;
Described stopping composition can be the composition of breeze, crop stalk seed powder or breeze and crop stalk seed powder.Breeze and crop stalk seed composition, both can any ratio combination.
Breeze of the present invention can be one or two or more kinds the composition in zeolite powder, wilkinite, medical stone powder, potter's clay, the kaolin, and described crop stalk seed powder can be one or two or more kinds the composition in rice chaff powder, rice bran, bean cake powder, wheat bran, peanut meal, the dish dregs of rice, the corn cob meal.
Subtilis B115 among the present invention on August 20th, 2004 in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, deposit number is № 1210.
Subtilis separates from Pond Silt and obtains.Get Pond Silt, thin up, stirring and evenly mixing, boil postcooling, be diluted with water to again, get diluent and evenly be applied to by weight by Tryptones 1%, extractum carnis 0.5%, NaCl0.5%, agar 1.5%, surplus is a water, on the LB solid medium of forming (pH 7.0～7.2), the suspicious bacterium colony of picking (colony edge is irregular) inoculates on the LB solid medium of another above-mentioned composition, identify after the pure culture, obtain subtilis B115.
Below the invention will be further described.
1, the principal character of subtilis B115
The principal character of subtilis B115 is: Gram-positive, and shaft-like, can form gemma, the gemma ovalize, sporangium is not expanded, catalase-positive, VP tests positive, the methyl red feminine gender can be utilized not aerogenesis of glucose, can utilize wood sugar, pectinose, N.F,USP MANNITOL, starch, gelatin, Citrate trianion, the nitrate reduction positive, 50 ℃ of growths, pH5.7 growth, 7%NaCl growth.
2, the preservation of subtilis B115 and cultivation
Bacterial classification is stored in the gluey LB substratum in 4 ℃, and this culture medium prescription (W/W) is: Tryptones 1%, and extractum carnis 0.5%, NaCl0.5%, agar 1.5%, surplus is a water, pH 7.0～7.2.The prolonged preservation of bacteria culture adopts ice-cold drying means.
Choose subtilis B115 bacterium colony with inoculating needle, be inoculated in and contain the aqueous substratum of 3～5ml in vitro, this culture medium prescription (W/W) is: Tryptones 1%, and extractum carnis 0.25%, NaCl0.5%, surplus is a water, pH7.0～7.2; 200 rev/mins of shaking tables are cultivated, and 30 ℃, 18～20 hours; Again with the microbionation in the test tube in the Erlenmeyer flask that contains above-mentioned aqueous substratum 250mL, 200 rev/mins of shaking tables are cultivated, 30 ℃, 21～24h obtains bacterium liquid, wherein subtilis B115 concentration is not less than 5 * 10
9CFU/mL.
3, subtilis B115 solid fermentation
Solid fermentation adopts simply constructed tray formula fermentation container, form by an aseptic greenhouse and many movably pallets, fermention medium is the pallet of packing into after sterilization, cooling, inoculation, bed thickness is 1cm～2cm, pallet is placed on the shelf in greenhouse, and by the temperature and humidity of the humidifier adjusting greenhouse space of heating, the temperature in greenhouse is controlled at 28～35 ℃, constantly feed the low pressure sterile air simultaneously, make thalline be in the suitableeest growth conditions.
The prescription of fermention medium (W/W) is: 3.5% soybean cake powder, and 4% wheat bran, 5% Semen Maydis powder, surplus is a water.Fermention medium is through 120～125 ℃ of (0.1～0.15Mpa) high-temperature sterilization 30 minutes, the inoculations of cooling back.
By weight, press the amount of fermention medium 1%, above-mentioned bacterium liquid is inoculated into ready fermention medium respectively, mix, be tiled in the enterprising aerobe fermentation of acting charitably of enamel basin or stainless steel tray behind the mixing, time 1～2d obtains containing the bacterium paste.
4, the preparation of subtilis B115 preparation
With above-mentioned contain the bacterium paste with etc. behind the stopping composition mixing of the present invention of weight, in 40 ± 1 ℃ of blowing-type loft drier, dry, cross 18～20 mesh sieves, mix thoroughly, make pulverous preparation.
Press gemma content 〉=20 * 10 of the subtilis B115 strain of the present invention's preparation
8CFU/mL.
