CN109022322A - A kind of preparation method of bifidobacterium lactis freeze-dried vaccine powder - Google Patents
A kind of preparation method of bifidobacterium lactis freeze-dried vaccine powder Download PDFInfo
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Abstract
The present invention discloses a kind of preparation method of bifidobacterium lactis freeze-dried vaccine powder, and basal medium includes component: Yeast protein peptone, yeast extract, glucose, oligofructose, L MALIC ACID, purified water, pH value 7.2；Its Optimal Medium includes component: Yeast protein peptone, yeast extract, glucose, oligofructose, L MALIC ACID, lactose, calcium chloride, purified water, pH value 7.2, zymotic fluid viable count is up to 5.53 × 109Cfu/ml or more.Its freeze-dried vaccine powder protective agent includes component: trehalose, skimmed milk powder, sucrose, ascorbic acid, glucose, purified water, and total viable count can achieve 9.0 × 10 in bifidobacterium lactis freeze-dried vaccine powder11Cfu/g or more.The preparation method effectively achievees the purpose that bifidobacterium lactis High Density Cultivation, thallus high income, freeze-dried powder number of viable are big, is advantageously implemented large-scale industrial production.
Description
Technical field
The invention belongs to field of biotechnology, in particular to a kind of preparation method of bifidobacterium lactis freeze-dried vaccine powder.
Background technique
Bifidobacterium lactis belongs to Bifidobacterium, and Bifidobacterium is the beneficial bacterium being colonized in human body intestinal canal, is health
Dominant microflora in Intestinal.Bifidobacterium belongs to Actinomy cetaceae, Gram-positive, atrichia, does not move, anaerobism.Bifid
Bacillus, which is mainly colonized, forms biological barrier in ileum and colon, have the function of adjusting intestinal microecology flora, Ant agonism,
Multiple biological functions, its presence such as trophism, adjusting immune function of human body, antitumor action directly affect gastral
Health status has potential prevention and treatment disease to act on.Since Bifidobacterium belongs to obligate anaerobe, there are oxygen, Low acid
And in bile environment, slow growth and easy death, high production cost.Therefore the big rule of the high density of Bifidobacterium to be realized
There is also comparable difficulty, in vitro culture is extremely difficult to higher viable count for mould culture, limits the exploitation of Bifidobacterium product
And application.
Summary of the invention
The present invention discloses a kind of preparation method of bifidobacterium lactis freeze-dried vaccine powder, the High Density Cultivation suitable for bifidobacterium lactis
And its freeze-drying saves, can Effective multiplication bifidobacterium lactis, improve total viable count in fermentation liquid and freeze-dried vaccine powder, also help reality
Existing large-scale industrial production.
The technical solution adopted by the present invention are as follows:
A kind of preparation method of bifidobacterium lactis freeze-dried vaccine powder, includes the following steps:
1) strain of fermentation is prepared
1.1) scribing line culture: after bifidobacterium lactis bacterium powder is diluted with sterile water, subregion is drawn on basal medium in plate
Line, 39 DEG C Anaerobic culturel 46~50 hours；
1.2) level-one purifying culture: the single colonie on picking basal medium plate is seeded to equipped with 5mL basal medium
Test tube in, sealing, 39 DEG C of incubator constant temperature stand Anaerobic culturel 18~23 hours, obtain level-one purifying bacteria suspension；
1.3) second level purifying culture: pressing 3~6% inoculum concentrations, and cultured level-one is purified bacterial suspension inoculation to being equipped with base
In the 100mL triangular flask of basal culture medium, 80% liquid amount is sealed, and 39 DEG C of incubator constant temperature stand Anaerobic culturel 16~20 hours,
It obtains second level and purifies bacteria suspension；
1.4) bacterium mud preparation and preservation: second level purifying bacteria suspension is centrifuged, and 10000rpm centrifugation 10min obtains bacterium
Bacterium mud is dissolved in the mixed liquor of basal medium and 50% glycerite composition, obtains bacterium mud mixed liquor, bacterium mud is mixed by mud
