CN1844364A - Microecological preparation-tetragenous viable bacteria preparation and method for preparing same - Google Patents

Microecological preparation-tetragenous viable bacteria preparation and method for preparing same Download PDF

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CN1844364A
CN1844364A CN 200610040206 CN200610040206A CN1844364A CN 1844364 A CN1844364 A CN 1844364A CN 200610040206 CN200610040206 CN 200610040206 CN 200610040206 A CN200610040206 A CN 200610040206A CN 1844364 A CN1844364 A CN 1844364A
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bacterium powder
former bacterium
cfu
gram
viable count
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CN 200610040206
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CN100412187C (en
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陆茂林
孙梅
匡群
施大林
刘淮
何义进
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江苏省微生物研究所有限责任公司
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Abstract

This invention relates to a micro-ecological agent and the preparation method thereof, belonging to the micro-ecological agent technological field. The preparation of the active bacteria agent of this invention comprises: using L-calcii lactas and four useful micro-organisms of clostridium bytyricum, Lactobacillus casei, bacillus subtilis and saccharomycete as characteristic elements, cultured purely or mixedly by means of universal fermentator, producing active bacteria powder by freeze-drying, and acquiring the finished products by double crossing of the powder at a specific proportion. By using the anaerobic and aerobic characteristics of related bacterial strains, this agent has the following merits: playing the synergy effect in enhancing the disease resistance of marine animals and the water purification of aquaculture, substituting antibiotics fully or partly for the disease prevention and cure in fresh water aquiculture and mariculture, and improving the economic benefits of aquaculture. The L-calcii lactase component can accelerate the husking growth and prevent the disease of soft husk of shellfish marine animals like shrimp and crab. This agent is characterized of being nontoxic, free from side effects, nonpersistent, without secondary pollution and no drug tolerance, and will have a wide commercial application in the fresh water aquiculture mariculture.

Description

A kind of probiotics---tetragenous viable bacteria preparation and preparation method thereof
Technical field
A kind of probiotics---tetragenous viable bacteria preparation and preparation method thereof belongs to the probiotics technical field.
Technical background
In recent years, along with developing rapidly of China's culture fishery, the expansion day by day of the aquaculture scale of high-density, intensification, the aquatic animal movement of aquaculture water middle and high concentration, the throw something and feed pollution in the natural waters that any discharging of bait remnants that bait causes and sewage causes in a large number go from bad to worse the breeding ecological environment.Aquatic animal is Crustacean such as shrimp crab especially, function of immune system is low, main non-specific material such as the lectin N,O-Diacetylmuramidase that relies on various hemocytes to produce waits the non-specific immune function of bringing into play body, in the aquaculture water environment that worsens, be subject to the attack of malignant bacteria and virus, make all kinds of disease pilositys, reduce the culturing economic benefit.Aquatic animal disease especially virus disease major part all develops soon, and harm is big.The common method of preventing and treating be use some microbiotic and chemical synthetic drug to water body carry out disinfection, purifying treatment, to killing of germ in environment and the aquatic animal body, thereby reach the effect of control disease.This method comparatively seriously reaches infectation of bacteria for some breeding environment deterioration certain control action kou, but for most of nurseries, because the aquatic product sprout resistance against diseases is very low, and in case illly mass mortality will occur, make the no obvious effects that heals with medicine.
Microbiotic and chemical synthetic drug life-time service or abuse not only cause the resistance resistance of pathogenic bacteria more and more obvious, and dosage also increases thereupon, result of use worse and worse, even invalid, and cause medicine residual in fishery products, reduced the quality of fishery products.In addition, microbiotic and chemical synthetic drug are when killing the pathogeny bacterium, also disturbed in the aquatic animal body and the breeding of the normal growth of aquaculture water beneficial microbe colony, weaken the anti-disease ability of aquatic animal self, make pollutent and residual bait at the bottom of water body and the pond can not get decomposing timely and effectively, water quality runs down, and destroys the eubiosis of water body, thereby brings out new round disease.Therefore, microbiotic and pharmaceutical chemicals are a kind of passive diseases prevention and treatment ways, can not fundamentally improve the resistance against diseases of aquatic animal, and when it controlled disease as a kind of emergent means, counter productive was conspicuous.
The control of carrying out the aquatic animal disease with probiotics (Microbial ecological agent) is a kind of novel method that developed recently gets up, have nontoxic, have no side effect, the advantage of noresidue.The microniological proudcts that probiotics is based on the microecological balance theory, the theoretical and little bionomic control of little ecoalimental is theoretical and grow up, it is controls of a kind of active form to the control of Animal diseases, adjust aspects such as the inherent nutrition of animal body, immunity, growth-stimulating, biological antagonist by profitable strain in the preparation and bringing into play the microecological balance of important effect, suppress the growth of pathogenic bacterium, promote the growth of animal self beneficial microorganism, strengthen the capacity of self-regulation of its little ecology, the generation of control and minimizing disease improves the host health level.The probiotics of using in aquaculture can also improve water quality by to the decomposition of water body objectionable impurities, the inhibition of pathogenic bacteria and the cultivation of water body profitable strain, keeps the water ecology balance.
The originally diseases that are used for the treatment of human body of probiotics more, probiotics begins to be used for the diseases prevention and treatment of cultivated animals subsequently, and the microbe species that is used to prepare feeding micro-ecological preparation is also increasing, is widely used.FDA (FDA) in 1989 and U.S. feed control official association have announced can directly feed and be commonly considered as safe microbial strains list, has 41 kinds, and year usage quantity is more than 8000 tons.China Ministry of Agriculture also announced 15 kinds of feed level microbe additive bacterial classifications that can directly use such as lactobacterium casei, subtilis, yeast saccharomyces cerevisiae in 2003.At present, domestic probiotics is mainly used in the livestock cultures such as pig ox chicken, and the application in the fishery products animal cultivation is in the starting stage.
The probiotics of using in aquaculture can be divided into two big classes by the quantity of contained microbe species, and promptly single bacterial strain type and many bacterial strains are compound.Single bacterial strain type such as photosynthetic bacteria preparation, bacillus preparation etc. only contain a kind of microorganism, do not have the advantage of many synergistic bacteriums effect, comparatively speaking the existence effect single, act on unabiding limitation.Therefore, the application of many bacterial strains slurriable combination in aquaculture is more, and common have employing genus bacillus, nitrobacteria or photosynthetic bacterium etc. to be mixed in proportion the slurriable combination that forms.Concerning the preparation of this type of slurriable combination, the selection of microbe species, compatibility and preparation technology are crucial, directly influence the effect of preparation.