5, to the water quality improvement effect
(1) the subtilis B115 preparation of different concns is to indoor static water body improved effect
After subtilis B115 preparation uses, dissolved oxygen, the pH of tank interior water there is not tangible influence.
Tank interior water is after using subtilis B115 preparation, and 0.1ppm group ammonia nitrogen reduces by 5.62～28.51%, and the most degradation peak value appears at 4d, and nitrite nitrogen reduces by 1.52～10.61%, and the most degradation peak value appears at 3-4d; Sulfide reduces by 2.17～20.65%, and the most degradation peak value appears at 4d, and COD reduces by 0.00～13.83%; 0.5ppm the group ammonia nitrogen reduces by 6.93～40.26%, the most degradation peak value appears at 4d, and nitrite nitrogen reduces by 7.01～21.66%, and the most degradation peak value appears at 3d; Sulfide reduces by 17.19～31.25%, and the most degradation peak value appears at 4d, and COD reduces-0.380～15.22%; 1.0ppm the group ammonia nitrogen reduces by 6.28～47.83%, the most degradation peak value appears at 5-7d, and nitrite nitrogen reduces by 5.11～24.82%, and the most degradation peak value appears at 4d; Sulfide reduces by 2.91～43.69%, and the most degradation peak value appears at 4d, and COD reduces by 3.98～20.15%.And do not use the variable quantity of the contrast pond ammonia nitrogen of subtilis B115 preparation is-2.43～13.11%, the variable quantity of nitrite nitrogen is-5.08～8.47%, the variable quantity of sulfide is-6.41～12.82%, does not have tangible degradation peak and occurs, and the COD variable quantity is-1.95～-7.06%.After showing that subtilis B115 preparation uses, can obviously reduce ammonia nitrogen concentration, nitrite nitrogen, the sulfide concentration of Chi Shui.Because this experiment carries out in the Static Water environment, so the COD of control group also is downward trend, but compares with test group, during 7d, only for using below 50% of bacillus preparation group, the result shows that subtilis B115 has the reduction effect to organic matter to fall.
0.1ppm, after the subtilis B115 preparation of 0.5ppm, 1.0ppm uses, all pond water quality there is different improving effects, improved effect and concentration are proportionate in this concentration range, but after 0.1ppm subtilis B115 preparation uses, the ammonia nitrogen degradation rate is 28.51% among the Chi Shui, nitrite nitrogen is 10.61%, sulfide is 20.65%, and after the use of the subtilis B115 preparation of 0.5ppm and 1.0ppm, ammonia nitrogen degradation rate＞40%, nitrite nitrogen＞20%, sulfide＞30%.Though 1.0ppm uses the back effect to be better than the result of use of 0.5ppm, but both compare, the effect difference is not very big, so taking all factors into consideration from aspects such as use cost and results of use, as the microecological water quality improving agent, when subtilis B115 concentration was 2,000,000,000/gram, the using dosage of 0.5ppm was more rational.
(2) subtilis B115 preparation is to the water quality improvement effect measuring of cultivation fish pond
Test is carried out in No. 3, No. 4 of the safe aquaculture of Huzhou City gold company limited and No. 5 ponds, and wherein No. 3 and No. 4 ponds are test tank, and No. 5 ponds are for contrasting the pond, the pond is adjacent, area is 8 mu, 1.5 meters of the depth of waters, No. 3 and the Taihu Lake whitefish is mainly cultured in No. 5 ponds, mandarin fish is mainly cultured in No. 4 ponds.No. 3 and No. 4 ponds are by subtilis B115 preparation operation instruction, and every pond is with subtilis B115 preparation 4000g full pool spilling head, the 3rd after the use, gather water sample analysis on the 7th and the 15th day and measure.Subtilis B115 preparation 4000g full pool spilling head is used in the test pond for the second time in the time of the 15th day.
