CN108179130A - A kind of preparation method of high activity Enterococcus faecalis microorganisms preparation dry powder - Google Patents
A kind of preparation method of high activity Enterococcus faecalis microorganisms preparation dry powder Download PDFInfo
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Abstract
The invention discloses a kind of preparation methods of high activity Enterococcus faecalis microorganisms preparation dry powder; this method optimizes fermentation medium; and by protectant a large amount of screenings; level-one, second order fermentation are carried out under suitable conditions; it is inoculated into wheat bran solid medium with secondary seed solution again; it is controlled to carry out highdensity fermentation under suitable conditions; segmented drying is carried out after fermentation; high activity Enterococcus faecalis microorganisms preparation dry powder is finally prepared, every gram of microorganism formulation dry powder contains 130~18,000,000,000 viable bacterias.Present invention advantageously solves the problem of production equipment input in existing Freeze Drying Technique is big, high energy consumption, high production cost；Solves the problem of pure liquid state fermentation mode viable count is low, and transportation cost is high, and addition is inconvenient, and the shelf-life is short simultaneously.
Description
Technical field
The present invention relates to technical field of microbial fermentation, and in particular to a kind of high activity Enterococcus faecalis microorganisms preparation dry powder
Preparation method.
Background technology
Enterococcus faecalis (Enterococcus faecalis) is the permission of the Ministry of Agriculture of China as additive for microbe feedstuff
One of strain, Application of Direct-fed Microbials is developed by scientific research institution more and more.Enterococcus faecalis can generate natural antibiotics,
Be conducive to body health；The antibacterial substances such as bacteriocin are generated simultaneously, inhibit the growth of the pathogens such as Escherichia coli and salmonella,
Improve intestinal microenvironment；It can also inhibit the breeding of urease-producing bacterium and spoilage organisms in enteron aisle, reduce enteron aisle urease and endogenous toxic material
The content of element declines ammonia and endotoxic content in blood, at the same can be formed in animal intestinal tract biofilm be attached to it is dynamic
On object intestinal mucosa, and develop, grow and breed.Enterococcus faecalis can soften the fiber in feed, improve turning for feed
Rate！Therefore it is a green, safe and healthy selection high activity enterococcus faecalis to be added in feed.
Current enterococcus faecalis technology of preparing, mostly using Conventional cryogenic Freeze Drying Technique, the technique productions equipment investment is big,
High energy consumption during liquid state fermentation is concentrated freeze-dried, production cost is high, is unfavorable for the product large-scale use；Or pure liquid hair
Ferment, market can only use aqua, but aqua viable count is low, and transportation cost is high, and addition is inconvenient, while the shelf-life is short.
Invention content
The invention discloses a kind of preparation method of high activity Enterococcus faecalis microorganisms preparation dry powder, to solve existing skill
The problem of equipment investment present in art is big, production cost is high, number of viable is low, addition is inconvenient, the shelf-life is short.
To solve the above-mentioned problems, the present invention adopts the following technical scheme that：
A kind of preparation method of high activity Enterococcus faecalis microorganisms preparation dry powder, includes the following steps：
(1) enterococcus faecalis of activation (Enterococcus faecalis) slant strains are transferred in sterile water, form kind
Sub- liquid, a concentration of the 5.0 × 10 of the seed liquor8Cfu/ml~1.0 × 109cfu/ml；
(2) seed liquor that step (1) obtains is transferred to 4%~6% inoculum concentration containing one grade fermemtation culture medium
In fermentation tank, 37 ± 1 DEG C, stir speed (S.S.) 0rpm, ventilatory capacity 0V/Vmin of fermentation temperature, tank presses 0.02~0.03Mpa, and dissolved oxygen is dense
Degree 5~10% when cultivating by 12~16 hours, adds protective agent 1, and fermentation time is 16~20 hours, make pH value be down to 3.6~
4.2 are made primary seed solution, and a concentration of the 1.3 × 10 of the primary seed solution9Cfu/ml~1.6 × 109cfu/ml；
(3) primary seed solution that step (2) obtains is transferred to 6~10% inoculum concentration containing second order fermentation culture medium
Fermentation tank in carry out second order fermentation, 37 ± 1 DEG C, stir speed (S.S.) 0rpm, ventilatory capacity 0V/Vmin of fermentation temperature, tank pressure 0.04
~0.05Mpa, oxyty 5~10%, when cultivating by 14~18 hours, addition protective agent 2, fermentation time 18~24 hours,
PH is made to be down to 3.6~4.2 values, is made secondary seed solution, a concentration of the 1.8 × 10 of the secondary seed solution9Cfu/ml~2.4 ×
109cfu/ml；
(4) it in gnotobasis, after the secondary seed solution addition protective agent 3 that step (3) is obtained, is connect with 8~10%