At present common used for aquiculture probiotics such as photosynthetic bacteria preparation, bacillus preparation, nitrobacteria preparation etc., mainly be utilize remaining bait excessive in the microbiological degradation water bodys such as photosynthetic bacterium, genus bacillus, nitrobacteria, drain refuse, plant and animal residues etc. is to the deleterious organic and inorganic substance of aquatic animal, inhibition growth of pathogenic bacteria, the improvement by the aquaculture water environment reaches the prevention effect to disease.In addition, preparation that has such as photosynthetic bacteria preparation, nutritive substances such as its microorganism self rich in proteins by being eaten to animal supplements the nutrients, promote growth, strengthen the disease resistivity.Therefore, these products are by realizing from the angle of improving water body environment or extra-nutrition to the control of aquatic animal disease, not directly by participating in the adjustment of microecological balance in the aquatic animal body, suppress the growth of pathogenic bacterium, promote the growth of animal self beneficial microorganism, strengthen the capacity of self-regulation of its little ecology, improve aquatic animal self resistance against diseases.Thus, based on above-mentioned background, have adjustment, the promotion growth of participating in microecological balance in the aquatic animal body directly concurrently if can develop, can improve simultaneously the probiotics of water quality again, be applied to the diseases prevention and treatment of aquaculture, then to reducing the use of microbiotic and chemicals, production pollution-free green fishery products, it is significant to improve the culturing economic benefit.
Summary of the invention
The objective of the invention is to prescription composition that obtains a kind of used for aquiculture microecological preparation-tetragenous viable bacteria preparation and preparation method thereof.
Technical scheme of the present invention: " tetragenous viable bacteria preparation " is many bacterial strains mixed type active bacteria formulation, with clostridium butyricum, lactobacterium casei, subtilis, four kinds of beneficial microorganisms of yeast and biologically actived calcium-L-calcium lactate is that characteristic component carries out compatibility, adopt four kinds of microorganisms of purebred cultivation of general fermentor tank or mixed culture, cryodrying prepares the former bacterium powder of viable bacteria, forms by specific proportioning is composite.
This tetragenous viable bacteria preparation is by clostridium butyricum (Clostridium butyricum) CGMCCNo.1647, lactobacterium casei (Lactobacillus casei) CGMCC 1.539, subtilis (Bacillus subtilis) CGMCC 1.1414 and yeast (Sacccharomycescerevisiae) CGMCC 2.614 four kinds of probiotic bacteriums and L-calcium lactates and zeolite powder is composite forms; Total viable count is respectively in the preparation: subtilis total viable count 〉=1 * 10 7The CFU/ gram, clostridium butyricum total viable count 〉=1 * 10 8The CFU/ gram, lactobacterium casei total viable count 〉=1 * 10 9The CFU/ gram, yeast total viable count 〉=1 * 10 6The CFU/ gram, the mass content of L-calcium lactate is 6%-10% in the preparation, the mass content of zeolite powder is 40%-45%.
This tetragenous viable bacteria preparation is to prepare and get with the former bacterium powder that the purebred cultivation of microorganism obtains, and its quality per distribution ratio (1) is:
The former bacterium powder of subtilis 2-2.5
The former bacterium powder of clostridium butyricum 20-25
The former bacterium powder of lactobacterium casei 5-6.5
The former bacterium powder of yeast 20-25
L-calcium lactate 6-10
Zeolite powder 40-45
Wherein:
Subtilis viable count 〉=5 * 10 in the former bacterium powder of subtilis 8The CFU/ gram
Clostridium butyricum viable count 〉=5 * 10 in the former bacterium powder of clostridium butyricum 8The CFU/ gram
Lactobacterium casei viable count 〉=2 * 10 in the former bacterium powder of lactobacterium casei 10The CFU/ gram
Yeast viable count 〉=5 * 10 in the former bacterium powder of yeast 6The CFU/ gram.
Or this tetragenous viable bacteria preparation is the former bacterium powder that obtains with the purebred cultivation of subtilis and mixes former bacterium powder and prepare and get with saccharomycetic with contain clostridium butyricum, lactobacterium casei that clostridium butyricum, lactobacterium casei and saccharomycetic mixed culture obtain that its quality per distribution ratio (2) is:
The former bacterium powder of subtilis 2-2.5
Mix former bacterium powder 45-52
L-calcium lactate 6-10
Zeolite powder 40-45
Wherein:
Subtilis viable count 〉=5 * 10 in the former bacterium powder of subtilis 8The CFU/ gram mixes clostridium butyricum viable count 〉=2.4 * 10 in the former bacterium powder 8The CFU/ gram, lactobacterium casei viable count 〉=2.4 * 10 9The CFU/ gram, yeast viable count 〉=2.4 * 10 6The CFU/ gram.
The preparation method of this tetragenous viable bacteria preparation is by clostridium butyricum (Clostridiumbutyricum) CGMCC No.1647, lactobacterium casei (Lactobacillus casei) CGMCC 1.539, subtilis (Bacillus subtilis) CGMCC 1.1414 and yeast (Sacccharomycescerevisiae) CGMCC 2.614 four kinds of probiotic bacteriums and L-calcium lactates and zeolite powder is composite forms.
A. the preparation of the cultivation of active thalline and former bacterium powder:
(1) the former bacterium powder of preparation subtilis
Actication of culture
The freeze-drying lactobacillus of subtilis, streak inoculation were cultivated 18-24 hour for 30-40 ℃ in the nutrient broth agar inclined-plane, line is transferred in nutrient broth agar eggplant bottle inclined-plane then, cultivates microscopy 20-24 hour for 30-40 ℃, to 90% above thalline formation gemma, be maturation.
Seed culture
Ripe bacterium mud scraped with sterilized water wash, the aseptic triangular flask of glaze pearl in packing into, vibration obtains uniform bacteria suspension, bacteria suspension heat 5-15 minute in 70-90 ℃ of water-bath, with the inoculum size access 0.1m of volume ratio 2%-10% 3Seeding tank, loading amount coefficient are the 60%-70% volume ratio.
Seed culture medium consists of: glucose 3-5g, and peptone 5-10g, yeast extract paste 3-5g, NaCl 5g, tap water is settled to 1000ml, pH7.0-7.5, culture condition is: mixing speed 100-200r/min, air flow 1-2m 3/ h, culture temperature is 30-40 ℃, incubation time is 16-20h.
Fermentor cultivation
Seed culture fluid inserts 1m with the inoculum size of volume ratio 5%-20% 3Fermentor tank, loading amount coefficient are the 60%-70% volume ratio, and the substratum of fermentor cultivation is made up of carbon source, nitrogenous source and inorganic salt, and carbon source is 5-15g/L with glucose or starch, content; Nitrogenous source peptone, yeast extract paste, soybean cake powder or groundnut meal, content is 10-20g/L, and inorganic salt are NaCl 5g/L, and culture condition is: mixing speed 100-200r/min, air flow 1-2m 3/ h, culture temperature is 30-40 ℃, and incubation time is 16-20h, and microscopy when the thalline more than 90% has formed gemma, is put and jar is finished to cultivate.
Microorganism collection
Nutrient solution pumps into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains wet thallus.
The preparation of the former bacterium powder of subtilis
Collect wet thallus, with dry starch with mass ratio 1: 1-5 mixes, and at 40-50 ℃ of dry 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, with viable count 〉=5 * 10 of dry starch adjusting subtilis 8The CFU/ gram is the former bacterium powder of subtilis.