At duration of test, the variable quantity of two test fishpond pH is respectively-0.26～0.40% and-0.39～0.66%, and contrast fishpond pH variable quantity is-0.39～0.13%; The velocity of variation of two test fishpond dissolved oxygens is-0.53～5.20% and-0.239～0.13%; Contrast fishpond dissolved oxygen velocity of variation is 0.51～5.68%, shows that the bacillus subtilis preparation uses the back that cultivation fish pond dissolved oxygen and pH are not had tangible influence.The test fishpond appears at 3d in the most degradation peak after using subtilis B115 preparation, ammonia nitrogen reduces by 49.45% and 48.57% respectively, nitrite nitrogen reduces by 13.86% and 13.22% respectively, sulfide reduces by 25.81% and 16.49% respectively, every afterwards hydration index raises gradually, during to 15d, two test fishpond ammonia nitrogens only reduce by 3.3% and 11.43%; Nitrite nitrogen reduces by 4.95% and 4.13%, and sulfide reduces by 14.52% and 2.06%.Therefore when 15d, continue subtilis B115 preparation full pool spilling head with 0.5ppm, most degradation peak after using for the second time still appears at 3d, ammonia nitrogen reduces by 45.60% and 50.29% respectively, nitrite nitrogen reduces by 18.81% and 11.57%, sulfide reduces by 30.65% and 19.59%, level before every hydration index rises to gradually again and uses during to 30 days.Contrast fishpond ammonia nitrogen variation range is-3.09%～13.58%, nitrite nitrogen-46.65%～11.63%, and sulfide can reduce-3.95%～11.63%, does not have tangible degradation peak and occurs.The COD in No. 4 and No. 3 test ponds is respectively and reduces by 0.59～6.00% and 0.28～3.92%, and the COD in contrast pond increases by 0.00～4.09%.After the result shows that subtilis B115 preparation uses, pond ammonia nitrogen, nitrite nitrogen, sulfide and organism had tangible Degradation, the degraded peak occurs with about the 3d of back, holding time about 15d in the pond, ammonia nitrogen can appear in 3d once more that append after the use, nitrite nitrogen and sulfide degraded peak.Therefore suggestion after subtilis B115 preparation once uses, continued to use with the planted agent at 15 days aborning, to improve the environment of cultivating pool effectively, reduced the amount of hazardous and noxious substances in the pond, improved culture benefit.
(3) subtilis B115 preparation is to the water quality improvement effect measuring in cultivated crabs pond
Test is opened the east 1 and eastern 2 ponds of new rivers crab plant and is carried out in Huzhou City green hill township, the pond is adjacent, and area is respectively 15 mu and 7 mu, and average depth is 0.6m.1 pond, east is a test tank, mainly to raise into crab.Use 3000g subtilis B115 preparation full pool spilling head during test.2 ponds, east are the contrast pond.
At duration of test, the variable quantity of test crab pond pH is respectively 0.57%～1.58%, and the velocity of variation of dissolved oxygen is-1.22%～2.91%, and contrast crab pond pH variable quantity is 0.14%～0.42%; The dissolved oxygen velocity of variation is 0.63%～3.30%, shows that subtilis B115 preparation uses the back that cultivated crabs pond dissolved oxygen and pH are not had tangible influence.Test crab pond is the 3rd day appearance degraded peak after using subtilis B115 preparation, and ammonia nitrogen reduces by 38.25%; Nitrite nitrogen reduces by 17.05%, and sulfide reduces by 14.29%; Every afterwards hydration index raises gradually.It is 33.90% that total coli group then maximum reduction value occurred at the 7th day, has all kept lower value in 3-15 days.
In a word, after three kinds of different water body subtilis B115 preparations use, dissolved oxygen and pH all there is not tangible influence.And to ammonia nitrogen most degradation peak value appear at after 3～4d, maximum average rate of decrease is 45.40 ± 5.06%, the most degradation peak value of nitrite nitrogen appears at and uses back 3d, maximum average rate of decrease is 16.03 ± 3.82%, the most degradation peak value of sulfide appears at and uses back 3～4d, and maximum average rate of decrease is 23.01 ± 7.27%; And the ammonia nitrogen of each control group, nitrite nitrogen, sulfide are fluctuation and change, and time of occurrence is also fixing, and both have notable difference (P＜0.1) statistical study.Therefore after can judging that subtilis B115 agent is used, ammonia nitrogen, nitrite nitrogen and sulfide in the water body there is tangible improving effect.