Kind amount access sterilizing after wheat bran solid medium in, be uniformly mixed, pack into culture room culture；During cultivating room culture, control
Ventilation quantity processed is：3~10 rates of ventilation/day, 34~38 DEG C of temperature, humidity 80%~90% are cultivated 24~28 hours, culture period
Between stir wheat bran solid medium 1~2 time, the Enterococcus faecalis microorganisms preparation of moisture content, the moisture content is thus prepared
Enterococcus faecalis microorganisms preparation water content for 35~40%, living bacteria count is 1.5 × 1010Cfu/g~2.0 ×
1010cfu/g；
(5) the Enterococcus faecalis microorganisms preparation immigration hot air circulation drying oven for the moisture content for obtaining step (4), first 46
It is handled under the conditions of~48 DEG C 1.5~2.0 hours, then in 40~43 DEG C of drying of temperature, it is micro- that high activity enterococcus faecalis is prepared
Biological agent dry powder, the water content of the high activity Enterococcus faecalis microorganisms preparation dry powder is 8~10%, and living bacteria count is
1.3×1010Cfu/g~1.8 × 1010cfu/g。
Further, the raw material of the one grade fermemtation culture medium described in step (2) and preparation method are：Every liter containing western red
160~175mL of persimmon juice, 5.0~8.0g of tryptone, 5.3~6.5g of beef extract, 3.3~5.0g of yeast extract, diammonium hydrogen citrate
1.4~2.5g, 16.8~19.5g of sucrose, 0.7~1.0mL of Tween 80,4.3~5.5g of sodium acetate trihydrate, three water phosphoric acid hydrogen two
1.3~2.5g of potassium, 0.28~0.48g of epsom salt, 0.16~0.24g of manganese sulfate monohydrate；Above-mentioned each raw material is uniformly mixed,
PH is adjusted as 6.2~6.8 to get one grade fermemtation culture medium.
Further, the raw material of the second order fermentation culture medium described in step (3) and preparation method are：Every liter containing western red
120~140mL of persimmon juice, 4.0~6.0g of casein peptone, 5.3~6.5g of beef extract powder, 3.3~5.0g of dusty yeast, cornstarch 35
~45g, 1.4~2.5g of anhydrous sodium acetate, 3.3~5.0g of sodium citrate, Dextran 60~65g, 8.5~10.0g of lactose, sulfuric acid
0.16~0.24g of manganese, 4.3~5.5g of sodium citrate, 0.28~0.48g of anhydrous magnesium sulfate；Above-mentioned each raw material is uniformly mixed, is adjusted
PH is saved as 6.5~7.0 to get second order fermentation culture medium.
Further, the wheat bran solid culture based raw material described in step (4) and preparation method are：As mass fraction by
Following components forms：Wheat bran 43%~55%, ammonium sulfate 0.1%~0.5%, epsom salt 0.58~0.62%, a water sulphur
Sour manganese 0.32~0.38%, surplus are water；Above-mentioned each raw material is uniformly mixed, under the conditions of 115~121 DEG C, sterilizing 40~45
Minute is to get wheat bran solid medium.
Further, the protective agent 1 is trehalose, and additive amount is 7~10%.
Further, the protective agent 2 is soluble starch, and additive amount is 5.5~7.5%.
Further, the protective agent 3 is skimmed milk power, and additive amount is 12~15%.
Compared with prior art, beneficial effects of the present invention are：The high activity Enterococcus faecalis microorganisms preparation of the present invention is done
The preparation method of powder adds Tomato juice, sucrose in one grade fermemtation culture medium, and when bacterium solution culture was by 12~16 hours, adds
Add 1 trehalose of protective agent early period；Tomato juice, cornstarch, glucan are added in second order fermentation culture medium, and cultivate to 14~
At 18 hours, 2 soluble starch of addition protective agent early period；The secondary seed solution that step (3) is obtained adds 3 defatted milk of protective agent
Powder.Using three grade fermemtation mode, fermentation medium is optimized, and a large amount of screening early period has been done to protective agent, it is particularly western red
Collocation between persimmon juice and sucrose and protective agent 1, the collocation between Tomato juice, cornstarch, glucan and protective agent 2, with
And the collocation between protective agent 1, protective agent 2 and protective agent 3, and level-one, second order fermentation are carried out under suitable conditions, then with two
Grade seed liquor is inoculated into wheat bran solid medium, it is controlled to carry out highdensity fermentation under suitable conditions, is used after fermentation
Segmented drying mode, advantageously solves in existing freeze-drying that big production equipment input, high energy consumption, production cost is high asks
Topic；Solves the problem of pure liquid state fermentation mode viable count is low, and transportation cost is high, and addition is inconvenient, and the shelf-life is short simultaneously.Through inspection
It surveys, the high activity Enterococcus faecalis microorganisms preparation dry powder being prepared according to the method for the present invention, every gram of microorganism formulation dry powder contains
130~18,000,000,000 viable bacterias.
Description of the drawings
Fig. 1 is enterococcus faecalis colony determination programme diagram.
Specific embodiment
The present invention is further illustrated, but the present invention is not limited to these embodiments with reference to specific embodiment.