(2) the former bacterium powder of preparation clostridium butyricum
Actication of culture
The freeze-drying lactobacillus of clostridium butyricum inserts activation medium, leaves standstill anaerobism and cultivates, and activation medium is formed: peptone 10g, beef extract 10g, yeast extract 3g, glucose 10g, Zulkovsky starch 1g, NaCl5g, NaAc 3g, cysteine hydrochloride 0.5g, CaCO 33g, distilled water is settled to 1000ml, and pH 7.0, cultivate 24-48h for 30-40 ℃, transfer in fresh activation medium with the inoculum size of volume ratio 5%-15%, cultivate 24-48h for 30-40 ℃, and microscopy during to 90% above thalline formation gemma, is maturation.
Seed culture
Bacterium liquid heated 5-15 minute in 70-90 ℃ of water-bath, with the inoculum size access 0.1m of volume ratio 2%-10% 3Seeding tank, the loading amount coefficient is the 70%-80% volume ratio, seed culture medium consists of: peptone 5-10g, beef extract 5-10g, yeast extract 3-10g, glucose 5-10g, starch 0.5-1g, NaCl 5g, NaAcl-3g, CaCO 31-3g, distilled water is settled to 1000ml, and pH 7.0, and 30-40 ℃ leaves standstill anaerobism cultivation 16-24h.
Fermentor cultivation
Seed culture fluid inserts 1m with the inoculum size of volume ratio 5%-20% 3Fermentor tank, loading amount coefficient are the 70%-80% volume ratio, and the substratum of fermentor cultivation consists of: peptone 5-10g, yeast extract 3-10g, glucose 5-15g, starch 0.5-1g, NaCl 5g, NaAc 1-3g, CaCO 33-10g, tap water is settled to 1000ml, and pH 7.0, and 30-40 ℃ leaves standstill anaerobism and cultivates 24-48h, and microscopy when 90% above thalline forms gemma, is promptly put and jar is finished cultivation.
Microorganism collection
Nutrient solution pumps into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains wet thallus.
The preparation of the former bacterium powder of clostridium butyricum
With clostridium butyricum wet thallus and dry starch with mass flow ratio 1: 1-5 mixes, 40-50 ℃ of dry 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, regulates viable count 〉=5 * 10 of clostridium butyricum with dry starch 8The CFU/ gram is the former bacterium powder of clostridium butyricum.
(3) the former bacterium powder of preparation lactobacterium casei
Actication of culture
The freeze-drying lactobacillus of lactobacterium casei inserts activation medium, leaves standstill anaerobism and cultivates, and activation medium consists of: peptone 10g, beef extract 10g, yeast extract 5g, K 2HPO 42g, dibasic ammonium citrate 2g, NaAc 5g, glucose 20g, tween-80 1g, MgSO 47H 2O 0.2g, MnSO 4H 2O 0.05g, CaCO 320g distilled water is settled to 1000ml, and pH6.8 cultivates 24-48h for 30-40 ℃, transfers in fresh activation medium with the inoculum size of volume ratio 5%-15%, cultivates 24-48h for 30-40 ℃.
Seed culture
Activation bacterium liquid inserts 0.1m with the inoculum size of volume ratio 2%-10% 3Seeding tank, loading amount coefficient are the 70%-80% volume ratio, and seed culture medium is formed: peptone 5-10g, yeast extract 3-10g, glucose 5-10g, CaCO 35-10g, KH 2PO 41-5g, distilled water is settled to 1000ml, pH6.5-7.0,30-40 ℃ leaves standstill anaerobism cultivation 24-48h.
Fermentor cultivation
Seed culture fluid inserts 1m with the inoculum size of volume ratio 10%-20% 3Fermentor tank, loading amount coefficient are the 70%-80% volume ratio, and the substratum of fermentor cultivation is formed: glucose 10-20g, peptone 5-10g, yeast extract 3-10g, KH 2PO 41-5g, (NH 4) 2SO 42-5g, CaCO 310-20g, tap water is settled to 1000ml, and pH 7.0, and 30-40 ℃ leaves standstill anaerobism cultivation 24-48h.
Microorganism collection
Nutrient solution pumps into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains wet thallus.
The preparation of the former bacterium powder of lactobacterium casei
Lactobacterium casei wet thallus and dry starch are with mass ratio 1: 1-5 mixes, 35-45 ℃ of dry 20-24h, and pulverizer is pulverized, and crosses 40 mesh sieves, regulates viable count 〉=2 * 10 of lactobacterium casei with dry starch 10The CFU/ gram is the former bacterium powder of lactobacterium casei.
(4) the former bacterium powder of preparation yeast
Actication of culture
The yeast freeze-drying lactobacillus inserts activation medium, leaves standstill anaerobism and cultivates, and activation medium consists of: the 12Brix. wort, cultivate 20-24h for 30-40 ℃, and transfer in fresh activation medium with the inoculum size of volume ratio 5%-15%, cultivate 16-20h for 30-40 ℃.
Seed culture
Activation bacterium liquid inserts 0.1m with the inoculum size of volume ratio 2%-10% 3Seeding tank, the loading amount coefficient is the 70%-80% volume ratio, seed culture medium consists of: yeast extract 3g, glucose 5-10g, (NH 4) 2SO 43g, MgSO 47H 2O0.2g, NaCl 0.1g, KH 2PO 42g, tap water is settled to 1000ml, pH4-6, mixing speed 100-150r/min, air flow 0.5-1m 3/ h cultivates 16-20h for 28-30 ℃
Fermentor cultivation
Seed culture fluid inserts 1m with the inoculum size of volume ratio 5%-10% 3Fermentor tank, the loading amount coefficient is the 70%-80% volume ratio, fermentation tank culture medium consists of: yeast extract 3g, glucose 10-15g, (NH 4) 2SO 43g, KH 2PO 42g, MgSO 47H 2O 0.2g, NaCl 0.1g, tap water is settled to 1000ml, pH4-6, mixing speed 100-200r/min, air flow 1-2m 3/ h cultivates 20-24h for 28-30 ℃.
Microorganism collection
Nutrient solution pumps into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains wet thallus.
The preparation of the former bacterium powder of yeast
With yeast wet thallus and dry starch with mass ratio 1: 1-5 mixes, 35-45 ℃ of dry 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, regulates saccharomycetic viable count 〉=5 * 10 with dry starch 6The CFU/ gram is the former bacterium powder of yeast.
(5) preparation clostridium butyricum, lactobacterium casei mixes former bacterium powder with saccharomycetic
Fermentor cultivation
Clostridium butyricum, lactobacterium casei and saccharomycetic seed culture fluid are inserted 1m with the inoculum size of volume ratio 5%-10%, 10%-15%, 2%-6% respectively 3Fermentor tank, the loading amount coefficient is the 70%-80% volume ratio, fermentation tank culture medium consists of: glucose or brown sugar 20-40g, peptone 3-8g, yeast extract 2-6g, KH 2PO 41-5g, (NH 4) 2SO 42-5g, NaCl 3g, CaCO 31-5g, NaAc 1-3g, tap water is settled to 1000ml, pH 6.5-7.0,30-40 ℃ leaves standstill anaerobism cultivation 24-48h.