6, subtilis B115 probiotics is to the influence of common pathogenic microorganism
Filter out the stronger bacterial strain B115 of anti-microbial activity.B115 bacterium liquid is cultivated and after centrifugal, its culturing filtrate has bacteriostatic activity to common fish-pathogenic bacteria, bacterial septicemia cause of disease (BSK-10), streptococcus aureus, large yellow croaker pathogenic bacterium (Vibrio parahaemolyticus, Vibrio harveyi) can be suppressed strongly, but Trionyx sinensis (Wiegmann) pathogenic bacterium (TL970424) can not be suppressed.Inhibition zone to BSK-10 is 14mm, is 15mm to the inhibition zone of large yellow croaker Vibrio harveyi, is 13mm to the inhibition zone of large yellow croaker Vibrio parahaemolyticus, is 13mm to the inhibition zone of river crab pathogenic bacterium C199920, is 10mm to the inhibition zone of streptococcus aureus.Illustrate that the B115 bacterium can secrete a kind of antimicrobial substance in culturing process, its scope of restraining fungi is to comprise Gram-negative bacteria and gram-positive microorganism quite widely, but not all bacterium.
(1) to the effect of BSK-10
When B115 and BSK-10 mixed culture, the growth of BSK-10 is not subjected to the inhibition of B115 during the consistent 5000CFU/ml of being with B115 of the starting point concentration of BSK-10, the content of BSK-10 is consistent with BSK-10 pure culture concentration in the 24h cultured products, the starting point concentration of BSK-10 be B115 1/10 and 1/100 o'clock, count results shows during 6h: the growth of BSK-10 is not subjected to the influence of B115 substantially, but do not detect BSK-10 (the minimum value of detecting is 100CFU/ml) during to 24h, illustrate that the BSK-10 of B115 centering, lower concentration has tangible killing action; But the BSK-10 of high density is inhibited to the growth of B115, and the propagation multiple of B115 is 1/1200 of contrast during 24h, in, the BSK-10 of lower concentration do not have influence substantially to the growth of B115.
(2) to the effect of C199920
B115 and C199920 mixed culture press down sterilization effect, test-results shows that B115 (concentration is 5000CFU/ml) has the obvious suppression effect to the C199920 of middle and high concentration (5000 and 500CFU/ml), and when 6h, just show, the C199920 of middle and high concentration its proliferative amount when 6h is respectively 1/5 and 1/12 of contrast, is 1/600 and 1/300 of contrast during to 24h; B115 has tangible killing action to the C199920 of lower concentration (50CFU/ml), all do not detect the existence of C199920 when 6h and 24h, be that initial concentration is that the C199920 of 50CFU/ml does not breed to 100CFU/ml, the B115 of visible 5000 CFU/ml has killing action to the C199920 of 50CFU/ml; And C199920 does not have influence substantially to the growth of B115, the C199920 of three concentration and B115 mixed culture to 24h, the content of B115 with the contrast no significant difference.
Aeromonas is distributed widely in the water body environment, can cause the serious disease of special aquatic products animals such as common cultured fishes such as crucian carp, bream, silver carp, flathead and shrimp, crab, soft-shelled turtle, the frog.The result of B115 and pathogenic Aeromonas mixed culture shows, the B115 of 5000CFU/ml can kill the fish Aeromonas septicemia pathogenic bacteria BSK-10 of 500CFU/ml, 50CFU/ml and the river crab Aeromonas pathogenic bacterium C199920 of 50CFU/ml, can suppress the growth of the C199920 of 5000 CFU/ml and 500CFU/ml.But B115 is to the pathogenic Aeromonas TL97424 of soft-shelled turtle unrestraint effect.Therefore, in aquaculture water, use the B115 preparation disease of preventing and treating some pathogenic Aeromonas and causing is had certain effect.
7, the toxicity of subtilis B115 probiotics and pathogenic
(1) toxicity test of genus bacillus
A, to the acute toxicity test of crucian
After 10ppm, 100ppm subtilis B115 preparation used, the test fish ingested, activity is normal, and surviving rate is 100%; After 1000ppm used, it is muddy that Chi Shui becomes slightly, but crucian ingests, the activity normal, and surviving rate is 100%; After 4000ppm uses 24h, water turbidity, test fish anoxic is raised the nose above water to breathe, and average mortality is 35% in the 96h.Subtilis B115 strain is to 96 hours LD of crucian
50＞4000ppm.