Embodiment 1
In superclean bench, pipe strain is lyophilized according to sterile behaviour in enterococcus faecalis (Enterococcus faecalis)
Opening is required, enough sterile waters is added in into freeze-drying pipe, with aseptic inoculation ring by after its mixing, chooses a ring bacterial suspension inoculation
In MRS agar test tubes medium slants, biochemical culture is put it into, 37 ± 1 DEG C are cultivated 20 hours, then by test tube slant seed
Be transferred in MRS agar eggplant-shape bottle slant mediums, 37 DEG C cultivate 18 hours, then with suitable sterile water by eggplant-shape bottle into
Ripe bacterium mud scrapes, loading 250ml triangular flasks, shaken well, formation seed liquor, and a concentration of 1.0 × 109cfu/ml.On
The raw material and preparation method for stating MRS agar mediums be：Every liter containing peptone 10.0g, beef extract 10.0g, yeast extract 5.0g,
Diammonium hydrogen citrate [(NH4)2HC6H5O7] 2.0g, glucose (C6H12O6·H2O) 20.0g, Tween 80 1.0mL, sodium acetate
(CH3COONa·3H2O) 5.0g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 2.0g, magnesium sulfate (MgSO4·7H2O) 0.58g, sulfuric acid
Manganese (MnSO4·H2O) 0.25g, agar 20.0g, surplus are distilled water；Above-mentioned each raw material is uniformly mixed, it is 6.6 to adjust PH,
Sterilize 20min at a temperature of 121 DEG C.
Seed liquor is transferred to 6% inoculum concentration in the 5L fermentation tanks containing one grade fermemtation culture medium, packing factor
70%, 37 ± 1 DEG C, stir speed (S.S.) 0rpm, ventilatory capacity 0V/Vmin of fermentation temperature, tank pressure maintains 0.02~0.03Mpa, dissolved oxygen
Concentration maintains 6~8%, when cultivating by 12 hours, when having arrived at exponential phase through growth curve detection thalline, passes through hair
Fermentation tank material-feeding port according to the requirement of fermentation tank feed operation, adds 1 trehalose of protective agent, additive amount 10%, continues fermentation to 20 small
When, pH value is down to 4.2 and is made primary seed solution, and a concentration of 1.6 × 109cfu/ml；The raw material of above-mentioned one grade fermemtation culture medium and
Preparation method is：Every liter contains Tomato juice 160mL, tryptone 8.0g, beef extract 6.5g, yeast extract 5.0g, hydrogen citrate
Diammonium 2.5g, sucrose 19.5g, Tween 80 1.0mL, sodium acetate trihydrate 5.5g, three water dipotassium hydrogen phosphate 2.5g, epsom salt
0.48g, manganese sulfate monohydrate 0.24g；Above-mentioned each raw material is uniformly mixed, adjusts the 20min that sterilizes at a temperature of PH is 6.8,121 DEG C.
Primary seed solution is transferred to 10% inoculum concentration in the 50L fermentation tanks containing second order fermentation culture medium and carries out two
Grade fermentation, 37 ± 1 DEG C, stir speed (S.S.) 0rpm, ventilatory capacity 0V/Vmin of fermentation temperature, tank pressure maintains 0.04~0.05Mpa,
Oxyty maintains 8~10%, when cultivating by 14 hours, when having arrived at exponential phase through growth curve detection thalline,
It by fermentation tank material-feeding port, is required according to fermentation tank feed operation, addition 2 soluble starch of protective agent, additive amount 7.5%, after
Supervention ferment was to 24 hours, when PH is down to 4.1, was made secondary seed solution, and a concentration of 2.4 × 109cfu/ml；Above-mentioned second order fermentation training
Support base raw material and preparation method be：Every liter contains Tomato juice 140mL, casein peptone 6.0g, beef extract powder 6.5g, dusty yeast
5.0g, cornstarch 45g, anhydrous sodium acetate 2.5g, sodium citrate 5.0g, glucan 65g, lactose 10.0g, manganese sulfate 0.24g,
Sodium citrate 5.5g, anhydrous magnesium sulfate 0.48g；Above-mentioned each raw material is uniformly mixed, adjusts at a temperature of PH is 6.5,121 DEG C and sterilizes
20min。
Prepare before inoculation：It is first carried out disinfection with peracetic acid disinfectant docking inter-species, culture room, production apparatus etc., then
The ultraviolet lamp disinfection 30min in culture room is opened again, prepares sterile culture environment, area 58m between inoculation2, cultivate room area 32m2,
In gnotobasis, 3 skimmed milk power of protective agent, additive amount 12%, after mixing, with 8% are added into secondary seed solution
It in wheat bran solid medium after inoculum concentration access sterilizing, is uniformly mixed, fills ventilative mesh bag into culture room culture.Cultivate room culture
Period, control ventilation quantity are：8~10 rates of ventilation/day, 37 ± 1 DEG C of temperature, room humidity 85%~90% are cultivated 26 hours,
Wheat bran solid medium is stirred during culture 2 times, the Enterococcus faecalis microorganisms preparation of moisture content 40% is thus prepared, effectively
Viable count is 1.7 × 1010cfu/g.The raw material and preparation method of above-mentioned wheat bran solid medium be：By total mass fraction 100%
Meter, is made of wheat bran 55%, ammonium sulfate 0.5%, epsom salt 0.62%, manganese sulfate monohydrate 0.38%, water 43.5%；It will be upper
State each raw material to be uniformly mixed, sterilize 45 minutes under the conditions of 115~121 DEG C, into sterile Air cooler be cooled to 40 DEG C it is spare.