Liquid is divided in sterilized cryopreservation tube, sealed membrane sealing, freeze in -80 DEG C of low temperature refrigerators, obtain bifidobacterium lactis bacterium mud freeze
Pipe is deposited, it is spare；
2) strain fermentation
2.1) it freezes bacterium mud recovery: taking the bifidobacterium lactis bacterium mud cryopreservation tube being stored in low temperature refrigerator, be immediately placed in 37 DEG C
Strain recovery is carried out in water-bath, is all melted until freezing liquid in pipe；Every cryopreservation tube bacterium solution is seeded to the training of the basis containing 10mL
In the test tube for supporting base, 39 DEG C of 16~20h of standing sealing culture；
2.2) strain expands culture: the basic culture solution recovered is seeded to according to 3~6% inoculum concentration equipped with basis
In the 100mL triangular flask of culture medium, liquid amount 80%, 39 DEG C of standing sealings are cultivated 16~20 hours；
2.3) strain one grade fermemtation: according to 3~6% inoculum concentrations, strain is expanded into the bacterial suspension inoculation that culture terminates and is extremely filled
Have in the 100mL triangular flask of Optimal Medium, liquid amount 80%, 39 DEG C of standing sealings are cultivated 6~10 hours；Start in fermentation
After 3.5 hours, it is 5.6 ± 0.5 that the permanent pH of bacterium solution is maintained by stream plus food-grade NaOH solution, until monitoring bacterium solution OD600
Value stops increasing, and stops fermentation immediately；
2.4) strain second order fermentation: according to 5~8% inoculum concentrations, the bacterial suspension inoculation of strain one grade fermemtation is excellent to being equipped with
In the fermentor for changing culture medium, Optimal Medium dress liquid product accounts for the 80% of fermenter volume, 39 DEG C of constant temperature agitation cultures 6~10
Hour；Mixing speed 80rpm, ventilatory capacity 0 are maintained after fermentation starts 3.5 hours by stream plus food-grade NaOH solution
The permanent pH of bacterium solution is 5.6 ± 0.5, until monitoring bacterium solution OD600Value stops increasing, and stops fermentation immediately；
3) preparation of freeze-dried powder
3.1) fermentation liquid is centrifuged: after fermentation, fermentor pot temperature being turned down, when broth temperature is lower than 20 in tank
DEG C when, preparation be centrifuged using centrifugation apparatus, after centrifugation, collect centrifugation apparatus rotary drum in bacterium mud；
3.2) it is freeze-dried: after bacterium mud is collected, according to bacterium mud: freeze drying protectant=1:1~2 volume ratio, to bacterium
Freeze drying protectant is added in mud, and stirs, be uniformly mixed, is dispensed into freeze drier pallet, then by freeze drier pallet
It is put into the freeze-drying for carrying out bacterium powder in freeze drier, keeps 0~1.0Pa of vacuum degree, is persistently lyophilized 39~69 hours；
3.3) freeze-dried powder is collected, is packed after being crushed by quality requirement.
The preparation method, the volume ratio of basal medium and 50% glycerite described in step 1.4) are 4:6, base
The volume ratio of second level purifying bacteria suspension is 1:10 before the mixed liquor and centrifugation of basal culture medium and 50% glycerite composition.
The preparation method, step 1) and 2) in basal medium preparation, formula: 25~40g/L of Yeast protein peptone,
20~30g/L of yeast extract, 15~25g/L of glucose, 3~7g/L of oligofructose, 3~7g/L of L MALIC ACID, surplus are pure
Change water, pH value 7.2；Each component is weighed according to formula rate, is mixed, is dissolved by heating, sterilize 30min at 115 DEG C.
The preparation method, the preparation of Optimal Medium in step 2), formula: 25~40g/L of Yeast protein peptone, yeast
10~20g/L of extract, 10~20g/L of glucose, 3~7g/L of oligofructose, 3~7g/L of L MALIC ACID, 6~12g/L of lactose,
0.3~1g/L of calcium chloride, surplus are purified water, pH value 7.2；Each component is weighed according to formula rate, is mixed, is dissolved by heating, 115
Sterilize 30min at DEG C.
The preparation method, the step 3.1) centrifugation apparatus use tube centrifuge, steam before centrifugation to rotary drum
Vapour sterilizing 30min, revolving speed 13000rpm.