B, to the acute toxicity test of fresh water freshwater shrimp
After 10ppm, 100ppm subtilis B115 preparation used, the test shrimp ingested, activity is normal, and surviving rate is 100%; After 1000ppm subtilis B115 preparation used, it is muddy that Chi Shui becomes slightly, and raising the nose above water to breathe appears in the fresh water freshwater shrimp, and average survival is 60% in the 96h; After 2000ppm used, it is muddy that Chi Shui becomes, and behind the 24h, the severe depletion of oxygen of fresh water freshwater shrimp is raised the nose above water to breathe, all dead in the 48h.Calculate the 96hLD of subtilis B115 preparation with method of linear interpolation to the fresh water freshwater shrimp
50Be 1350ppm.
C, to the small white mouse toxicity test
The small white mouse random packet, 10 every group, male and female half and half, 10 in every cage is weighed before the administration.Test divides mouthful irritates an administration, mixes two groups of bait administrations, and each group is by high, medium and low three dosed administrations, and wherein high dose group mouthful is irritated respectively and mixed bait and give bacterium 5 * 10
10CFU/, middle dosage group mouth is irritated and is mixed bait and give bacterium 1 * 10
10CFU/, the low dosage mouth is irritated and is mixed bait and give bacterium 0.5 * 10
10Be administered once CFU/ every day.Establish test control group and blank group simultaneously.
After each is organized mouse and throws something and feeds with subtilis B115 preparation, observe the situation such as appearance, behavior, diet of small white mouse every day.Off-test in 30 days 24h after the last administration, hematology is measured in blood sampling, blood biochemical is learned index, and each is organized the disconnected neck in small white mouse blood sampling back and puts to death, and dissects rapidly, examines the heart, liver, spleen, lung, kidney, stomach, brain, uterus, ovary, testis pathology.
Off-test in 30 days 24h after the last administration, eye socket is got blood, measures red corpuscle sum, oxyphorase, white corpuscle and lymphocyte with automatic blood analyzer; Measure glutamic-oxal(o)acetic transaminase, gpt, AST and ALK from the biochemical analysis instrument entirely with BackMan, the data of measuring are done variance test
After the off-test in 30 days, the activity of each test group and control group small white mouse, color gloss, breathe all normally, the nasal cavity end sees that secretory product is excessive, and ight soil is shaped, and surviving rate is 100%.30 days feed intakes and the equal indifference of small white mouse body weight (P＞0.1), the heart, liver, spleen, lung, kidney, stomach, brain, uterus, ovary, testis are all normal.The hematology of test group and control group small white mouse and blood biochemical learn index through the t assay show the small white mouse mouth irritate and mix bait throw raise subtilis B115 preparation after, the t value all＞0.1, both indifferences show that subtilis B115 strain is to the small white mouse nontoxicity.
(2) the pathogenic test of subtilis B115 preparation
(1) the crucian mouth is irritated the pathogenic test of subtilis B115 strain
Subtilis B115 preparation is diluted to 50 * 10
8Cfu/mL and with 4 ℃, the centrifugal 10min of 6000rpm gets bacterium mud, is diluted to 200 * 10 with physiological saline
8Behind the cfu/mL, with homemade mouthful of filling device, pour into 1ml respectively, 0.5ml and subtilis B115 culture and the bacterium mud diluent of 0.1ml, mouth was irritated once in per two days, mouthful filling is three times continuously, establish a mouthful filling 1ml sterilization LB medium controls simultaneously, mouth is irritated and is finished the continuous observation in back 15 days, each death all occurs after organizing test group and control group crucian mouth filling subtilis B115 preparation, surviving rate is 100%, comparing body weight with control group does not have obvious decline, and body surface and internal organs anatomic observation do not occur unusually, shows that mouthful filling subtilis B115 strain is to the crucian no pathogenicity.