The assay method of enterococcus faecalis bacterium number
1st, instrument and equipment：Constant incubator：36±1℃；Refrigerator：0~4 DEG C；Water bath with thermostatic control：46±1℃；Electric furnace：It can
Mode；Suction pipe：Capacity is 1,10,25ml；Wide-mouth bottle or triangular flask：Capacity is 500ml；Plate：A diameter of 9cm；Test tube：18×
180mm；Microscope；High-pressure sterilizing pot；Baking oven.
2nd, culture medium and reagent：Sodium oxide molybdena, gram staining liquid, 3% hydrogenperoxide steam generator, indole reagent, nitrate
Culture medium, nitrate reagent, gelatin culture medium, Modified MC agar (Modified Chalmers culture mediums).
The raw material and preparation method of above-mentioned Modified MC agar (Modified Chalmers culture mediums) be：Tomato soaks juice
50g (500g/L), soy peptone 5g, beef extract powder 5g, yeast extract powder 5g, glucose 20g, lactose 20g, calcium carbonate 10g, fine jade
Fat 15g, dimethyl diaminophenazine chloride 0.05g；Above-mentioned each raw material is uniformly mixed, it is 6.0 ± 0.2 to adjust final PH, 115 DEG C of sterilizings of high steam
30min。
In addition to especially indicating, agents useful for same is analytical reagents in experiment, and water is distilled water or the water of respective degrees.
3rd, enterococcus faecalis colony determination program：As shown in Figure 1.
4th, operating procedure：
(1) the sterilizing wide-mouth bottle that 225ml sterile salines will be put by the sample 25g fully shaken up with sterile working
It is interior, shake well 20min, 1:10 uniform dilution；
(2) 1 is drawn with 1ml sterilized straws:10 dilution 1ml along tube wall inject and contain 9ml sterile salines slowly
(notice that pipette tip not touch in-line dilution liquid) in test tube；
(3) 1ml sterilized straws separately are taken, by aforesaid operations sequence, makees 10 times of incremental dilutions, be so often incremented by once, i.e.,
Use a 1ml sterilized straw instead；
(4) 2~3 acceptable diluent degree is selected (generally to take 10-4、10-5、10-6), with sterilizing 100ul micropipettors, respectively
0.1ml is taken to be inoculated in the improvement MC agar plates prepared, each dilution makees 3 tablets, uniform with sterile spreading rod
It is coated on whole surface；Simultaneously draw 0.1ml sterile salines in the improvement MC agar plates prepared with sterile coating
Rod coating uniformly does blank control；
(5) after agar solidification, tablet is overturn, 72 ± 2h of culture in 36 ± 1 DEG C of constant incubators is put and takes out, observe bacterium colony
Feature is chosen tablet of the clump count between 30~300 and is counted.
5th, suspicious colony identification：It selects 5 bacterium colonies at random to be identified, be seen including colonial morphology, thalline shape microscope
It examines and physiology is identified.
6th, qualification result is evaluated：
Colonial morphology：Tablet bottom is pink, and bacterium colony is smaller, round, red, neat in edge, can there is light swoon.
Thalline shape：Bacterium shape circle or oval can extend, 0.5~1.0 micron of diameter along the direction of chain, it is most of in pairs or
Short catenation, does not move usually.
Gram's staining：Gram-positive.
Biochemical identification：Negative catalase does not restore nitrate, and do not liquefy gelatin, does not generate indole and hydrogen sulfide,
Ferment sorbierite, azymic arabinose.
7th, bacterium colony counts
(1) selection of flat-plate bacterial colony number：Tablet of the clump count between 30~300 is selected to measure as total plate count to mark
Standard, a dilution should use two tablet averages, one of bacterium colony has larger sheet bacterium colony to grow using two tablets
When, then it should not use, and should be using the tablet that no sheet bacterium colony is grown as the clump count of the dilution, if sheet bacterium colony is less than flat
The half of plate, and bacterium colony distribution is very uniform in remaining half, you can multiply 2 after calculating half of tablet to represent full ware clump count.