The preparation method, frozen-dried protective agent prescription in step 3.2) are as follows: 50~200g/L of trehalose, skimmed milk power 50
~200g/L, 0.5~5g/L of sucrose, 0.5~5g/L of sodium ascorbate, 0.5~5g/L of glucose, surplus are purified water.
The preparation method, freeze drying protectant the preparation method comprises the following steps:
A accurately weighs skimmed milk power by formula rate, and part purified water in formula is taken to be used to be completely dissolved skimmed milk power, in
Sterilize 30min at 115 DEG C, obtains solution A；
B other components are weighed according to formula rate, are mixed, and are dissolved with the purified water of remainder in formula, at 115 DEG C
Sterilize 30min, obtains solution B；
C sterilizing terminates to place to after room temperature, is uniformly mixed solution A and solution B to obtain freeze drying protectant in sterile, will
The freeze drying protectant prepared is refrigerated in spare in -4 DEG C of refrigerators.
The preparation method, purifying the mass ratio of water consumption in purifying water consumption and b in step a is 1~2:3.
The preparation method, when the middle use freeze drier of step 3.2) carries out the freeze-drying of bacterium powder, the temperature of freeze drier
Degree and freeze-drying time are arranged by section, specifically, the 1st section: freeze-drying 2~4h of duration, temperature are set as -40 DEG C；2nd section:
24~36h of duration is lyophilized, temperature is set as -25 DEG C；3rd section: freeze-drying 2~6h of duration, temperature are set as -15 DEG C；4th area
Section: freeze-drying 2~6h of duration, temperature are set as -5 DEG C；5th section: freeze-drying 1~3h of duration, temperature are set as 0 DEG C；6th section:
2~4h of duration is lyophilized, temperature is set as 15 DEG C；7th section: freeze-drying 2~4h of duration, temperature are set as 25 DEG C；8th section: freeze
Dry 4~6h of duration, temperature are set as 30 DEG C.
The present invention has the advantages that following prominent:
1, the fermentation liquid for the bifidobacterium lactis that the present invention obtains, zymotic fluid viable count is up to 5.53 × 109Cfu/ml or more；
The bifidobacterium lactis freeze-dried vaccine powder total viable count that the present invention obtains is up to 9.0 × 1011Cfu/g or more.
2, the present invention uses the Optimal Medium for being more suitable for thalli growth for the strain fermentation of bifidobacterium lactis, excellent
To change and add lactose and calcium chloride in culture medium, is not only suitable for bifidobacterium lactis thalli growth, reproduction speed is fast, and number of viable is big,
And reduce the cost of culture medium.
3, the component raw material used in the present invention for the culture medium of bifidobacterium lactis is food-grade, is suitable for extensive
Bifidobacterium lactis freeze-dried powder of the industrialized production as food additives.
4, expanding training method using classification promotes thalline quantity to quickly increase, and is controlled by stream plus food-grade NaOH solution
PH is constant in fermentation process processed, a large amount of thallus is obtained in the short time, and strain is stablized, and variation is degenerated less.
5, bifidobacterium lactis bacterium mud is separated and collected using tube centrifuge, for bacterium mud yield up to 1.5% or more, viable count is high
Up to 1011Cfu/g or more, and the rotary drum of tube centrifuge can use steam sterilizing, effectively prevent the microbiological contamination in centrifugal process.
4, using Freeze Drying Technique production active bacteria formulation, there are many prominent advantages: micro- 1. due to drying at low temperature
Biology will not lose bioactivity；2. the growth of microorganism and the effect of enzyme can not almost carry out, energy during low temperature drying
Best keep the original character for being frozen dry matter；3. volume, shape are basically unchanged after dry；4. being frozen dried product in sponge
Shape, not drying shrinkage, thus while dissolving, are big with water engaging surface, can restore original character rapidly；5. 95%-99% in substance can be removed
Moisture, extend the storage life of product；6. reducing and ging up using temperature gradient, reduce the damage to thallus.
