CN107245467A - One kind cultivation microorganism formulation and preparation method thereof - Google Patents
One kind cultivation microorganism formulation and preparation method thereof Download PDFInfo
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Abstract
The invention discloses one kind cultivation microorganism formulation and preparation method thereof, it by weight ratio is 1 that it, which is,:10 composite fluid strain and nutrient solution by be formulated；The nutrient solution is prepared from by the raw material of following parts by weight：75 85 parts of glucose, 45 55 parts of brown sugar, 0.4 0.6 parts of potassium dihydrogen phosphate, 0.8 1.2 parts of urea, 46 parts of multi-hydroxy carboxy acid's titanium, 990 1100 parts of boiling water；The composite fluid strain be by weight ratio be 1:10 composite bacteria liquid and bacteria culture fluid is prepared from.
Description
Technical field
The invention belongs to cultivate formulation art, more particularly to a kind of cultivation microorganism formulation and preparation method thereof.
Background technology
As aquaculture industrialization, scale are continued to develop, demand of the people to meat product is greatly met, also carry
It is high and improve life.After the life of people is improved and improved constantly, there is higher chase after to the quality and local flavor of meat
Ask.Carnivorous quality and local flavor when always wanting to give for change the foster method of farmers''s soil.Under inscribing before this, cultivation industry is in order to meet people to meat
The demand that matter quality and local flavor are improved, part plant does not stint extension feeding time to improve to reduce the aquaculture model of family
The quality and local flavor of meat.Taking for having puts the method with free-ranging in a suitable place to breed to improve the quality and local flavor of meat.Satisfaction masses are tried to achieve to meat
The pursuit of food matter and local flavor.But the change of aquaculture model, also think the increase of aquaculture cost, be finally that carnivorous price is remote
Far beyond common carnivorous price, what is had exceeds several times, it is difficult to meet vast public demand.
Industrialization, the scale pattern for being badly in need of solution aquaculture at present are constant, deliver speed for sale and do not extend, do not increase and cultivate into
This is the animal meat quality quality that animal meat quality quality reaches and exceeds the foster method of soil simultaneously, makes to butcher rear livestock meat without urine smell taste, mouth
Feel chewy, soup meat overflows the fresh perfume (or spice) of nature.The price of meat and the price of common meat maintain an equal level, and greatly to meet masses to high-quality meat
The demand of food and local flavor.
The content of the invention
The present invention seeks to a kind of cultivation microorganism formulation and its preparation side are provided for above-mentioned the deficiencies in the prior art
Method.
The present invention realizes that above-mentioned purpose is adopted the technical scheme that：One kind cultivation microorganism formulation, it is by weight ratio
For 1:10 composite fluid strain and nutrient solution, which spreads cultivation, to be formed；The nutrient solution be by following parts by weight raw material prepare and
Into：75-85 parts of glucose, 45-55 parts of brown sugar, 0.4-0.6 parts of potassium dihydrogen phosphate, 0.8-1.2 parts of urea, multi-hydroxy carboxy acid's titanium 4-
6th, boiling water 990-1100 parts；The composite fluid strain be by weight ratio be 1:10 composite bacteria liquid and bacteria culture fluid spreads cultivation
Form, the bacteria culture fluid is prepared from by the raw material of following parts by weight：75-85 parts of glucose, 45-55 parts of brown sugar,
3-5 parts of peptone, 2-3 parts of yeast extract, 0.3-0.7 parts of magnesium sulfate, 0.7-0.8 parts of potassium dihydrogen phosphate, 0.8-1.2 parts of urea, chlorine
Change 1-2 parts of sodium, 0.02-0.03 parts of ferrous sulfate, 0.008-0.012 parts of manganese sulfate, 1 milliliter of multi-hydroxy carboxy acid's titanium, distilled water
990-1100 parts；（Heat sterilization is to without offscum）！, the following strain of composite bacteria liquid, which is mixed, to be formed：Rhizopus oryzae, yeast
Bacterium, Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc mesenteroides, Pediococcus parvulus, streptococcus fecalis, bacillus licheniformis, short and small brood cell's bar
Bacterium.