(2) selection of dilution：Dilution of the average colony between 30~300 should be selected, is multiplied by extension rate report
It；If there are two dilution, the clump count of growth is between 30~300, then depending on how to determine between the two；If it compares
Value is less than or equal to 2, should report its average；If more than 2, then wherein smaller number is reported；If all dilutions are averaged
Clump count is all higher than 300, then should be multiplied by extension rate by the highest average colony number of dilution reports it；If all dilutions
Average colony number is respectively less than 30, then should be multiplied by extension rate by the minimum average colony number of dilution reports it.
(3) report of clump count：After bacterium colony counts, random 5 bacterium colonies of picking are identified, according to turning out to be enterococcus faecalis
Clump count calculates the bacterium number in the ware, then multiplies its extension rate up to this bacterium number in every gram of sample, calculates as follows：
A=B × C/5 × f × 10
In formula：A be measure enterococcus faecalis clump count, unit cfu/g；B repeats suspicious three times for same dilution
Enterococcus faecalis average colony number；C is the number that enterococcus faecalis is confirmed as in the bacterium colony of 5 identifications；F is extension rate；10 is turn
Change the factor.
The Enterococcus faecalis microorganisms preparation of moisture content 40% is moved into hot air circulation drying oven, first 48 ± 1 DEG C of processing 1.5
Hour, 41 ± 1 DEG C of drying of latter temperature, the high activity Enterococcus faecalis microorganisms preparation that moisture content 10% is thus prepared is done
Powder, living bacteria count are 1.8 × 1010Cfu/g, crushing packing is into finished product, after detection is qualified, as commodity selling.
Embodiment 2：
In superclean bench, pipe strain is lyophilized according to sterile behaviour in enterococcus faecalis (Enterococcus faecalis)
Opening is required, suitable sterile water is added in into freeze-drying pipe, with aseptic inoculation ring by after its mixing, chooses a ring bacterial suspension inoculation
In MRS agar test tubes medium slants, the inclined-plane after inoculation is put into biochemical cultivation case, 37 ± 1 DEG C are cultivated 20 hours, then will training
The test tube slant kind supported is transferred in MRS agar eggplant-shape bottle slant mediums, and 37 DEG C are cultivated 17 hours, then with suitable nothing
Bacterium mud ripe on eggplant-shape bottle is scraped by bacterium water, is packed into 250ml triangular flasks, and shaken well forms seed liquor, a concentration of
9.0×108cfu/ml.Above-mentioned MRS agar mediums raw material and preparation method are：Every liter contains peptone 10.0g, beef extract
10.0g, yeast extract 5.0g, diammonium hydrogen citrate [(NH4)2HC6H5O7] 2.0g, glucose (C6H12O6·H2O) 20.0g, tween
80 1.0mL, sodium acetate (CH3COONa·3H2O) 5.0g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 2.0g, magnesium sulfate (MgSO4·
7H2O) 0.58g, manganese sulfate (MnSO4·H2O) 0.25g, agar 20.0g, surplus are distilled water；Above-mentioned each raw material is mixed equal
It is even, adjust the 20min that sterilizes at a temperature of PH is 6.6,121 DEG C.
Seed liquor is transferred to 5% inoculum concentration in the 5L fermentation tanks containing one grade fermemtation culture medium, packing factor
70%, 37 ± 1 DEG C, stir speed (S.S.) 0rpm, ventilatory capacity 0V/Vmin of fermentation temperature, tank pressure maintains 0.02~0.03Mpa, dissolved oxygen
Concentration maintains 6~8%, when cultivating by 11 hours, when having arrived at exponential phase through growth curve detection thalline, passes through hair
Fermentation tank material-feeding port according to the requirement of fermentation tank feed operation, adds 1 trehalose of protective agent, additive amount 9%, continues fermentation to 19 small
When, pH value is down to 4.1 and is made primary seed solution, and a concentration of 1.5 × 109cfu/ml；The raw material of above-mentioned one grade fermemtation culture medium and
Preparation method is：Every liter contains Tomato juice 160mL, tryptone 8.0g, beef extract 6.5g, yeast extract 5.0g, hydrogen citrate
Diammonium 2.5g, sucrose 19.5g, Tween 80 1.0mL, sodium acetate trihydrate 5.5g, three water dipotassium hydrogen phosphate 2.5g, epsom salt
0.48g, manganese sulfate monohydrate 0.24g；Above-mentioned each raw material is uniformly mixed, adjusts the 20min that sterilizes at a temperature of PH is 6.8,121 DEG C.