Microorganism collection
Nutrient solution pumps into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains to mix wet thallus.
Mix the preparation of former bacterium powder
To mix wet thallus and dry starch with mass ratio 1: 1-5 mixes, 35-45 ℃ of dry 20-24h, and pulverizer is pulverized, and crosses 40 mesh sieves, regulates clostridium butyricum viable count 〉=2.4 * 10 with dry starch 8The CFU/ gram, lactobacterium casei viable count 〉=2.4 * 10 9The CFU/ gram, yeast viable count 〉=2.4 * 10 6The CFU/ gram is and mixes former bacterium powder.
B. the preparation of tetragenous viable bacteria preparation
After L-calcium lactate, zeolite powder are crossed 40 mesh sieves, prepare burden by proportioning (1) or proportioning (2), mix thoroughly, make subtilis total viable count 〉=1 * 10 in the finished product with feed mixing machine with former bacterium powder 7The CFU/ gram, clostridium butyricum total viable count 〉=1 * 10 8The CFU/ gram, lactobacterium casei total viable count 〉=1 * 10 9The CFU/ gram, yeast total viable count 〉=1 * 10 6CFU/ gram, finished product adopt aluminium foil packed.
Proportioning (1) mass percent is:
The former bacterium powder of subtilis 2-2.5
The former bacterium powder of clostridium butyricum 20-25
The former bacterium powder of lactobacterium casei 5-6.5
The former bacterium powder of yeast 20-25
L-calcium lactate 6-10
Zeolite powder 40-45
Wherein:
Subtilis viable count 〉=5 * 10 in the former bacterium powder of subtilis 8The CFU/ gram
Clostridium butyricum viable count 〉=5 * 10 in the former bacterium powder of clostridium butyricum 8The CFU/ gram
Lactobacterium casei viable count 〉=2 * 10 in the former bacterium powder of lactobacterium casei 10The CFU/ gram
Yeast viable count 〉=5 * 10 in the former bacterium powder of yeast 6The CFU/ gram.
Proportioning (2) mass percent is:
The former bacterium powder of subtilis 2-2.5
Mix former bacterium powder 45-52
L-calcium lactate 6-10
Zeolite powder 40-45
Wherein:
Subtilis viable count 〉=5 * 10 in the former bacterium powder of subtilis 8The CFU/ gram mixes clostridium butyricum viable count 〉=2.4 * 10 in the former bacterium powder 8CFU/ gram, lactobacterium casei viable count 〉=2.4 * 10 9CFU/ gram and yeast viable count 〉=2.4 * 10 6The CFU/ gram.
Beneficial effect of the present invention:
This preparation has the not characteristics of aerobic decomposing organic matter, and contained microorganism activates soon in the preparation, and fecundity is strong, easily forms useful dominant bacteria in aquaculture water.Utilize the anaerobism of wherein contained clostridium butyricum, lactobacterium casei, subtilis, four kinds of microorganisms of yeast, good oxygen characteristic, performance synergy aspect the purification of water quality that strengthens aquatic animal resistance against diseases and aquaculture water, can substitute or partly substitute the diseases prevention and treatment that microbiotic is applied to fresh water and seawater aquaculture, raising aquaculture economic benefit.The L-calcium lactate has the effect that promotes shelling growth, prevention soft shell disease in the preparation to crustaceans aquatic animals such as aquatic animals especially shrimp crab.The all natural nature that is present in of the component of this preparation, nontoxic, have no side effect, noresidue and secondary pollution, do not produce resistance, in freshwater aquiculture and sea farming, have vast market and use.
Culture whole process and all can use this preparation, in aquaculture water, splash or interpolation tetragenous viable bacteria preparation in the bait of aquatic animal,, splash this preparation 100-1000 gram or add this preparation 0.1%-1% in bait in mass of every mu every meter depth of water is by eating of aquatic animal, enter in the aquatic animal body, adjust the beneficial flora balance of himself, improve immunity function, strengthen anti-infection ability, safeguard microecological balance in the aquatic animal body, the generation of disease preventing and treating; The yeast thalline contains multivitamin and somatomedin, and aquatic animal is had trophism; The L-calcium lactate is the calcium salt of L-lactic acid, L-lactic acid is natural to be present in the animal body, its calcium salt-L-calcium lactate is required when calcareous at additional aquatic animal to grow, easier absorbing, and crustaceans aquatic animals such as prawn crab have the effect that promotes shelling growth, prevention soft shell disease.
This preparation of splashing in aquaculture water pollutes, drains refuse, remaining bait, plant and animal residues etc. to the aquatic animal objectionable impurities at the bottom of efficiently decomposing the pool, purifies water, and improves the aquaculture water environment, suppresses growth of pathogenic bacteria, strengthens the prevention effect to disease.
Biological material specimens preservation and source explanation
Clostridium butyricum (Clostridium butyricum) JSIM-MCB20040312: be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, be called for short CGMCC, preservation date is on March 10th, 2006, and preserving number is CGMCC No.1647.
Subtilis (Bacillus subtilis): available from CGMCC, strain number 1.1414, referring to the CGMCC bacterial classification catalogue third edition, 1997, bacterium P12, the 3rd kind reciprocal.
Lactobacterium casei (Lactobacillus casei): available from CGMCC, strain number 1.539 is referring to the CGMCC bacterial classification catalogue third edition, 1997, bacterium P43, the 2nd kind.
Yeast (Sacccharomyces cerevisiae): available from CGMCC, strain number 2.614 is referring to the CGMCC bacterial classification catalogue third edition, 1997, yeast P80, the 7th kind.
Description of drawings
Fig. 1 tetragenous viable bacteria preparation process thereof block diagram.
Specific embodiments
Embodiment 1: the purebred cultivation of viable bacteria body
(1) the purebred cultivation of subtilis (Bacillus subtilis CGMCC1.1414)
Actication of culture
The freeze-drying lactobacillus of aseptic unlatching subtilis, streak inoculation are cultivated 18-24h for 37 ℃ in test tube nutrient broth agar inclined-plane, line is transferred in nutrient broth agar eggplant bottle inclined-plane then, cultivates 20-24h, microscopy for 37 ℃, when 90% above thalline forms gemma, be maturation, prepare culture transferring.
Seed culture
Prepare the aseptic triangular flask of an interior glaze pearl, with sterilized water the ripe bacterium mud on the eggplant bottle inclined-plane is scraped and washed, the triangular flask of packing into, vibrating dispersion bacterium mud obtains uniform bacteria suspension.Bacteria suspension was heated 5 minutes in 85 ℃ of water-baths, with the inoculum size access 0.1m of volume ratio 5% 3Seeding tank. the loading amount coefficient of seeding tank is 60% (v/v).