A kind of method for preparing the cultivation microorganism formulation, the described method comprises the following steps：
（One）Rhizopus oryzae and saccharomycete spread cultivation
Test tube bacterium is accessed into the Czapek's medium that 94 centimeters of culture dishes of diameter are prepared by Rhizopus oryzae and saccharomycete respectively, it is each per bacterium
4 wares.Put 30 DEG C of insulating box culture 72h, the vigorous 2 healthy and strong culture dishes of growth selection, by two bacterium respectively with 30 milliliters of sterilized waters
Ready bottled 200 milliliters general propagation fluid nutrient mediums of 1000 milliliters of triangles in advance are eluted to, insulating box is put into and is increased
Value spreads cultivation 72h, and culture to bacterium solution muddiness has an obvious thalline, and bacterium solution pH value is down to 3.5-4 and terminates standby, and bacterium number is examined up to 10 hundred million in border
To be qualified.Two bacterium are pressed 1 again:1 ratio respectively takes 20 milliliters to be put into 1000 milliliters of 400 milliliters of the Multiplying culture prepared in advance liquid
Interior culture, microscopy bacterium number puts safe standby up to 10 hundred million.
（Two）Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc mesenteroides, Pediococcus parvulus, streptococcus fecalis spread cultivation
A, will spread cultivation Nutrient medium：5 grams of peptone, 20 grams of tryptone, 5 grams of yeast extract, 200 milliliters of Tomato juice, liver extract, glucose
3 grams, 3 grams of lactose, 0.05 gram of Tween-80,1 liter of distilled water, the dissolving sterilizing of 20 grams of heating stirrings of agar, it is sterile under pour and sterilized
Culture dish, more than sterile access each test tube slant bacterium after solidification accesses 94 centimeters of wares of plate 4 of diameter, insulating box 30 per bacterium
DEG C culture is covered with to bacterium colony；
B, prepare 2000 milliliters by above culture medium and do not put agar, sterilize cool to 25 DEG C, be divided in 500 milliliters of sterilized triangles
In bottle, 200 milliliters every bottle, with sterile purified water, 30 milliliters select 5 kinds of bacterium that training has grown in gnotobasis, respectively with sterilizing brush
Son elution bacterium colony respective nutrient solution into corresponding nutrient solution, puts 30 DEG C of insulating box cultures to bacterium solution muddiness, when pH value is to 3.5-4
Termination is standby, and microscopy bacterium number reaches that more than 1,000,000,000 be qualified.1000 milliliters are prepared by former culture medium, is dispensed to the three of three 1000
Sterilized in the bottle of angle, gnotobasis takes 5 kinds of different each 10 milliliters of additions of bacterium, 3 triangular flasks respectively, puts 28 DEG C of insulating box cultures to bacterium
Liquid is muddy, and pH value is down to 4, and microscopy bacterium number is qualified up to more than 1,000,000,000, puts safe standby.
（Three）Bacillus licheniformis, bacillus pumilus spread cultivation
A, spread cultivation Nutrient medium：5 grams of peptone, beef leaching cream, 5 grams of sodium chloride, 5 milligrams of manganese sulfate, 15 grams of agar, distilled water
1000ml.PH value 7.Agar is dissolved by heating, sterilizing is poured to sterilized 10 culture dishes in gnotobasis while hot（Specification 94cm）,
After solidification access test tube bacterium, put 35 DEG C of of insulating box cultivate to be covered with culture dish terminate；
B, the Nutrient medium that will spread cultivation remove agar, and other nutrients are dissolved in 1000ml distilled water, are gone out in 3 triangular flasks of packing
Bacterium, sterilize rearmounted cool to 25 DEG C of gnotobasis, takes off culture dish bacterium colony into the liquid that spreads cultivation with sterilized water 30 and sterile scrub, puts 35 DEG C
Incubator is muddy to bacterium solution, and PH is down to 4, and it is qualified up to more than 1,000,000,000 that bacterium number is examined in border, puts safe standby
（Four）The culture of composite fluid strain
A, with multi-hydroxy carboxy acid's titanium and distillation aquathermolysis glucose, brown sugar, peptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate,
Urea, sodium chloride, ferrous sulfate,（Calcium chloride）Manganese sulfate, adjustment solution pH value is 7；
B, by the solvent nutrient solution average mark of upper step be mounted in each 300ml sterilization treatments of 3 1000ml triangular flasks, in gnotobasis
It is lower to incite somebody to action Step 1: two, three cultured liquid bacterias are mixed and shaken up, by 1:10 ratio is separately added into three triangular flask nutrient solutions
In, multilayer aseptic paper sealing, jog is even, puts constant incubator culture, cultivates 3-5 days, and bacterium solution is muddy, and pH value is down to less than 4, border
It is qualified that bacterium number, which is examined, up to respectively more than 1,000,000,000, and preservation is standby.