(2) the pathogenic test of injection subtilis B115 strain
The centrifugal back of subtilis B115 bacterium mud is diluted to 200 * 10 with physiological saline
8Behind the cfu/mL, abdominal injection crucian 10 tail/groups, every tail injects 0.5 and 0.1ml respectively.Control group crucian injection sterile saline, every endnote is penetrated 0.5ml.Mitten crab is injected 0.1ml, every injecting normal saline 0.1ml of control group for every in 10 of the 3rd step base portion injections; Penaeus vannamei is injected 10 tails, and every endnote is penetrated 0.1ml, and the every endnote of control group is penetrated 0.1ml physiological saline; 10/group of mice by intraperitoneal injection, 10/group of subcutaneous injection is injected 0.5ml and 0.1ml respectively for every, and every 2d injection is once injected every injecting normal saline 0.5ml of control group 3 times continuously.Observed 15 days continuously.The duration of test water temperature is 25 ± 1 ℃, and the pond water-soluble oxygen is more than 5mg/L, and pH7.35, temperature are 30 ± 3 ℃.In 15 days behind each test group and control group crucian, mitten crab and the Penaeus vannamei injection subtilis B115, all do not occur dead, surviving rate is 100%, and comparing body weight with control group does not have obvious decline, and body surface and internal organs anatomic observation do not occur unusually.The activity of each test group and control group small white mouse, fur color gloss, breathe all normally, the nasal cavity end sees that secretory product is excessive.Subcutaneous injection partly has caking, and the small white mouse ascites of abdominal injection increases, and internal organs anatomic observations such as liver,spleen,kidney, intestines, sexual gland are normal.Therefore preliminary judge this laboratory from Pond Silt isolating subtilis B115 strain to common aquatic animal and small white mouse no pathogenicity.
On probation showing, improver of water quality of the present invention, pond ammonia nitrogen, nitrite nitrogen, sulfide and organism had tangible Degradation, can effectively improve the environment of cultivating pool, reduce the amount of hazardous and noxious substances in the pond, and, can effectively improve culture benefit to fish nontoxicity and pathogenic.Preparation of the present invention can be a solid dosage, helps storing, packs, transports.
Embodiment
Embodiment 1
Get Pond Silt, add the sterilized water dilution of 10 times of weight, stirring and evenly mixing boiled 8 minutes, cooling, and portioning is diluted to 10 respectively with sterilized water again
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8Get different dilution liquid 0.1ml, evenly be applied to by weight by Tryptones 1% extractum carnis 0.5%, NaCl0.5%, agar 1.5%, surplus are water, on the LB solid medium of composition (pH 7.0～7.2), the suspicious bacterium colony of picking (colony edge is irregular), inoculate on the LB solid medium of another above-mentioned composition, identify after the pure culture, obtain subtilis B115.
Withered genus bacillus B115 is inoculated in contains 4ml by weight by Tryptones 1%, extractum carnis 0.25%, NaCl0.5%, surplus is a water, the aqueous substratum of composition (pH7.0～7.2) is in vitro, again with the microbionation in the test tube in the Erlenmeyer flask that contains above-mentioned aqueous substratum 250mL, 200 rev/mins of shaking tables are cultivated, and 30 ℃, 21 hours, obtain bacterium liquid, subtilis B115 concentration is not less than 5 * 10 in this bacterium liquid
9CFU/mL.
Behind the water mixing with wheat bran 4%, soybean cake powder 3.5%, Semen Maydis powder 5% and surplus, autoclaving is made fermention medium after the cooling by weight.The above-mentioned bacterium liquid of fermention medium weight 1% is added fermention medium, be tiled in enamel holder basin behind the mixing, bed thickness is 1.5cm, pallet is placed on the shelf in greenhouse, by the temperature and humidity of the humidifier adjusting greenhouse space of heating, constantly feed the low pressure sterile air simultaneously, make thalline carry out aerobic fermentation, 24 hours time, must contain the bacterium paste.
Press 1: 1 mixed by weight by zeolite powder and rice chaff powder, get stopping composition.With etc. weight described stopping composition and contain bacterium paste mixing after, in 40 ± 1 ℃ of blowing-type loft drier, dry, cross 18～20 mesh sieves, mix thoroughly, make pulverous preparation.The Bacillus subtillis B115 of said preparation is 8.2 * 10 after measured
9Individual/g; The described stopping composition that adds 3 times of weight of contained stopping composition is again made subtilis B115 preparation, wherein contains subtilis B115 21 * 10
8Individual/g.