5, to avoid causing the damage of bacterium cell membrane in freezing dry process, freeze drying protectant is added, in freeze drying protectant
Middle skimmed milk power mainly plays protective layer in cell surface；Sucrose and glucose can be in freeze-drying process due to containing hydroxyl group
It is middle to replace the original moisture subbase group of cell membrane, hydrogen bond is formed with phosphatide and protein, to stabilize the integrality of cell membrane；
Trehalose forms a kind of glassy state in freezing dry process, and viscosity is high under glassy state, thus coefficient of molecular diffusion is very low,
Make macromolecular substances retard motion, the permeability of cell wall reduces, to play a protective role；Sodium ascorbate has anti-oxidant
Effect.Every kind of protective agent ingredient all plays respective effect in freezing dry process in compounding protective agent system, while mutually
Between again have synergistic effect.
6, the skimmed milk power in freeze drying protectant takes independent sterilization method, adequately protect its active ingredient not by peracid,
Alkali or high temperature are crossed, protective effect can be preferably played in bacterium powder freeze-drying process, improves freeze drying viable microorganism rate；And it is made
Freeze-dried powder color it is shallower, conducive to improving a poor appetite.
Specific embodiment
A kind of preparation method of the bifidobacterium lactis freeze-dried powder of embodiment 1
1) strain of fermentation is prepared
1.1) scribing line culture: after bifidobacterium lactis bacterium powder is diluted with sterile water, subregion is drawn on basal medium in plate
Line, 39 DEG C Anaerobic culturel 49 hours；
1.2) level-one purifying culture: the single colonie on picking basal medium plate is seeded to equipped with 5mL basal medium
Test tube in, sealing, 39 DEG C of incubator constant temperature stand Anaerobic culturel 22 hours, obtain level-one purifying bacteria suspension；
1.3) second level purifying culture: 4% inoculum concentration is pressed, by cultured level-one bacterial suspension inoculation to being equipped with basal medium
100mL triangular flask in, 80% liquid amount, sealing, 39 DEG C of incubator constant temperature stand Anaerobic culturel 18 hours, obtain second level purifying bacterium
Suspension；
1.4) bacterium mud preparation and preservation: second level purifying bacteria suspension is centrifuged, and 10000rpm centrifugation 10min obtains bacterium
Bacterium mud is dissolved in mixed liquor (wherein basal medium and 50% glycerol of basal medium and 50% glycerite composition by mud
The volume ratio of solution is 4:6；Second level purifies bacteria suspension before the mixed liquor and centrifugation of basal medium and 50% glycerite composition
Volume ratio be 1:10) in, obtain bacterium mud mixed liquor, bacterium mud mixed liquor be divided in sterilized cryopreservation tube, sealed membrane sealing,
It freezes in -80 DEG C of low temperature refrigerators, obtaining bifidobacterium lactis bacterium mud cryopreservation tube, it is spare；
2) strain fermentation
2.1) it freezes bacterium mud recovery: taking the bifidobacterium lactis bacterium mud cryopreservation tube being stored in low temperature refrigerator, be immediately placed in 37 DEG C
Strain recovery is carried out in water-bath, is all melted until freezing liquid in pipe；Every cryopreservation tube bacterium solution is seeded to the training of the basis containing 10mL
In the test tube for supporting base, 39 DEG C of standing sealing culture 18h；
2.2) strain expands culture: the basic culture solution recovered is seeded to according to 3% inoculum concentration equipped with basis training
In the 100mL triangular flask for supporting base, liquid amount 80%, 39 DEG C of standing sealings are cultivated 18 hours；
2.3) according to 3% inoculum concentration, it is excellent to being equipped with that strain strain one grade fermemtation: is expanded into the bacterial suspension inoculation that culture terminates
In the 100mL triangular flask for changing culture medium, liquid amount 80%, 39 DEG C of standing sealings are cultivated 8 hours；After fermentation starts 3.5 hours,
It is 5.6 ± 0.5 that the permanent pH of bacterium solution is maintained by stream plus food-grade NaOH solution, until monitoring bacterium solution OD600Value stops increasing
It is long, stop fermentation immediately.
2.4) strain second order fermentation: according to 5% inoculum concentration, the bacterial suspension inoculation of strain one grade fermemtation is trained to optimization is equipped with
In the fermentor for supporting base, Optimal Medium dress liquid product accounts for the 80% of fermenter volume, and 39 DEG C of constant temperature agitations are cultivated 8 hours；It stirs
Speed 80rpm is mixed, ventilatory capacity 0 maintains bacterium solution by stream plus food-grade NaOH solution after fermentation starts 3.5 hours
Permanent pH is 5.6 ± 0.5, until monitoring bacterium solution OD600Value stops increasing, and stops fermentation immediately.