C, the liquid making method that spreads cultivation by compound bacteria prepare 3000ml sterilizings, load each in 5 1000ml triangular flasks that sterilized
Three of the above, is prepared Mixed Microbes by 1 by 600ml:10 ratio puts 30 DEG C of case trainings of culture respectively with adding in each triangular flask
Support, 3-5 days pH values of culture are up to 4, and it is qualified up to more than 1,000,000,000 that total bacteria count is examined in border.As co-incubation composite fluid strain.
（Five）Production is spread cultivation with bacterium：
By except the clout mixed dissolution of the nutrient solution of multi-hydroxy carboxy acid's titanium, water temperature adds multi-hydroxy carboxy acid's titanium when being down to 25 DEG C；Will
Composite fluid strain and nutrient solution are 1 by volume:10 ratio mixing, is cultivated three days in the environment of putting 25 DEG C, cultured
The vigor holding 90% in 1 year of strain preparation sealing preserve, it is as qualified.
The liquid that spreads cultivation of breeding is formulated by following raw material：5 grams of peptone, 2 grams of yeast extract, 10 grams of glucose, phosphorus
1 gram of acid dihydride potassium, 1 gram of magnesium sulfate, 1 milliliter of titanium hydroxycarboxylate, 1000 milliliters of pure water.
The present invention principle and beneficial effect be：Current intensive culture uses perfect compound feed, cultivates close
Degree is big, and breeding cycle is short, and the additive added in feed is wide in variety, and the anti-disease medicines of addition are more.Intensive culture it is wide in variety
It is the adventive introduced.Many factors cause the scatol in breeding process in livestock and poultry body to accumulate used in remote super native foster method
The accumulation of the factors such as feed, environment, activity formation scatol.This just forms the foster livestock and poultry of soil and the livestock and poultry of intensive culture
There is difference on edible flavor.The foster activity of soil is big, and breeding cycle is long, the close chewy of muscle.The meat mouthful of intensive culture
Feel soft tasteless.(2), influence of the scatol to meat.As long as the livestock and poultry eubolism in cultivation livestock and poultry, just there is scatol generation,
Severe contamination air and surrounding enviroment, also have a strong impact on livestock birds health when it is excreted with metabolin.In livestock and poultry internal deposition
Different smell is produced in muscle, internal another substances-androstenone is the same with scatol to be deposited in fat.Scatol and androstene
Ketone additionally depends on cycle and body weight to the influence degree of meat different smell, and animal reaches that deposition increases before sexal maturity, sexal maturity
After deposit it is more.Scatol results from the colon of animal, and the content from large intestine to straight field scatol is continuously increased.Except entering liver
Part afterwards, which is metabolized, drains, and remaining retains in liver, kidney, blood, muscle and fat.This is to send raw meat in animal meat quality
The main cause of shy taste.(3), scatol can cause the damage of lung tissue in blood, and the meeting excreted endangers exhaling for livestock and poultry
Function and physiological function are inhaled, emergency reaction can be caused during low concentration, livestock and poultry production performance is reduced, is having the environment of scatol when long
In can change the neuroendocrine function of livestock and poultry.During high concentration, livestock and poultry acute poisoning can be caused, maldevelopment, allergy, machine occur
Can fail etc..It is the main reason that severe contamination breeding environment and livestock and poultry body surface volatilize excrement stink.(4), livestock and poultry feeding is to containing
Scatol can be changed into the exogenous diet of scatol and enter internal, and scatol inevitably will disappear in vivo with livestock and poultry
Change enzyme to have an effect, it plays inhibitory action to a- amylase, pepsin, the trypsase in livestock and poultry body.Have a strong impact on
Digestion and utilization to feed.
To solve the problem of these are present, solved using the distinctive effect of microorganism, acquired results are almost eliminated
The common problem that current livestock and poultry cultivation is present.The flora that its method selection has mould, saccharomycete and lactic acid bacteria symbiosis is cultivated,
By cultured liquid flora, 0.5-1% is added in mash feed in proportion.Also 1 can be compared by example:100 are added in drinking-water.Not
Disconnected feeding and drink enters bacterium in water.Solids Anaerobic culturel compound bacteria, the good rear and perfect compound feed of culture, by 5% can also be used
Or 10% different livestock and poultry of feeding.Flora enters livestock and poultry digestive system, the function produced by it in constantly searching for food and drinking water
Show several aspects.