Primary seed solution is transferred to 10% inoculum concentration in the 50L fermentation tanks containing second order fermentation culture medium and carries out two
Grade fermentation, 37 ± 1 DEG C, stir speed (S.S.) 0rpm, ventilatory capacity 0V/Vmin of fermentation temperature, tank pressure maintains 0.04~0.05Mpa,
Oxyty maintains 8~10%, when cultivating by 13 hours, when having arrived at exponential phase through growth curve detection thalline,
It by fermentation tank material-feeding port, is required according to fermentation tank feed operation, addition 2 soluble starch of protective agent, additive amount 7.5%, after
Supervention ferment was to 22 hours, when PH is down to 4.1, was made secondary seed solution, and a concentration of 2.2 × 109cfu/ml；Above-mentioned second order fermentation training
Support base raw material and preparation method be：Every liter contains Tomato juice 140mL, casein peptone 6.0g, beef extract powder 6.5g, dusty yeast
5.0g, cornstarch 45g, anhydrous sodium acetate 2.5g, sodium citrate 5.0g, glucan 65g, lactose 10.0g, manganese sulfate 0.24g,
Sodium citrate 5.5g, anhydrous magnesium sulfate 0.48g；Above-mentioned each raw material is uniformly mixed, adjusts at a temperature of PH is 6.5,121 DEG C and sterilizes
20min。
Prepare before inoculation：It is first carried out disinfection with peracetic acid disinfectant docking inter-species, culture room, production apparatus etc., then
The ultraviolet lamp disinfection 30min in culture room is opened again, prepares sterile culture environment, area 58m between inoculation2, cultivate room area 32m2,
In gnotobasis, 3 skimmed milk power of protective agent, additive amount 12%, after mixing, with 8% are added into secondary seed solution
It in wheat bran solid medium after inoculum concentration access sterilizing, is uniformly mixed, fills ventilative mesh bag into culture room culture.Cultivate room culture
Period, control ventilation quantity are：7~9 rates of ventilation/day, 37 ± 1 DEG C of temperature, room humidity 85%~90% are cultivated 24 hours,
Wheat bran solid medium is stirred during culture 2 times, the Enterococcus faecalis microorganisms preparation of moisture content 39% is thus prepared, effectively
Viable count is 1.9 × 1010cfu/g.The raw material and preparation method of above-mentioned wheat bran solid medium be：By total mass fraction 100%
Meter, is made of wheat bran 55%, ammonium sulfate 0.5%, epsom salt 0.62%, manganese sulfate monohydrate 0.38%, water 43.5%；It will be upper
State each raw material to be uniformly mixed, sterilize 45 minutes under the conditions of 115~121 DEG C, into sterile Air cooler be cooled to 40 DEG C it is spare.
The Enterococcus faecalis microorganisms preparation of moisture content 39% is moved into hot air circulation drying oven, first 48 ± 1 DEG C of processing 1.5
Hour, 41 ± 1 DEG C of latter temperature is dried, and the high activity Enterococcus faecalis microorganisms preparation dry powder of moisture content 8% is thus prepared,
Living bacteria count is 1.7 × 1010Cfu/g, crushing packing is into finished product, after detection is qualified, as commodity selling.
Embodiment 3：
In superclean bench, pipe strain is lyophilized according to sterile behaviour in enterococcus faecalis (Enterococcus faecalis)
Opening is required, after adding in suitable sterile water mixing, is chosen on a ring bacterial suspension inoculation to MRS agar test tubes medium slants,
It is put into biochemical cultivation case, 37 ± 1 DEG C are cultivated 20 hours, then that cultured test tube slant kind is transferred to MRS agar eggplant-shape bottles is oblique
In the culture medium of face, 37 DEG C are cultivated 18 hours, are added in appropriate amounts of sterilized water and are scraped by bacterium mud ripe on eggplant-shape bottle, are packed into 250ml
Triangular flask, shaken well, formation seed liquor, a concentration of 8.0 × 108cfu/ml.The raw material and system of above-mentioned MRS agar mediums
Preparation Method is：Every liter contains peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, diammonium hydrogen citrate [(NH4)2HC6H5O7]
2.0g, glucose (C6H12O6·H2O) 20.0g, Tween 80 1.0mL, sodium acetate (CH3COONa·3H2O) 5.0g, phosphoric acid hydrogen two
Potassium (K2HPO4·3H2O) 2.0g, magnesium sulfate (MgSO4·7H2O) 0.58g, manganese sulfate (MnSO4·H2O) 0.25g, agar
20.0g, surplus are distilled water；Above-mentioned each raw material is uniformly mixed, adjusts the 20min that sterilizes at a temperature of PH is 6.6,121 DEG C.
Seed liquor is transferred to 5% inoculum concentration in the 5L fermentation tanks containing one grade fermemtation culture medium, packing factor
70%, 37 ± 1 DEG C, stir speed (S.S.) 0rpm, ventilatory capacity 0V/Vmin of fermentation temperature, tank pressure maintains 0.02~0.03Mpa, dissolved oxygen
Concentration maintains 6~8%, when cultivating by 14 hours, has arrived at exponential phase through growth curve detection thalline, passes through fermentation
Tank material-feeding port according to the requirement of fermentation tank feed operation, adds 1 trehalose of protective agent, and additive amount 9% continued fermentation to 19 hours,
PH value is down to 3.6 and is made primary seed solution, and a concentration of 1.3 × 109cfu/ml；Above-mentioned one grade fermemtation culture medium：Every liter contains west
Red persimmon juice 160mL, it tryptone 8.0g, beef extract 6.5g, yeast extract 5.0g, diammonium hydrogen citrate 2.5g, sucrose 19.5g, spits
Warm 801.0mL, sodium acetate trihydrate 5.5g, three water dipotassium hydrogen phosphate 2.5g, epsom salt 0.48g, manganese sulfate monohydrate 0.24g；
Above-mentioned each raw material is uniformly mixed, adjusts the 20min that sterilizes at a temperature of PH is 6.8,121 DEG C.