Embodiment 2
What be different from example 1 is that this routine fermention medium consists of soybean cake powder 4% by its weight, wheat bran 5%, the water of Semen Maydis powder 5% and surplus; Stopping composition is by weight by zeolite powder: rice bran is to mix at 2: 1 to form, stopping composition with contain the bacterium paste and mix back 41 ℃ of oven dry, its viable bacteria content of mensuration powder preparation is 9.8 * 10
9Individual/g, add 3 times of described stopping composition of weight of contained stopping composition again, making bacterial content is 24 * 10
8The preparation of individual/g.Other step is identical with example.
Embodiment 3
This routine fermention medium consists of soybean cake powder 4% by its weight, wheat bran 4%, the water of Semen Maydis powder 3% and surplus, fermentation culture 24 hours; Stopping composition is by weight by kaolin: corn cob meal is that mixing in 2: 1 is formed, and measuring its viable bacteria content of powder preparation is 9.2 * 10
9Individual/g, add 3 times of described stopping composition of weight of contained stopping composition again, making bacterial content is 22 * 10
8The preparation of individual/g.
Embodiment 4
This routine fermention medium is formed soybean cake powder 4%, wheat bran 2%, the water of Semen Maydis powder 4% and surplus by its weight; Stopping composition is by weight by wilkinite: wheat bran is to mix at 4: 1, stopping composition with contain the bacterium paste and mix back 40 ℃ of oven dry, its viable bacteria content of mensuration powder preparation is 7.8 * 10
9Individual/g, add 2.5 times of described stopping composition of weight of contained stopping composition again, making bacterial content is 21 * 10
8The preparation of individual/g.
Embodiment 5
This routine fermention medium consists of soybean cake powder 3% by its weight, wheat bran 5%, the water of Semen Maydis powder 3% and surplus; Stopping composition is by weight by potter's clay: bean cake powder is to mix at 3: 1, stopping composition with contain the bacterium paste and mix back 41 ℃ of oven dry, its viable bacteria content of mensuration powder preparation is 7.6 * 10
9Individual/g, add 2.5 times of described stopping composition of weight of contained stopping composition again, making bacterial content is 20 * 10
8The preparation of individual/g.
Embodiment 6
This routine fermention medium consists of soybean cake powder 2% by its weight, the water of wheat bran 3% and surplus; Stopping composition is by weight by the medical stone powder: peanut meal is to mix at 3: 1, stopping composition with contain the bacterium paste and mix back 40 ℃ of oven dry, its viable bacteria content of mensuration powder preparation is 6.8 * 10
9Individual/g, add the zeolite powder of 2 times of weight of contained stopping composition again, making bacterial content is 22 * 10
8The preparation of individual/g.
Embodiment 7
This routine fermention medium consists of wheat bran 5% by its weight, the water of Semen Maydis powder 4% and surplus; Stopping composition is by weight by zeolite powder: the dish dregs of rice are to mix at 3: 1, stopping composition with contain the bacterium paste and mix back 41 ℃ of oven dry, its viable bacteria content of mensuration powder preparation is 6.6 * 10
9Individual/g, add the zeolite powder of 2 times of weight of contained stopping composition again, making bacterial content is 21 * 10
8The preparation of individual/g.
Embodiment 8
This routine fermention medium consists of soybean cake powder 3% by its weight, the water of wheat bran 4% and surplus; Stopping composition is that zeolite powder mixes, stopping composition with contain the bacterium paste and mix back 40 ℃ of oven dry, its viable bacteria content of mensuration powder preparation is 6.3 * 10
9Individual/g, add the zeolite powder of 2 times of weight of contained stopping composition again, making bacterial content is 20 * 10
8The preparation of individual/g.
Embodiment 9
This routine fermention medium consists of soybean cake powder 3% by weight, the water of Semen Maydis powder 4% and surplus; Stopping composition is the rice chaff powder, stopping composition with contain the bacterium paste and mix back 41 ℃ of oven dry, measuring its viable bacteria content of powder preparation is 9.8 * 10
8Individual/g, add the big chaff of 3 times of weight of contained stopping composition again, making bacterial content is 3 * 10
8The preparation of individual/g.