1. gastrointestinal tract of livestock and fowls pH value is adjusted, composite flora is when nutrient solution pH value is down to 3.5, and flora enters dormancy shape
State, when the livestock and poultry stomach and intestinal environment that enter pH value and proper temperature, it can comparatively fast bring back to life and carry out metabolism lactic acid, propionic acid, second
Acid, butyric acid etc., can help livestock and poultry to adjust intestines and stomach pH value in its metabolic activity, when gastrointestinal tract of livestock and fowls is down to PH4-5,
The intestines and stomach energy activator protein matter of this level digestive ferment relevant with starch.Relieve in livestock and poultry body to alpha-amylase, stomach cardia
The suppression of enzyme, trypsase.Empirical tests, as gastrointestinal tract of livestock and fowls PH>Pepsin is inactivated when 6, is at this moment conducive to harmful large intestine
The survival and metabolism of bacillus.PH≤5 is conducive to beneficial lactic acid bacteria and streptococcus, Bifidobacterium survival and is metabolized when acid.Give
Livestock and poultry supplement compound lactobacillus group is assisted for the purpose of livestock and poultry reduction intestines and stomach pH value, and energy is digested and assimilated to feed to improving livestock and poultry
Power and reduction aquaculture cost are significant.
2. empirical tests, as gastrointestinal tract of livestock and fowls PH ﹦ 8, about 78% tryptophan transfer turns to scatol, and 22% tryptophan transfer is Yin
Diindyl, the tryptophan transfer that 42% is there are about during PH ﹦ 6.5 turns to scatol, and 58% tryptophan transfer is indoles, there was only 16% tryptophan during PH ﹦ 5
It is converted into scatol and 84% tryptophan transfer turns to indoles.The generation of scatol is diet and endogenous protein in livestock and poultry caecum
Lower generation is acted on large intestine anaerobe, the corruption production such as Escherichia coli, clostridium, proteus is especially harmful to
The L-Trp formation indole-3-acetic acid decarboxylation that can degrade of ammonia bacterium forms scatol.Gastrointestinal tract of livestock and fowls pH value is adjusted with composite flora,
Effectively accumulation of the control scatol in livestock and poultry liver, kidney, blood, muscle and fat can be played.It is that tryptophan is more converted
For indoles.Indoles is a composition in spices, and livestock and poultry Meat Flavor, which is improved, in micro concentration larger help.Indoles
Heteroauxin is combined into as a kind of growth promoter with acetoxylation, and its growth to livestock and poultry is that have promotion and help.
3. composite flora can produce many benefit materials, produce in addition to the functions such as stomach invigorating, adjustment enteron aisle in its metabolic process
Raw lactein function is special.The antibacterial functions of lactein mainly use the different loci electric charge on its protein structure
Several differences, can press down and kill harmful Escherichia coli, salmonella etc. and cause object bacteria membrane structure part to change, with similar clear
Clean dose of function.In general, its bactericidal mechanism is first to adsorb on the cell membrane of object bacteria, invade again in film and form through-hole
Road, causes harmful bacteria dead to cause material change in film.The similar antibiotic of effect of lactein, anti-medicine is not produced completely
Property and toxicity, and be difficult to be destroyed by small intestine trypsase, it can effectively suppress the growth of pathogen.It can be risen with substitute antibiotics
It is the more green no drug residue of meat of livestock and poultry to the effect of defending and fighting against diseases.
By to adding composite bacteria agent in animal and fowl fodder and drinking-water system continuously supplements composite bacteria agent,（It is produced
2-hydracrylic acid, it is a kind of carboxylic acid）, the pH value of gastrointestinal tract of livestock and fowls is maintained in compared with low state, it is suppressed that scatol
Excess generation, is that the quality and local flavor of livestock meat are changed and lifted.Become with the quality that the pig slaughtering formed experiences meat
Change.The pig delivered for sale, which plucks, to be butchered, and be not pungent shy taste when entering having one's hair waved hair, is overflowed after cutting open the chest without fishy smell, flatulence, shy taste.Liver is fresh
Red bright, lung toner is red clean, and intestinal wall is relatively thin.1., meat is scarlet, and pH value 5.7-6.2 is identical with control group.2., meat pressurizes
Moisture holding capacity crop is higher than control group by 7.5%, stage casing is higher by 7.8%, back leg and is higher by 3.1%.3., fatty fusing point is higher by control group>
1.5 DEG C, fat oil is melted out less, and fat meat fries out mouthfeel embrittlement non-greasy.4., aliphatic acid C20 in meat:4 (n -6) arachidonic acids are higher than
Control group, this composition is the precursor substance of carnivorous fragrance.Aliphatic acid C22:6 (n -3) DHA are higher than control group, are commonly called as that " brain is white
Gold ".5., hepatocuprein in meat（SOD）﹥ 0.1% is higher than control group.This can slow down lipid oxidation, play protection meat
Effect.6., water boils fresh meat clear soup without fishy smell, cooks meat and is not added with seasoning.Directly eat the fresh fragrant, chewiness of meat.There is the edible soil of recurrence
The taste of stock keeping poultry, but 7., after fresh meat freezing thawed again yellowish pink constant, meat beyond the raise pigs fresh fragrant and chewiness of meat of soil
Constant, mouthfeel is constant.