Seed culture medium consists of: glucose 3g, and peptone 5g, yeast extract paste 3g, NaCl 5g, tap water is settled to 1000ml, and pH 7.0-7.5 cultivates 17h, mixing speed 150r/min, air flow 1-2m for 37 ℃ 3/ h.
Fermentor cultivation
Seed culture fluid inserts 1m with the inoculum size of volume ratio 10% 3Fermentor tank, loading amount coefficient are 65% (v/v), and the substratum of fermentor cultivation consists of: glucose 10g, and soybean cake powder 10g, inorganic salt are NaCl 5g, tap water is settled to 1000ml, cultivates 20h, mixing speed 150r/min, air flow 1.5m for 37 ℃ 3/ h.In culturing process,, add soya-bean oil or bubble enemy and carry out froth breaking, prevent to cause the situation of living contaminants because of the foam fermentor tank of escaping out according to the surging situation of foamy.Get fermented liquid and carry out microscopy, when the thalline more than 90% has formed gemma, promptly put a jar end and cultivate.
Microorganism collection
Nutrient solution is pumped into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains wet thallus, for the former bacterium powder of preparation Bacillus subtilus.
(2) the purebred cultivation of clostridium butyricum (Clostridium butyricum JSIM-MCB-20040312)
Actication of culture
The freeze-drying lactobacillus of aseptic unlatching clostridium butyricum inserts the test tube that activation medium is housed, and leaves standstill anaerobism and cultivates.Activation medium consists of: peptone 10g, beef extract 10g, yeast extract 3g, glucose 10g, Zulkovsky starch 1g, NaCl 5g, NaAc 3g, cysteine hydrochloride 0.5g, CaCO 33g, distilled water is settled to 1000ml, and pH7.0 cultivates 24h for 37 ℃, transfers in fresh activation medium with the inoculum size of volume ratio 10% then, cultivates 35h for 37 ℃, and microscopy is promptly ripe when 90% above thalline forms gemma, prepares culture transferring.
Seed culture
Activation bacterium liquid heated 5 minutes in 85 ℃ of water-baths, with the inoculum size access 0.1m of volume ratio 10% 3Seeding tank, loading amount coefficient are 80% (v/v).Seed culture medium consists of: peptone 5g, beef extract 10g, yeast extract 3g, glucose 5g, starch 0.5g, NaCl 5g, NaAc 3g, CaCO 31g, distilled water is settled to 1000ml, pH7.0,37 ℃ leave standstill anaerobism cultivation 20h.
Fermentor cultivation
Seed culture fluid inserts 1m with the inoculum size of volume ratio 10% 3Fermentor tank, loading amount coefficient are 80% (v/v), and fermentation tank culture medium consists of: peptone 7g, yeast extract 3g, glucose 5g, Zulkovsky starch 1g, NaCl 5g, NaAc 3g, CaCO 33g, tap water is settled to 1000ml, and pH leaves standstill anaerobism for 7.0,37 ℃ and cultivates 40h, and microscopy when 90% above thalline forms gemma, is promptly put a jar end and is cultivated.
Microorganism collection
Nutrient solution is pumped into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains wet thallus, for the former bacterium powder of preparation clostridium butyricum.
(3) lactobacterium casei (the purebred cultivation of (Lactobacillus casei CGMCC1.539)
Actication of culture
The freeze-drying lactobacillus of aseptic unlatching lactobacterium casei inserts the test tube that activation medium is housed, and leaves standstill anaerobism and cultivates.
Activation medium consists of: peptone 10g, beef extract 10g, yeast extract 5g, K 2HPO 42g, dibasic ammonium citrate 2g, NaAc 5g, glucose 20g, tween-80 1g, MgSO 47H 2O 0.2g, MnSO 4H 2O0.05g, CaCO 320g distilled water is settled to 1000ml, and pH6.8 cultivates 30h for 30 ℃, transfers in fresh activation medium with the inoculum size of volume ratio 8% then, cultivates 24h for 30 ℃.
Seed culture
Activation bacterium liquid inserts 0.1m with the inoculum size of volume ratio 5% 3Seeding tank.The loading amount coefficient of seeding tank is 80% (v/v), and seed culture medium consists of: peptone 10g, yeast extract 5g, glucose 5g, CaCO 37g, KH 2PO 42g, distilled water is settled to 1000ml, pH6.5-7.0,30 ℃ leave standstill anaerobism cultivation 30h.
Fermentor cultivation
Seed culture fluid inserts 1m with the inoculum size of volume ratio 15% 3Fermentor tank. fermentor tank loading amount coefficient is 80% (v/v), and the substratum of fermentor cultivation consists of: glucose 10g, peptone 5g, yeast extract 5g, KH 2PO 42g, (NH 4) 2SO 43g, CaCO 315g, tap water is settled to 1000ml, and pH leaves standstill anaerobism for 7.0,30 ℃ and cultivates 36h.
Microorganism collection
Nutrient solution is pumped into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains wet thallus, for the former bacterium powder of preparation lactobacterium casei.
(4) the purebred cultivation of yeast (Sacccharomyces cerevisiae CGMCC2.614)
Actication of culture
Aseptic unlatching zymic freeze-drying lactobacillus inserts the test tube that activation medium is housed, and leaves standstill anaerobism and cultivates.
Activation medium consists of: the 12Brix. wort, cultivate 24h for 30 ℃, and transfer in fresh activation medium with the inoculum size of volume ratio 5% then, cultivate 16h for 30 ℃.
Seed culture
Activation bacterium liquid inserts 0.1m with the inoculum size of volume ratio 5% 3Seeding tank, loading amount coefficient are 70% (v/v), and seed culture medium consists of: yeast extract 3g, glucose 5g, (NH 4) 2SO 43g, MgSO 47H 2O 0.2g, NaCl 0.1g, KH 2PO 42g, tap water is settled to 1000ml, and pH5 cultivates 18h, mixing speed 100r/min, air flow 0.5m for 30 ℃ 3/ h.
Fermentor cultivation
Seed culture fluid meets 1m with the inoculum size of volume ratio 5% 3Fermentor tank, loading amount coefficient are 70% (v/v), and fermentation tank culture medium consists of: yeast extract 3g, glucose 10-15g, (NH 4) 2SO 43g, KH 2PO 42g, MgSO 47H 2O0.2g, NaCl 0.1g, tap water is settled to 1000ml, and pH cultivates 20h, mixing speed 100r/min, air flow 1m for 5,30 ℃ 3/ h.
Microorganism collection
Nutrient solution is pumped into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains wet thallus, for the former bacterium powder of preparation yeast.
Embodiment 2: the mixed culture of clostridium butyricum, lactobacterium casei and yeast viable bacteria body
Actication of culture
The activation of clostridium butyricum, lactobacterium casei, yeast freeze-drying lactobacillus is with identical described in the embodiment 1.
Seed culture
Clostridium butyricum, lactobacterium casei, saccharomycetic seed culture are with identical described in the embodiment 1.
Fermentor cultivation
Clostridium butyricum, lactobacterium casei, saccharomycetic seed culture fluid are inserted 1m with 6%, 12%, 2% inoculum size respectively 3Fermentor tank, loading amount coefficient are 80% (v/v).