Primary seed solution is transferred to 10% inoculum concentration in the 50L fermentation tanks containing second order fermentation culture medium and carries out two
Grade fermentation, 37 ± 1 DEG C, stir speed (S.S.) 0rpm, ventilatory capacity 0V/Vmin of fermentation temperature, tank pressure maintains 0.04~0.05Mpa,
Oxyty maintains 8~10%, when cultivating by 14 hours, when having arrived at exponential phase through growth curve detection thalline,
It by fermentation tank material-feeding port, is required according to fermentation tank feed operation, addition 2 soluble starch of protective agent, additive amount 7.5%, after
Supervention ferment was to 22 hours, when pH is down to 4.1, was made secondary seed solution, and a concentration of 1.8 × 109cfu/ml；Above-mentioned second order fermentation training
Support base raw material and preparation method be：Every liter contains Tomato juice 140mL, casein peptone 6.0g, beef extract powder 6.5g, dusty yeast
5.0g, cornstarch 45g, anhydrous sodium acetate 2.5g, sodium citrate 5.0g, glucan 65g, lactose 10.0g, manganese sulfate 0.24g,
Sodium citrate 5.5g, anhydrous magnesium sulfate 0.48g；Above-mentioned each raw material is uniformly mixed, adjusts at a temperature of PH is 6.5,121 DEG C and sterilizes
20min。
Prepare before inoculation：It is first carried out disinfection with peracetic acid disinfectant docking inter-species, culture room, production apparatus etc., then
The ultraviolet lamp disinfection 30min in culture room is opened again, prepares sterile culture environment, area 58m between inoculation2, cultivate room area 32m2,
In gnotobasis, 3 skimmed milk power of protective agent, additive amount 12%, after mixing, with 8% are added into secondary seed solution
It in wheat bran solid medium after inoculum concentration access sterilizing, is uniformly mixed, fills ventilative mesh bag into culture room culture.Cultivate room culture
Period, control ventilation quantity are：3~6 rates of ventilation/day, 37 ± 1 DEG C of temperature, room humidity 85%~90% are cultivated 26 hours,
Wheat bran solid medium is stirred during culture 1 time, the Enterococcus faecalis microorganisms preparation of moisture content 36% is thus prepared, effectively
Viable count is 1.6 × 1010cfu/g.The raw material and preparation method of above-mentioned wheat bran solid medium be：By total mass fraction 100%
Meter, is made of wheat bran 55%, ammonium sulfate 0.5%, epsom salt 0.62%, manganese sulfate monohydrate 0.38%, water 43.5%；It will be upper
State each raw material to be uniformly mixed, sterilize 45 minutes under the conditions of 115~121 DEG C, into sterile Air cooler be cooled to 40 DEG C it is spare.
The Enterococcus faecalis microorganisms preparation of moisture content 36% is moved into hot air circulation drying oven, first 48 ± 1 DEG C of processing 1.5
Hour, 41 ± 1 DEG C of latter temperature is dried, and the high activity Enterococcus faecalis microorganisms preparation dry powder of moisture content 9% is thus prepared,
Living bacteria count is 1.3 × 1010Cfu/g, crushing packing is into finished product, after detection is qualified, as commodity selling.
The foregoing is merely the better embodiments of the present invention, and the invention is not limited in the above embodiments, are implementing
In the process there may be the small structural modification in part, if the various changes or modifications of the present invention are not departed from the essence of the present invention
God and range, and belong within the scope of the claim and equivalent technologies of the present invention, then the present invention is also intended to comprising these changes
And modification.