By meat obtained by this method cultivation broiler chicken, meat duck without urine smell taste, chewiness, with original naturally fresh perfume (or spice).This method
Solve can not change the quality and local flavor of animal meat quality again to sacrifice large-scale cultivation, that is, eliminates suppression livestock and poultry and digest and assimilate
Injurious factor, improve prouctiveness, improve breeding environment.Demand of the people to high-quality livestock meat is solved, is stabilized
Valency is high and away from the market imbalance between supply and demand.
Embodiment
The present invention is further described below with reference to embodiment.
Embodiment 1
The present invention cultivation microorganism formulation, it be by weight ratio be 1:10 composite fluid strain and nutrient solution, which spreads cultivation, to be formed；
The nutrient solution is prepared from by the raw material of following parts by weight：76 parts of glucose, 47 parts of brown sugar, 0.4 part of potassium dihydrogen phosphate,
0.9 part of urea, 4 parts of multi-hydroxy carboxy acid's titanium, 995 parts of boiling water；The composite fluid strain be by weight ratio be 1:10 compound bacteria
Plant liquid and bacteria culture fluid is prepared from, the bacteria culture fluid is prepared from by the raw material of following parts by weight：Glucose
77 parts, 46 parts of brown sugar, 3 parts of peptone, 2 parts of yeast extract, 0.4 part of magnesium sulfate, 0.73 part of potassium dihydrogen phosphate, 0.9 part of urea, chlorination
1 part of sodium, 0.023 part of ferrous sulfate, 0.009 part of manganese sulfate, 1 milliliter of multi-hydroxy carboxy acid's titanium, 998 parts of distilled water；The compound bacteria
The following strain mixed culture of liquid is planted to form：Rhizopus oryzae, saccharomycete, Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc mesenteroides, small pieces
Coccus, streptococcus fecalis, bacillus licheniformis, bacillus pumilus.
The preparation method of the upper cultivation microorganism formulation comprises the following steps：
（One）Rhizopus oryzae and saccharomycete spread cultivation
Rhizopus oryzae and saccharomycete are spread cultivation with Czapek's medium respectively, the liquid that spread cultivation with increment of the Rhizopus oryzae and saccharomycete after spreading cultivation is put
Enter insulating box progress increment to spread cultivation, culture to bacterium solution muddiness has obvious thalline, bacterium solution pH value is down to 3.5-4 and terminates standby, border inspection
Bacterium number；
（Two）Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc mesenteroides, Pediococcus parvulus, streptococcus fecalis spread cultivation
A, will spread cultivation Nutrient medium：5 grams of peptone, 20 grams of tryptone, 5 grams of yeast extract, 200 milliliters of Tomato juice, liver extract, glucose
3 grams, 3 grams of lactose, 0.05 gram of Tween-80,1 liter of distilled water, the dissolving sterilizing of 20 grams of heating stirrings of agar, it is sterile under pour and sterilized
Culture dish, more than sterile access each each 4 ware of test tube slant bacterium after solidification, 30 DEG C of insulating box culture 36-72h to bacterium colony are covered with；
B, by the above culture medium 1000ml is prepared, do not put agar, sterilize cool to 25 DEG C, assign to 5 sterilized 500ml triangular flasks
In, every bottle of 200ml is best by the extraction growth of cultured 5 kinds of bacterium, is respectively washed bacterium colony with the sterile washings of 30ml and sterilizing brush
Culture dish in corresponding nutrient solution is taken off, 30 DEG C of insulating box culture 72h are put to bacterium solution muddiness, when pH value is to 3.5-4, microscopy bacterium number
>=10 hundred million, terminate standby.
1000ml is prepared by former culture medium, divides to each 500ml sterilizings of 2 1000ml triangular flasks, is accessed in gnotobasis
5 kinds of bacterium are cultivated, take 10ml to be added to same triangular flask per bacterium, the triangular flask for having added bacterium is sealed with aseptic paper multilayer, put
30 DEG C of insulating box culture 72h, diseases caused by external factors bacterium solution is muddy, surveys pH value 3.5-4,4 DEG C of safes are put standby in border inspection bacterium number >=10 hundred million.