Fermentation tank culture medium consists of: brown sugar 20g, peptone 5g, yeast extract 3g, KH 2PO 41g, (NH 4) 2SO 42g, NaCl 3g, CaCO 33g, NaAc 3g, tap water 1000ml, pH leave standstill anaerobism for 7.0,35 ℃ and cultivate 48h.
Microorganism collection
Nutrient solution is pumped into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains wet thallus, for preparation clostridium butyricum, lactobacterium casei and the former bacterium powder of saccharomycetic mixing.
Embodiment 3: the preparation of the former bacterium powder of microorganism pure strain
(1) the former bacterium powder of subtilis
The subtilis wet thallus of collecting after the purebred cultivation is mixed with mass ratio with dry starch at 1: 2, at 40-50 ℃ of following cryodrying 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, regulates total viable count 〉=5 * 10 of subtilis in the former bacterium powder with dry starch 8The CFU/ gram.
(2) the former bacterium powder of clostridium butyricum
The clostridium butyricum wet thallus of collecting after the purebred cultivation is mixed with mass ratio with dry starch at 1: 2.5, at 40-50 ℃ of following cryodrying 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, regulates total viable count 〉=5 * 10 of clostridium butyricum in the former bacterium powder with dry starch 8The CFU/ gram.
(3) the former bacterium powder of lactobacterium casei
The lactobacterium casei wet thallus of collecting after the purebred cultivation is mixed with mass ratio with dry starch at 1: 2, and at 35-45 ℃ of following cryodrying 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, regulates total viable count 〉=2 * 10 of lactobacterium casei in the former bacterium powder with dry starch 10The CFU/ gram.
(4) the former bacterium powder of yeast
The clostridium butyricum wet thallus of collecting after the purebred cultivation is mixed with mass ratio with dry starch at 1: 5, and at 35-45 ℃ of following cryodrying 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, regulates saccharomycetic total viable count 〉=5 * 10 in the former bacterium powder with dry starch 6The CFU/ gram.
Embodiment 4: mix the preparation of former bacterium powder
The mixing wet thallus of collecting after clostridium butyricum, lactobacterium casei and the yeast mixed culture is mixed with mass ratio with dry starch at 1: 2, at 35-45 ℃ of following cryodrying 20-24h, pulverizer is pulverized, cross 40 mesh sieves, regulate with dry starch and mix clostridium butyricum total viable count 〉=2.4 * 10 in the former bacterium powder 8The CFU/ gram, lactobacterium casei total viable count 〉=2.4 * 10 9The CFU/ gram, yeast total viable count 〉=2.4 * 10 6The CFU/ gram.
Embodiment 5: the preparation of tetragenous viable bacteria preparation finished product
After L-calcium lactate, zeolite powder are crossed 40 mesh sieves,, mix thoroughly, make subtilis total viable count 〉=1 * 10 in the finished product with feed mixing machine with the listed proportioning of former bacterium powder according to the form below 7The CFU/ gram, clostridium butyricum total viable count 〉=1 * 10 8The CFU/ gram, lactobacterium casei total viable count 〉=1 * 10 9The CFU/ gram, yeast total viable count 〉=1 * 10 6The CFU/ gram.Finished product adopts aluminium foil packed, every packed amount 1kg, and 15 bags is 1 packed in cases, carton material adopts fluting board.
Table 1 is by 5 routine quality percentage compositions of proportioning (1)
Component Proportioning (1)
The quality percentage composition
The former bacterium powder of subtilis ??2.0% ??2.2% ??2.0% ??2.5% ??2.0%
The former bacterium powder of clostridium butyricum ? ??22.5% ? ??25.0% ? ??20.0% ? ??20.0% ? ??20.0%
The former bacterium powder of lactobacterium casei ??6.5% ??5.0% ??6.0% ??6.5% ??6.0%
The former bacterium powder of yeast ??20.0% ??20.0% ??22.0% ??25.0% ??21.0%
The L-calcium lactate ??8.0% ??6.0% ??10.0% ??6.0% ??6.0%
Zeolite powder ??41.0% ??41.8% ??40.0% ??40.0% ??45.0%
Add up to ??100.0% ? ??100.0% ? ??100.0% ? ??100.0% ? ??100.0% ?
Table 2 is by 4 routine quality percentage compositions of proportioning (2)
Component Proportioning (2)
The quality percentage composition
The former bacterium powder of subtilis ??2.0% ??2.2% ??2.5% ??2.0%
Mix former bacterium powder ??52.0% ??47.8% ??45.0% ??48.0%
The L-calcium lactate ??6.0% ??8.0% ??7.5% ??10.0%
Zeolite powder ??40.0% ??42.0% ??45.0% ??40.0%
Add up to ??100.0% ??100.0% ??100.0% ??100.0%

Claims (5)

1. tetragenous viable bacteria preparation is characterized in that by clostridium butyricum (Clostridiumbutyricum) CGMCC No.1647, lactobacterium casei (Lactobacillus casei) CGMCC 1.539, subtilis (Bacillus subtilis) CGMCC 1.1414 and yeast (Sacccharomycescerevisiae) CGMCC 2.614 four kinds of probiotic bacteriums and L-calcium lactates and zeolite powder is composite forms; Total viable count is respectively in the preparation: subtilis total viable count 〉=1 * 10 7The CFU/ gram, clostridium butyricum total viable count 〉=1 * 10 8The CFU/ gram, lactobacterium casei total viable count 〉=1 * 10 9The CFU/ gram, yeast total viable count 〉=1 * 10 6The CFU/ gram, the mass content of L-calcium lactate is 6%-10% in the preparation, the mass content of zeolite powder is 40%-45%.
2. tetragenous viable bacteria preparation according to claim 1 is characterized in that preparing and getting with the former bacterium powder that the purebred cultivation of microorganism obtains, and its quality per distribution ratio (1) is:
The former bacterium powder of subtilis 2-2.5
The former bacterium powder of clostridium butyricum 20-25
The former bacterium powder of lactobacterium casei 5-6.5
The former bacterium powder of yeast 20-25
L-calcium lactate 6-10
Zeolite powder 40-45
Wherein:
Subtilis viable count 〉=5 * 10 in the former bacterium powder of subtilis 8The CFU/ gram
Clostridium butyricum viable count 〉=5 * 10 in the former bacterium powder of clostridium butyricum 8The CFU/ gram
Lactobacterium casei viable count 〉=2 * 10 in the former bacterium powder of lactobacterium casei 10The CFU/ gram
Yeast viable count 〉=5 * 10 in the former bacterium powder of yeast 6The CFU/ gram.