3) preparation of freeze-dried powder
3.1) fermentation liquid is centrifuged: after fermentation, fermentor pot temperature being turned down, when broth temperature is lower than 20 in tank
DEG C when, preparation be centrifuged using centrifugation apparatus, after centrifugation, collect centrifugation apparatus rotary drum in bacterium mud；The centrifugation apparatus
For tube centrifuge, steam sterilizing 30min, revolving speed 13000rpm are carried out to rotary drum before centrifugation.
3.2) it is freeze-dried: after bacterium mud is collected, according to bacterium mud: freeze drying protectant=1:1.2 volume ratio, to bacterium mud
Middle addition freeze drying protectant, and stir, be uniformly mixed, it is dispensed into freeze drier pallet, then puts freeze drier pallet
The freeze-drying for entering to carry out bacterium powder in freeze drier, keeps vacuum degree 0.6Pa, is persistently lyophilized 54 hours；
The temperature and freeze-drying time of freeze drier are arranged by section, specifically, the 1st section: freeze-drying duration 3h, temperature are set
It is set to -40 DEG C；2nd section: freeze-drying duration 30h, temperature are set as -25 DEG C；3rd section: freeze-drying duration 4h, temperature is set as-
15℃；4th section: freeze-drying duration 4h, temperature are set as -5 DEG C；5th section: freeze-drying duration 2h, temperature are set as 0 DEG C；6th area
Section: freeze-drying duration 3h, temperature are set as 15 DEG C；7th section: freeze-drying duration 3h, temperature are set as 25 DEG C；8th section: when freeze-drying
Long 5h, temperature are set as 30 DEG C.
3.3) freeze-dried powder is collected, is packed after being crushed by quality requirement.
The preparation of basal medium, formula: Yeast protein peptone 35g/L, yeast extract 25g/L, glucose 24g/L, low
Fructooligosaccharides 6g/L, L MALIC ACID 6g/L, surplus are purified water, pH value 7.2；Each component is weighed according to formula rate, is mixed, heating
It dissolves, sterilize 30min at 115 DEG C.
The preparation of Optimal Medium, formula: Yeast protein peptone 32g/L, yeast extract 15g/L, glucose 13g/L, low
Fructooligosaccharides 6g/L, L MALIC ACID 6g/L, lactose 11g/L, calcium chloride 0.6g/L, surplus are purified water, pH value 7.2；According to formula
Ratio weighs each component, mixes, and dissolves by heating, and sterilize 30min at 115 DEG C.
Frozen-dried protective agent prescription are as follows: trehalose 120g/L, skimmed milk power 100g/L, sucrose 3g/L, sodium ascorbate 1.5g/
L, glucose 1.5g/L, surplus are purified water.Preparation: a accurately weighs skimmed milk power by formula rate, takes 250mL in formula pure
Change water for being completely dissolved skimmed milk power, sterilize 30min at 115 DEG C, obtains solution A；B other components claim according to formula rate
Amount, mixing are dissolved with the purified water of remainder in formula, and sterilize 30min at 115 DEG C, obtains solution B；C sterilizing terminates to place
To room temperature, it is uniformly mixed solution A and solution B to obtain freeze drying protectant in sterile, the freeze drying protectant prepared is cold
It is hidden in spare in -4 DEG C of refrigerators.
The comparative experiments of 2 basal medium formulation of embodiment
Single factor exploration bifidobacterium lactis increases influence of the oligosaccharides to bacterium number living in bacteria factor.Most of oligosaccharide not by
Alimentary canal absorbs, but is just utilized by some probiotics in big enteral, thus has unique physiological function, can promote double
The growth of discrimination bacillus effectively improves the viable count of Bifidobacterium.
The comparative experiments of 1 bifidobacterium lactis basal medium of table
Basal medium is prepared respectively according to above-mentioned 1~4 group of formula, and surplus is purified water.By 4% inoculum concentration, will cultivate
Good level-one bacterial suspension inoculation is into the triangular flask of 100mL basal medium, sealing, and 39 DEG C of incubator constant temperature stand Anaerobic culturel
18 hours.Calculate the yield and viable count of bifidobacterium lactis.As shown in Table 1, the basis training that formula 1 is formulated using embodiment 1
Base is supported, yield and viable count are above other formula groups.