（Three）Bacillus licheniformis, bacillus pumilus spread cultivation
A, spread cultivation Nutrient medium：5 grams of peptone, beef leaching cream, 5 grams of sodium chloride, 5 milligrams of manganese sulfate, 15 grams of agar, distilled water 1
Rise.PH value 7.Agar is dissolved by heating, sterilizing is poured to sterilized culture dish in gnotobasis while hot, and test tube bacterium is accessed after solidification,
Put 35 DEG C of of insulating box cultivate to be covered with culture dish terminate；
B, the Nutrient medium that will spread cultivation remove agar, and other nutrients are dissolved in distilled water according to quantity, prepare 1000ml, assign to 4
Each 300ml sterilizings in 1000ml triangular flasks, sterilize rearmounted cool to 25 DEG C of gnotobasis, will cultivate culture dish sterilized water 30ml
De- culture dish bacterium colony is scrubbed to corresponding triangular flask with sterile, 35 DEG C of incubators are put to bacterium solution muddiness, and PH is down to 4, border inspection bacterium number
>=10 hundred million standby.
The nutrient solution used by triangular flask prepares 1000ml points to each 500ml sterilizings of 2 1000ml triangular flasks, in asepsis ring
Cool to 25 DEG C of border, two kinds of bacterium respectively take 25 milliliters to be added to same triangular flask, prepare two bottles and put 35 DEG C of insulating box culture 72h, bacterium solution
Muddiness, pH value 3.5-4,4 DEG C of refrigerating boxes are put standby in microscopy bacterium number >=1,000,000,000.
（Four）The culture of composite fluid strain
A, with multi-hydroxy carboxy acid's titanium and distillation aquathermolysis glucose, brown sugar, peptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate,
Urea, sodium chloride, ferrous sulfate, manganese sulfate, adjustment solution pH value are 7；
B, by the solvent nutrient solution average mark of upper step after sterilization treatment in three reservoirs, in an aseptic environment will
Step 1: two, three cultured liquid bacteria mixing shake up, by 1:10 ratio is separately added into three reservoirs, multilayer without
Bacterium paper is sealed, and jog is even, puts constant incubator culture, is cultivated 3 days, and bacterium solution is muddy, and pH value is down to 3.5-4, and bacterium number is examined up to ten in border
More than hundred million, which meet compounding, uses bacterium；
C, the compound method preparation sterilizing by above liquid medium, load triangular flask, three of the above bacterium are pressed 1:10 ratio
Respectively with adding in triangular flask, incubator culture is put, three days pH values of culture are 3.5-4, and border examines total bacteria count and is up to more than 1,000,000,000
Composite fluid strain；
（Five）Production is spread cultivation with bacterium：
By except the clout mixed dissolution of the nutrient solution of multi-hydroxy carboxy acid's titanium, water temperature adds multi-hydroxy carboxy acid's titanium when being down to 25 DEG C；Will
Composite fluid strain and nutrient solution are 1 by volume:10 ratio mixing, is cultivated three days in the environment of putting 25 DEG C, cultured
1 year vigor of strain preparation sealing preserve keeps 90%, as spreads cultivation and uses bacterium original seed.
The increment liquid that spreads cultivation is formulated by following raw material：5 grams of peptone, 2 grams of yeast extract, 10 grams of glucose, phosphorus
1 gram of acid dihydride potassium, 1 gram of magnesium sulfate, 1 milliliter of titanium hydroxycarboxylate, 1000 milliliters of pure water.
Embodiment 2
A kind of cultivation microorganism formulation of the present invention, it by weight ratio is 1 that it, which is,:10 composite fluid strain and nutrient solution spread cultivation and
Into；The nutrient solution is prepared from by the raw material of following parts by weight：80 parts of glucose, 50 parts of brown sugar, potassium dihydrogen phosphate 0.5
Part, 1 part of urea, 5 parts of multi-hydroxy carboxy acid's titanium, 1000 parts of boiling water；The composite fluid strain be by weight ratio be 1:10 it is compound
Strain liquid and bacteria culture fluid are prepared from, and the bacteria culture fluid is prepared from by the raw material of following parts by weight：Grape
80 parts of sugar, 50 parts of brown sugar, 4 parts of peptone, 2.5 parts of yeast extract, 0.5 part of magnesium sulfate, 0.75 part of potassium dihydrogen phosphate, 1 part of urea, chlorine
Change 2 parts of sodium, 0.025 part of ferrous sulfate, 0.01 part of manganese sulfate, 1 milliliter of multi-hydroxy carboxy acid's titanium, 1000 parts of distilled water；It is described compound
The following strain mixed culture of strain liquid is formed：It is Rhizopus oryzae, saccharomycete, Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc mesenteroides, small
Piece coccus, streptococcus fecalis, bacillus licheniformis, bacillus pumilus；Its preparation method be the same as Example 1.