Embodiment 10
This routine fermention medium consists of soybean cake powder 2% by weight, wheat bran 5%, the water of Semen Maydis powder 1.5% and surplus; Stopping composition is a corn cob meal, stopping composition with contain the bacterium paste and mix back 41 ℃ of oven dry, measuring its viable bacteria content of powder preparation is 168 * 10
8The preparation of individual/g adds the rice chaff powder of 1 times of weight of contained stopping composition again, and making bacterial content is 84 * 10
8The preparation of individual/g.
Claims (2)
1, bacillus subtilis formulation, it is characterized in that with its weight being that benchmark contains 1.4～4.0% following fermention medium solid substances, 86～90% following stopping composition and the water of surplus, subtilis B115 in the described preparation (Bacillus subtilis) B115 CGMCC N
o1210 content are 1～100 * 10
8Individual/gram;
Described fermention medium solid substance is two kinds or three kinds a composition in soybean cake powder, wheat bran, the Semen Maydis powder;
Described stopping composition is breeze, crop stalk seed powder or is the composition of breeze and crop stalk seed powder.
2, bacillus subtilis formulation as claimed in claim 1, it is characterized in that described breeze is one or more in zeolite powder, wilkinite, medical stone powder, potter's clay, the kaolin, described crop stalk seed powder is one or more in rice chaff powder, rice bran, bean cake powder, wheat bran, peanut meal, the dish dregs of rice, the corn cob meal.
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| CNB2004101010219A CN1321173C (en) | 2004-12-02 | 2004-12-02 | Bacillus subtilis preparation |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102199569A (en) * | 2011-04-21 | 2011-09-28 | 马鞍山科邦生态肥有限公司 | Bacillus subtilis submerged fermentation culture medium applied to microbial fertilizers |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101333499B (en) * | 2008-05-13 | 2010-06-09 | 昆山工研院华科生物高分子材料研究所有限公司 | Complex active bacterial biological water purifying a gent and method for preparing same |
| CN102181423A (en) * | 2011-03-18 | 2011-09-14 | 辽宁省农业科学院大连生物技术研究所 | Immobilized active probiotic and production method thereof |
| CN102220266B (en) * | 2011-05-12 | 2012-12-26 | 山东省农业科学院家禽研究所 | Production method and open fermentation method of bacillus subtilis culture preparation |
| CN102334606B (en) * | 2011-07-22 | 2013-04-24 | 广东省微生物研究所 | Preparation method for lactic acid-producing bacillus subtilis microbial preparation |
| CN102503690B (en) * | 2011-11-03 | 2013-07-10 | 广西得盛生物科技有限公司 | Granular biological organic fertilizer and preparation method thereof |
| CN102757277B (en) * | 2012-07-31 | 2013-12-04 | 长春华御实业集团有限公司 | Paddy biofertilizer, preparation method thereof and bacillus subtilis |
| CN103160453B (en) * | 2013-02-26 | 2014-07-16 | 武汉施瑞福生物技术有限公司 | Preparation method of microecologics for nitrite degradation, microecologics and use thereof |
| CN105441352A (en) * | 2015-09-16 | 2016-03-30 | 广州金水动物保健品有限公司 | Compound microorganism water stabilizing preparation |
| CN112106704A (en) * | 2020-10-28 | 2020-12-22 | 广东梁氏水产种业有限公司 | Application of bdellovibrio bacteriovorus in improvement of domestication rate of siniperca chuatsi artificial feed |
| CN118126882A (en) * | 2023-05-26 | 2024-06-04 | 江苏海洋大学 | Heterotrophic nitrifying bacteria JHD-Z2 and microbial inoculum, preparation method and application thereof |
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| CN102199569A (en) * | 2011-04-21 | 2011-09-28 | 马鞍山科邦生态肥有限公司 | Bacillus subtilis submerged fermentation culture medium applied to microbial fertilizers |
| CN102199569B (en) * | 2011-04-21 | 2013-01-16 | 马鞍山科邦生态肥有限公司 | Bacillus subtilis submerged fermentation culture medium applied to microbial fertilizers |
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Application publication date: 20050629 Assignee: Zhejiang Crown Technology Co., Ltd. Assignor: Zhejiang Institute of Fresh Water Aquatic Products Contract record no.: 2017330000021 Denomination of invention: Bacillus subtilis preparation and preparation method thereof Granted publication date: 20070613 License type: Common License Record date: 20170329 |