The comparative experiments of 3 optimization culture based formulas of embodiment
The comparative experiments of 2 bifidobacterium lactis Optimal Medium of table
Optimal Medium is prepared respectively according to above-mentioned I, II, III, IV, V, VI 6 kind of formula, and surplus is sterile water.According to
5% inoculum concentration, by the bacterial suspension inoculation of strain one grade fermemtation into the fermentor that Optimal Medium is housed, Optimal Medium fills liquid
Volume accounts for the 80% of fermenter volume, and 39 DEG C of constant temperature agitations are cultivated 8 hours；Mixing speed 80rpm, ventilatory capacity 0 are opened in fermentation
After beginning 3.5 hours, it is 5.6 ± 0.5 that the permanent pH of bacterium solution is maintained by stream plus food-grade NaOH solution.Calculate bifidobacterium lactis
Yield and viable count.As shown in Table 2, the Optimal Medium that formula II is formulated using embodiment 1, yield and viable count are high
In other formula groups.
The comparative experiments of 4 frozen-dried protective agent prescription of embodiment
The comparative experiments of 3 bifidobacterium lactis frozen-dried protective agent prescription of table
Freeze drying protectant is prepared respectively according to five kinds of above-mentioned A, B, C, D, E formulas, and surplus is sterile water, in identical freezing
Freeze-dried powder A, B, C, D, E are made under drying condition, is stored 12 months under the conditions of freeze-dried powder A, B, C, D, E are placed in -20 DEG C, examines
Survey its viable count and moisture content.As shown in Table 3: formula C, i.e. cream made from the freeze drying protectant using the formula of embodiment 1 are double
Discrimination bacillus freeze-dried powder, after storing 12 months under the conditions of -20 DEG C, thallus viable count is much higher than other 4 kinds of freeze drying protectants, and water
Divide relatively low.
Claims (9)
1. a kind of preparation method of bifidobacterium lactis freeze-dried vaccine powder, which comprises the steps of:
1) strain of fermentation is prepared
1.1) scribing line culture: after bifidobacterium lactis bacterium powder is diluted with sterile water, the sectional streak on basal medium in plate, 39
DEG C Anaerobic culturel 46~50 hours；
1.2) level-one purifying culture: the single colonie on picking basal medium plate is seeded to the examination equipped with 5mL basal medium
Guan Zhong, sealing, 39 DEG C of incubator constant temperature stand Anaerobic culturel 18~23 hours, obtain level-one purifying bacteria suspension；
1.3) second level purifying culture: pressing 3~6% inoculum concentrations, and cultured level-one is purified bacterial suspension inoculation to being equipped with basic training
In the 100mL triangular flask for supporting base, 80% liquid amount is sealed, and 39 DEG C of incubator constant temperature stand Anaerobic culturel 16~20 hours, obtains two
Grade purifying bacteria suspension；
1.4) bacterium mud preparation and preservation: second level purifying bacteria suspension is centrifuged, and 10000rpm centrifugation 10min obtains bacterium mud, will
Bacterium mud is dissolved in the mixed liquor of basal medium and 50% glycerite composition, obtains bacterium mud mixed liquor, by bacterium mud mixed liquor point
In sterilized cryopreservation tube, sealed membrane sealing is frozen in -80 DEG C of low temperature refrigerators, obtaining bifidobacterium lactis bacterium mud cryopreservation tube,
It is spare；
2) strain fermentation
2.1) it freezes bacterium mud recovery: taking the bifidobacterium lactis bacterium mud cryopreservation tube being stored in low temperature refrigerator, be immediately placed in 37 DEG C of water-baths
Strain recovery is carried out in pot, is all melted until freezing liquid in pipe；Every cryopreservation tube bacterium solution is seeded to basal medium containing 10mL
Test tube in, 39 DEG C of standings sealing culture, 16~20h；
2.2) strain expands culture: the basic culture solution recovered is seeded to according to 3~6% inoculum concentration equipped with basis culture
In the 100mL triangular flask of base, liquid amount 80%, 39 DEG C of standing sealings are cultivated 16~20 hours；