Embodiment 3
A kind of cultivation microorganism formulation of the present invention, it by weight ratio is 1 that it, which is,:10 composite fluid strain and nutrient solution spread cultivation and
Into；The nutrient solution is prepared from by the raw material of following parts by weight：85 parts of glucose, 55 parts of brown sugar, potassium dihydrogen phosphate 0.6
Part, 1.2 parts of urea, 6 parts of multi-hydroxy carboxy acid's titanium, 1100 parts of boiling water；The composite fluid strain be by weight ratio be 1:10 answer
Close strain liquid and bacteria culture fluid is prepared from, the bacteria culture fluid is prepared from by the raw material of following parts by weight：Portugal
75-85 parts of grape sugar, 55 parts of brown sugar, 5 parts of peptone, 3 parts of yeast extract, 0.7 part of magnesium sulfate, 0.8 part of potassium dihydrogen phosphate, urea 1.2
Part, 2 parts of sodium chloride, 0.03 part of ferrous sulfate, 0.012 part of manganese sulfate, 1 milliliter of multi-hydroxy carboxy acid's titanium, 1100 parts of distilled water；It is described
The following strain mixed culture of composite bacteria liquid is formed：The bright beading of Rhizopus oryzae, saccharomycete, Lactobacillus plantarum, Lactobacillus brevis, goldbeater's skin
Bacterium, Pediococcus parvulus, streptococcus fecalis, bacillus licheniformis, bacillus pumilus, its preparation method be the same as Example 1.
The selection of strain and source in above example：Chinese Academy of Sciences's Culture Collection（Numbering bi0), north receive biological bacteria
Plant collection（BNC、BNCC)：Rhizopus oryzae：BNCC-339462, saccharomycete：Bi0-64682, Lactobacillus plantarum:
BNC339515, Lactobacillus brevis：BNCC337373, Leuconostoc mesenteroides：Bi0-67753, Pediococcus parvulus：Bi0-67910, excrement chain
Coccus：Bi0-18763, bacillus licheniformis bi0-60464, bacillus pumilus:bi0-59404.
The present invention application method be：1st, powder-material：Material per ton adds 5 kilograms of preparations to stir；2nd, powdery wet-mixing material:Often
Ton material mixing thoroughly with suitable quantity of water with 5 kilograms of preparations；3rd, fed granules material:Add 1-2 kilograms of preparation in drinking-water per ton.4th, sent out with solid
Yeast-like fungi agent substitutes complete diet pellet by 5%-10%.
Claims (3)
1. one kind cultivation microorganism formulation, it is characterised in that：It is by weight ratio be 1:10 composite fluid strain and nutrient solution
It is formulated；The nutrient solution is prepared from by the raw material of following parts by weight：75-85 parts of glucose, 45-55 parts of brown sugar,
0.4-0.6 parts of potassium dihydrogen phosphate, 0.8-1.2 parts of urea, 4-6 parts of multi-hydroxy carboxy acid's titanium, 990-1100 parts of boiling water；The complex liquid
Body strain be by weight ratio be 1:10 composite bacteria liquid and bacteria culture fluid is prepared from, and the bacteria culture fluid is by following
The raw material of parts by weight is prepared from：75-85 parts of glucose, 45-55 parts of brown sugar, 3-5 parts of peptone, 2-3 parts of yeast extract, sulfuric acid
0.3-0.7 parts of magnesium, 0.7-0.8 parts of potassium dihydrogen phosphate, 0.8-1.2 parts of urea, 1-2 parts of sodium chloride, ferrous sulfate 0.02-0.03
Part, 0.008-0.012 parts of manganese sulfate, 1 milliliter of multi-hydroxy carboxy acid's titanium, 990-1100 parts of distilled water；Below the composite bacteria liquid
Strain mixed culture is formed：Rhizopus oryzae, saccharomycete, Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc mesenteroides, Pediococcus parvulus, excrement chain
Coccus, bacillus licheniformis, bacillus pumilus.