3. tetragenous viable bacteria preparation according to claim 1, it is characterized in that the former bacterium powder that obtains with the purebred cultivation of subtilis and mix former bacterium powder and prepare and get with saccharomycetic that its quality per distribution ratio (2) is with contain clostridium butyricum, lactobacterium casei that clostridium butyricum, lactobacterium casei and saccharomycetic mixed culture obtain:
The former bacterium powder of subtilis 2-2.5
Mix former bacterium powder 45-52
L-calcium lactate 6-10
Zeolite powder 40-45
Wherein:
Subtilis viable count 〉=5 * 10 in the former bacterium powder of subtilis 8The CFU/ gram mixes clostridium butyricum viable count 〉=2.4 * 10 in the former bacterium powder 8The CFU/ gram, lactobacterium casei viable count 〉=2.4 * 10 9The CFU/ gram, yeast viable count 〉=2.4 * 10 6The CFU/ gram.
4. preparation method as tetragenous viable bacteria preparation as described in the claim 1,2 or 3 is characterized in that by clostridium butyricum (Clostridium butyricum) CGMCC No.1647, lactobacterium casei (Lactobacillus casei) CGMCC 1.539, subtilis (Bacillus subtilis) CGMCC 1.1414 and yeast (Sacccharomyces cerevisiae) CGMCC 2.614 four kinds of probiotic bacteriums and L-calcium lactates and zeolite powder is composite forms;
A. the preparation of the cultivation of active thalline and former bacterium powder:
(1) the former bacterium powder of preparation subtilis
Actication of culture
The freeze-drying lactobacillus of subtilis, streak inoculation were cultivated 18-24 hour for 30-40 ℃ in the nutrient broth agar inclined-plane, line is transferred in nutrient broth agar eggplant bottle inclined-plane then, cultivates microscopy 20-24 hour for 30-40 ℃, to 90% above thalline formation gemma, be maturation;
Seed culture
Ripe bacterium mud scraped with sterilized water wash, the aseptic triangular flask of glaze pearl in packing into, vibration obtains uniform bacteria suspension, bacteria suspension heat 5-15 minute in 70-90 ℃ of water-bath, with the inoculum size access 0.1m of volume ratio 2%-10% 3Seeding tank, loading amount coefficient are the 60%-70% volume ratio,
Seed culture medium consists of: glucose 3-5g, and peptone 5-10g, yeast extract paste 3-5g, NaCl 5g, tap water is settled to 1000ml, pH7.0-7.5, culture condition is: mixing speed 100-200r/min, air flow 1-2m 3/ h, culture temperature is 30-40 ℃, incubation time is 16-20h;
Fermentor cultivation
Seed culture fluid inserts 1m with the inoculum size of volume ratio 5%-20% 3Fermentor tank, loading amount coefficient are the 60%-70% volume ratio, and the substratum of fermentor cultivation is made up of carbon source, nitrogenous source and inorganic salt, and carbon source is 5-15g/L with glucose or starch, content; Nitrogenous source peptone, yeast extract paste, soybean cake powder or groundnut meal, content is 10-20g/L, and inorganic salt are NaCl 5g/L, and culture condition is: mixing speed 100-200r/min, air flow 1-2m 3/ h, culture temperature is 30-40 ℃, and incubation time is 16-20h, and microscopy when the thalline more than 90% has formed gemma, is put and jar is finished to cultivate;
Microorganism collection
Nutrient solution pumps into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains wet thallus;
The preparation of the former bacterium powder of subtilis
Collect wet thallus, with dry starch with mass ratio 1: 1-5 mixes, and at 40-50 ℃ of dry 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, with viable count 〉=5 * 10 of dry starch adjusting subtilis 8The CFU/ gram is the former bacterium powder of subtilis;
(2) the former bacterium powder of preparation clostridium butyricum
Actication of culture
The freeze-drying lactobacillus of clostridium butyricum inserts activation medium, leaves standstill anaerobism and cultivates, and activation medium is formed: peptone 10g, beef extract 10g, yeast extract 3g, glucose 10g, Zulkovsky starch 1g, NaCl5g, NaAc 3g, cysteine hydrochloride 0.5g, CaCO 33g, distilled water is settled to 1000ml, and pH 7.0, cultivate 24-48h for 30-40 ℃, transfer in fresh activation medium with the inoculum size of volume ratio 5%-15%, cultivate 24-48h for 30-40 ℃, and microscopy during to 90% above thalline formation gemma, is maturation;
Seed culture
Bacterium liquid heated 5-15 minute in 70-90 ℃ of water-bath, with the inoculum size access 0.1m of volume ratio 2%-10% 3Seeding tank, the loading amount coefficient is the 70%-80% volume ratio, seed culture medium consists of: peptone 5-10g, beef extract 5-10g, yeast extract 3-10g, glucose 5-10g, starch 0.5-1g, NaCl 5g, NaAc1-3g, CaCO 31-3g, distilled water is settled to 1000ml, and pH 7.0, and 30-40 ℃ leaves standstill anaerobism cultivation 16-24h;
Fermentor cultivation
Seed culture fluid inserts 1m with the inoculum size of volume ratio 5%-20% 3Fermentor tank, loading amount coefficient are the 70%-80% volume ratio, and the substratum of fermentor cultivation consists of: peptone 5-10g, yeast extract 3-10g, glucose 5-15g, starch 0.5-1g, NaCl 5g, NaAc 1-3g, CaCO 33-10g, tap water is settled to 1000ml, and pH 7.0, and 30-40 ℃ leaves standstill anaerobism and cultivates 24-48h, and microscopy when 90% above thalline forms gemma, is promptly put and jar is finished cultivation;
Microorganism collection
Nutrient solution pumps into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains wet thallus;
The preparation of the former bacterium powder of clostridium butyricum
With clostridium butyricum wet thallus and dry starch with mass ratio 1: 1-5 mixes, 40-50 ℃ of dry 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, regulates viable count 〉=5 * 10 of clostridium butyricum with dry starch 8The CFU/ gram is the former bacterium powder of clostridium butyricum;
(3) the former bacterium powder of preparation lactobacterium casei
Actication of culture
The freeze-drying lactobacillus of lactobacterium casei inserts activation medium, leaves standstill anaerobism and cultivates, and activation medium consists of: peptone 10g, beef extract 10g, yeast extract 5g, K 2HPO 42g, dibasic ammonium citrate 2g, NaAc 5g, glucose 20g, tween-80 1g, MgSO 47H 2O 0.2g, MnSO 4H 2O 0.05g, CaCO 320g, distilled water is settled to 1000ml, and pH6.8 cultivates 24-48h for 30-40 ℃, transfers in fresh activation medium with the inoculum size of volume ratio 5%-15%, cultivates 24-48h for 30-40 ℃;
Seed culture
Activation bacterium liquid inserts 0.1m with the inoculum size of volume ratio 2%-10% 3Seeding tank, loading amount coefficient are the 70%-80% volume ratio, and seed culture medium is formed: peptone 5-10g, yeast extract 3-10g, glucose 5-10g, CaCO 35-10g, KH 2PO 41-5g, distilled water is settled to 1000ml, pH6.5-7.0,30-40 ℃ leaves standstill anaerobism cultivation 24-48h;