Claims (7)
1. a kind of preparation method of high activity Enterococcus faecalis microorganisms preparation dry powder, which is characterized in that include the following steps：
(1) the enterococcus faecalis slant strains of activation are transferred in sterile water, formation seed liquor, a concentration of the 5.0 of the seed liquor
×108Cfu/ml~1.0 × 109cfu/ml；
(2) seed liquor that step (1) obtains is transferred to the fermentation containing one grade fermemtation culture medium with 4%~6% inoculum concentration
In tank, 37 ± 1 DEG C, stir speed (S.S.) 0rpm, ventilatory capacity 0V/Vmin of fermentation temperature, tank 0.02~0.03Mpa of pressure, oxyty 5~
10%, when cultivating by 12~16 hours, protective agent 1 is added, fermentation time is 16~20 hours, and pH value is made to be down to 3.6~4.2 systems
Into primary seed solution, a concentration of the 1.3 × 10 of the primary seed solution9Cfu/ml~1.6 × 109cfu/ml；
(3) primary seed solution that step (2) obtains is transferred to the hair containing second order fermentation culture medium with 6~10% inoculum concentration
Carry out second order fermentation in fermentation tank, 37 ± 1 DEG C, stir speed (S.S.) 0rpm, ventilatory capacity 0V/Vmin of fermentation temperature, tank pressure 0.04~
0.05Mpa, oxyty 5~10% when cultivating by 14~18 hours, add protective agent 2, and fermentation time 18~24 hours makes
PH is down to 3.6~4.2 values, is made secondary seed solution, and a concentration of the 1.8 × 10 of the secondary seed solution9Cfu/ml~2.4 ×
109cfu/ml；
(4) in gnotobasis, after the secondary seed solution addition protective agent 3 that step (3) is obtained, with 8~10% inoculum concentration
It in wheat bran solid medium after access sterilizing, is uniformly mixed, packs into culture room culture；During cultivating room culture, control is logical
Air quantity is：3~10 rates of ventilation/day, 34~38 DEG C of temperature, humidity 80%~90% are cultivated 24~28 hours, are turned over during culture
It moves wheat bran solid medium 1~2 time, the Enterococcus faecalis microorganisms preparation of moisture content, the excrement of the moisture content is thus prepared
The water content of enterococcus microorganism formulation is 35~40%, and living bacteria count is 1.5 × 1010Cfu/g~2.0 × 1010cfu/g；
(5) the Enterococcus faecalis microorganisms preparation immigration hot air circulation drying oven for the moisture content for obtaining step (4), first 46~48
It is handled under the conditions of DEG C 1.5~2.0 hours, then in 40~43 DEG C of drying of temperature, high activity Enterococcus faecalis microorganisms is prepared
Preparation dry powder, the water content of the high activity Enterococcus faecalis microorganisms preparation dry powder are 8~10%, living bacteria count for 1.3 ×
1010Cfu/g~1.8 × 1010cfu/g。
2. the preparation method of high activity Enterococcus faecalis microorganisms preparation dry powder according to claim 1, which is characterized in that step
Suddenly the raw material of the one grade fermemtation culture medium described in (2) and preparation method are：Every liter contains 160~175mL of Tomato juice, pancreas egg
5.0~8.0g of white peptone, 5.3~6.5g of beef extract, 3.3~5.0g of yeast extract, 1.4~2.5g of diammonium hydrogen citrate, sucrose 16.8
~19.5g, 0.7~1.0mL of Tween 80,4.3~5.5g of sodium acetate trihydrate, three 1.3~2.5g of water dipotassium hydrogen phosphate, seven water sulphur
Sour 0.28~0.48g of magnesium, 0.16~0.24g of manganese sulfate monohydrate；Above-mentioned each raw material is uniformly mixed, it is 6.2~6.8 to adjust PH,
Up to one grade fermemtation culture medium.
3. the preparation method of high activity Enterococcus faecalis microorganisms preparation dry powder according to claim 1, which is characterized in that step
Suddenly the raw material of the second order fermentation culture medium described in (3) and preparation method are：Every liter contains 120~140mL of Tomato juice, junket egg
4.0~6.0g of white peptone, 5.3~6.5g of beef extract powder, 3.3~5.0g of dusty yeast, 35~45g of cornstarch, anhydrous sodium acetate 1.4
~2.5g, 3.3~5.0g of sodium citrate, Dextran 60~65g, 8.5~10.0g of lactose, 0.16~0.24g of manganese sulfate, lemon
Sour 4.3~5.5g of sodium, 0.28~0.48g of anhydrous magnesium sulfate；Above-mentioned each raw material is uniformly mixed, it is 6.5~7.0 to adjust PH, i.e.,
Obtain second order fermentation culture medium.
4. the preparation method of high activity Enterococcus faecalis microorganisms preparation dry powder according to claim 1, which is characterized in that step
Suddenly the wheat bran solid culture based raw material described in (4) and preparation method are：It is composed of the following components as mass fraction：Wheat bran
43%~55%, ammonium sulfate 0.1%~0.5%, epsom salt 0.58~0.62%, manganese sulfate monohydrate 0.32~0.38%,
Surplus is water；Above-mentioned each raw material is uniformly mixed, under the conditions of 115~121 DEG C, sterilizes 40~45 minutes to get wheat bran solid
Culture medium.
5. the preparation method of high activity Enterococcus faecalis microorganisms preparation dry powder according to claims 1 to 4, feature exist
In the protective agent 1 is trehalose, and additive amount is 7~10%.
6. the preparation method of high activity Enterococcus faecalis microorganisms preparation dry powder according to claims 1 to 4, feature exist
In the protective agent 2 is soluble starch, and additive amount is 5.5~7.5%.
7. the preparation method of high activity Enterococcus faecalis microorganisms preparation dry powder according to claims 1 to 4, feature exist
In the protective agent 3 is skimmed milk power, and additive amount is 12~15%.
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