2. a kind of method for preparing cultivation microorganism formulation as claimed in claim 1, it is characterised in that：Methods described includes following
Step：
（One）Rhizopus oryzae and saccharomycete spread cultivation
Rhizopus oryzae and saccharomycete are spread cultivation with Czapek's medium respectively, the liquid that spread cultivation with increment of the Rhizopus oryzae and saccharomycete after spreading cultivation is put
Enter insulating box progress increment to spread cultivation, culture to bacterium solution muddiness has obvious thalline, bacterium solution pH value is down to 3.5-4 and terminates standby, border inspection
Bacterium number；
（Two）Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc mesenteroides, Pediococcus parvulus, streptococcus fecalis spread cultivation
A, will spread cultivation Nutrient medium：5 grams of peptone, 20 grams of tryptone, 5 grams of yeast extract, 200 milliliters of Tomato juice, liver extract, glucose
3 grams, 3 grams of lactose, 0.05 gram of Tween-80,1 liter of distilled water, the dissolving sterilizing of 20 grams of heating stirrings of agar, it is sterile under pour and sterilized
Culture dish, more than sterile access each each 4 ware of test tube slant bacterium after solidification, 30 DEG C of insulating box culture to bacterium colonies is covered with；
B, by above culture medium 1 liter is prepared, do not put agar, sterilize cool to 25 DEG C, will be cultivated in gnotobasis with sterile purified water
5 kinds of good bacterium culture dishes bacterium colony under sterilizing brush and sterile washing, into nutrient solution, puts 30 DEG C of insulating box cultures muddy to bacterium solution
It is turbid, standby, microscopy bacterium number is terminated when pH value is to 3.5-4;
（Three）Bacillus licheniformis, bacillus pumilus spread cultivation
A, spread cultivation Nutrient medium：5 grams of peptone, beef leaching cream, 5 grams of sodium chloride, 5 milligrams of manganese sulfate, 15 grams of agar, distilled water 1
Rise, pH value 7, dissolve by heating agar, sterilizing is poured to sterilized culture dish in gnotobasis while hot, and test tube bacterium is accessed after solidification,
Put 35 DEG C of of insulating box cultivate to be covered with culture dish terminate；
B, the Nutrient medium that will spread cultivation remove agar, and other nutrients are dissolved in distilled water, put in triangular flask and sterilize, and sterilize rearmounted nothing
Cool to 25 DEG C of collarium border, takes off culture dish bacterium colony into the liquid that spreads cultivation with sterilized water and sterile scrub, puts 35 DEG C of incubators muddy to bacterium solution
Turbid, PH is down to 4, border inspection bacterium number；
（Four）The culture of composite fluid strain
A, with multi-hydroxy carboxy acid's titanium and distillation aquathermolysis glucose, brown sugar, peptone, yeast extract, magnesium sulfate, potassium dihydrogen phosphate,
Urea, sodium chloride, ferrous sulfate, manganese sulfate, adjustment solution pH value are 7；
B, by the solvent nutrient solution average mark of upper step mounted in sterilization treatment in three reservoirs, after in an aseptic environment will
Step 1: two, three cultured liquid bacteria mixing shake up, by 1:10 ratio is separately added into three reservoirs, multilayer without
Bacterium paper is sealed, and jog is even, puts constant incubator culture, is cultivated 3 days, and bacterium solution is muddy, and pH value is down to less than 4, and bacterium number is examined up to ten in border
More than hundred million, which meet compounding, uses bacterium；
C, the compound method preparation sterilizing by above liquid medium, load triangular flask, three of the above bacterium are pressed 1:10 ratio
Respectively with adding in triangular flask, incubator culture is put, three days pH values of culture are up to less than 4, and border examines total bacteria count and is up to more than 1,000,000,000
Composite fluid strain；
（Five）Production is spread cultivation with bacterium：
By except the clout mixed dissolution of the nutrient solution of multi-hydroxy carboxy acid's titanium, water temperature adds multi-hydroxy carboxy acid's titanium when being down to 25 DEG C；Will
Composite fluid strain and nutrient solution are 1 by volume:10 ratio mixing, is cultivated three days in the environment of putting 25 DEG C, cultured
1 year vigor of strain preparation sealing preserve keeps 80%,.
3. method according to claim 2, it is characterised in that：The increment liquid that spreads cultivation is formulated by following raw material：
5 grams of peptone, 2 grams of yeast extract, 10 grams of glucose, 1 gram of potassium dihydrogen phosphate, 1 gram of magnesium sulfate, 1 milliliter of titanium hydroxycarboxylate, pure water
1000 milliliters.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109652331A (en) * | 2019-01-03 | 2019-04-19 | 沧州临港海益水产养殖有限公司 | For regulating and controlling the probiotics of pH value and the method for administration in sea-farming |
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| CN103773721A (en) * | 2013-12-31 | 2014-05-07 | 陕西万源生物农业科技有限公司 | Composite probiotics for raising pigs |
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| CN109652331A (en) * | 2019-01-03 | 2019-04-19 | 沧州临港海益水产养殖有限公司 | For regulating and controlling the probiotics of pH value and the method for administration in sea-farming |
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