Fermentor cultivation
Seed culture fluid inserts 1m with the inoculum size of volume ratio 10%-20% 3Fermentor tank, loading amount coefficient are the 70%-80% volume ratio, and the substratum of fermentor cultivation is formed: glucose 10-20g, peptone 5-10g, yeast extract 3-10g, KH 2PO 41-5g, (NH 4) 2SO 42-5g, CaCO 310-20g, tap water is settled to 1000ml, pH7.0,30-40 ℃ leaves standstill anaerobism cultivation 24-48h;
Microorganism collection
Nutrient solution pumps into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains wet thallus;
The preparation of the former bacterium powder of lactobacterium casei
Lactobacterium casei wet thallus and dry starch are with mass ratio 1: 1-5 mixes, 35-45 ℃ of dry 20-24h, and pulverizer is pulverized, and crosses 40 mesh sieves, regulates viable count 〉=2 * 10 of lactobacterium casei with dry starch 10The CFU/ gram is the former bacterium powder of lactobacterium casei;
(4) the former bacterium powder of preparation yeast
Actication of culture
The yeast freeze-drying lactobacillus inserts activation medium, leaves standstill anaerobism and cultivates, and activation medium consists of: the 12Brix. wort, cultivate 20-24h for 30-40 ℃, and transfer in fresh activation medium with the inoculum size of volume ratio 5%-15%, cultivate 16-20h for 30-40 ℃;
Seed culture
Activation bacterium liquid inserts 0.1m with the inoculum size of volume ratio 2%-10% 3Seeding tank, the loading amount coefficient is the 70%-80% volume ratio, seed culture medium consists of: yeast extract 3g, glucose 5-10g, (NH 4) 2SO 43g, MgSO 47H 2O0.2g, NaCl 0.1g, KH 2PO 42g, tap water is settled to 1000ml, pH4-6, mixing speed 100-150r/min, air flow 0.5-1m 3/ h cultivates 16-20h for 28-30 ℃;
Fermentor cultivation
Seed culture fluid inserts 1m with the inoculum size of volume ratio 5%-10% 3Fermentor tank, the loading amount coefficient is the 70%-80% volume ratio, fermentation tank culture medium consists of: yeast extract 3g, glucose 10-15g, (NH 4) 2SO 43g, KH 2PO 42g, MgSO 47H 2O 0.2g, NaCl 0.1g, tap water is settled to 1000ml, pH4-6, mixing speed 100-200r/min, air flow 1-2m 3/ h cultivates 20-24h for 28-30 ℃;
Microorganism collection
Nutrient solution pumps into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains wet thallus;
The preparation of the former bacterium powder of yeast
With yeast wet thallus and dry starch with mass ratio 1: 1-5 mixes, 35-45 ℃ of dry 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, regulates saccharomycetic viable count 〉=5 * 10 with dry starch 6The CFU/ gram is the former bacterium powder of yeast;
(5) preparation clostridium butyricum, lactobacterium casei mixes former bacterium powder with saccharomycetic
Fermentor cultivation
Clostridium butyricum, lactobacterium casei and saccharomycetic seed culture fluid are inserted 1m with the inoculum size of volume ratio 5%-10%, 10%-15%, 2%-6% respectively 3Fermentor tank, the loading amount coefficient is the 70%-80% volume ratio, fermentation tank culture medium consists of: glucose or brown sugar 20-40g, peptone 3-8g, yeast extract 2-6g, KH 2PO 41-5g, (NH 4) 2SO 42-5g, NaCl 3g, CaCO 31-5g, NaAc 1-3g, tap water is settled to 1000ml, pH 6.5-7.0,30-40 ℃ leaves standstill anaerobism cultivation 24-48h;
Microorganism collection
Nutrient solution pumps into rotating speed, and to be that the continuous centrifugal machine of 15000r/min carries out centrifugal, abandons supernatant liquor, obtains to mix wet thallus;
Mix the preparation of former bacterium powder
To mix wet thallus and dry starch with mass ratio 1: 1-5 mixes, 35-45 ℃ of dry 20-24h, and pulverizer is pulverized, and crosses 40 mesh sieves, regulates clostridium butyricum viable count 〉=2.4 * 10 with dry starch 8The CFU/ gram, lactobacterium casei viable count 〉=2.4 * 10 9The CFU/ gram, yeast viable count 〉=2.4 * 10 6The CFU/ gram is and mixes former bacterium powder;
B. the preparation of tetragenous viable bacteria preparation
After L-calcium lactate, zeolite powder are crossed 40 mesh sieves, prepare burden by proportioning (1) or proportioning (2), mix thoroughly, make subtilis total viable count 〉=1 * 10 in the finished product with feed mixing machine with former bacterium powder 7The CFU/ gram, clostridium butyricum total viable count 〉=1 * 10 8The CFU/ gram, lactobacterium casei total viable count 〉=1 * 10 9The CFU/ gram, yeast total viable count 〉=1 * 10 6CFU/ gram, finished product adopt aluminium foil packed;
Proportioning (1) mass percent is:
The former bacterium powder of subtilis 2-2.5
The former bacterium powder of clostridium butyricum 20-25
The former bacterium powder of lactobacterium casei 5-6.5
The former bacterium powder of yeast 20-25
L-calcium lactate 6-10
Zeolite powder 40-45
Wherein:
Subtilis viable count 〉=5 * 10 in the former bacterium powder of subtilis 8The CFU/ gram
Clostridium butyricum viable count 〉=5 * 10 in the former bacterium powder of clostridium butyricum 8The CFU/ gram
Lactobacterium casei viable count 〉=2 * 10 in the former bacterium powder of lactobacterium casei 10The CFU/ gram
Yeast viable count 〉=5 * 10 in the former bacterium powder of yeast 6The CFU/ gram.
Proportioning (2) mass percent is:
The former bacterium powder of subtilis 2-2.5
Mix former bacterium powder 45-52
L-calcium lactate 6-10
Zeolite powder 40-45
Wherein:
Subtilis viable count 〉=5 * 10 in the former bacterium powder of subtilis 8The CFU/ gram mixes clostridium butyricum viable count 〉=2.4 * 10 in the former bacterium powder 8CFU/ gram, lactobacterium casei viable count 〉=2.4 * 10 9CFU/ gram and yeast viable count 〉=2.4 * 10 6The CFU/ gram.
5. a strain clostridium butyricum (Clostridium butyricum) JSIM-MCB20040312, the bacterial strain deposit number is CGMCC No.1647.
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CN104522648A (en) * 2014-12-18 2015-04-22 杭州龙达新科生物制药有限公司 Tetragenus probiotic preparation and application thereof
CN105795097A (en) * 2014-12-30 2016-07-27 付顺林 Aquaculture feed and preparation method thereof
CN104480052A (en) * 2015-01-05 2015-04-01 江苏省苏微微生物研究有限公司 Composite bacillus preparation containing three strains, preparation method of composite bacillus preparation and application of composite bacillus preparation to ecological breeding
CN105432514A (en) * 2015-11-09 2016-03-30 天津振邦水产养殖有限公司 Method for improving sea cucumber growth water area environment by adopting compound microbial ecological agent
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