2.3) according to 3~6% inoculum concentrations, it is excellent to being equipped with that strain strain one grade fermemtation: is expanded into the bacterial suspension inoculation that culture terminates
In the 100mL triangular flask for changing culture medium, liquid amount 80%, 39 DEG C of standing sealings are cultivated 6~10 hours；It is small to start 3.5 in fermentation
Shi Hou, it is 5.6 ± 0.5 that the permanent pH of bacterium solution is maintained by stream plus food-grade NaOH solution, until monitoring that bacterium solution OD600 value is stopped
Only increase, stops fermentation immediately；
2.4) strain second order fermentation: according to 5~8% inoculum concentrations, the bacterial suspension inoculation of strain one grade fermemtation is trained to optimization is equipped with
In the fermentor for supporting base, Optimal Medium dress liquid product accounts for the 80% of fermenter volume, and 39 DEG C of constant temperature agitation cultures 6~10 are small
When；Mixing speed 80rpm, ventilatory capacity 0 maintain bacterium by stream plus food-grade NaOH solution after fermentation starts 3.5 hours
The permanent pH of liquid is 5.6 ± 0.5, until monitoring that bacterium solution OD600 value stops increasing, stops fermentation immediately；
3) preparation of freeze-dried powder
3.1) fermentation liquid is centrifuged: after fermentation, fermentor pot temperature being turned down, when broth temperature is lower than 20 DEG C in tank
When, preparation is centrifuged using centrifugation apparatus, after centrifugation, collects the bacterium mud in centrifugation apparatus rotary drum；
3.2) it is freeze-dried: after bacterium mud is collected, according to bacterium mud: freeze drying protectant=1:1~2 volume ratio, into bacterium mud
Freeze drying protectant is added, and stirs, be uniformly mixed, freeze drier pallet is dispensed into, is then put into freeze drier pallet
The freeze-drying that bacterium powder is carried out in freeze drier, keeps 0~1.0Pa of vacuum degree, is persistently lyophilized 39~69 hours；
3.3) freeze-dried powder is collected, is packed after being crushed by quality requirement.
2. preparation method as described in claim 1, which is characterized in that basal medium described in step 1.4) and 50% glycerol
The volume ratio of solution is 4:6, and second level purifies bacteria suspension before the mixed liquor and centrifugation of basal medium and 50% glycerite composition
Volume ratio be 1:10.
3. preparation method as described in claim 1, which is characterized in that step 1) and 2) in basal medium preparation, formula:
25~40g/L of Yeast protein peptone, 20~30g/L of yeast extract, 15~25g/L of glucose, 3~7g/L of oligofructose, L- apple
3~7g/L of tartaric acid, surplus are purified water, pH value 7.2；Each component is weighed according to formula rate, is mixed, is dissolved by heating, at 115 DEG C
Sterilize 30min.
4. preparation method as described in claim 1, which is characterized in that the preparation of Optimal Medium in step 2), formula: yeast
25~40g/L of peptone, 10~20g/L of yeast extract, 10~20g/L of glucose, 3~7g/L of oligofructose, L MALIC ACID 3
~7g/L, 6~12g/L of lactose, 0.3~1g/L of calcium chloride, surplus are purified water, pH value 7.2；Each group is weighed according to formula rate
Point, it mixes, dissolves by heating, sterilize 30min at 115 DEG C.
5. preparation method as described in claim 1, which is characterized in that the step 3.1) centrifugation apparatus is centrifuged using tubular type
Machine carries out steam sterilizing 30min, revolving speed 13000rpm to rotary drum before centrifugation.
6. preparation method as described in claim 1, which is characterized in that frozen-dried protective agent prescription in step 3.2) are as follows: trehalose
50~200g/L, 50~200g/L of skimmed milk power, 0.5~5g/L of sucrose, 0.5~5g/L of sodium ascorbate, glucose 0.5~
5g/L, surplus are purified water.
7. preparation method as described in claim 1, which is characterized in that freeze drying protectant the preparation method comprises the following steps:
A accurately weighs skimmed milk power by formula rate, takes part purified water in formula to be used to be completely dissolved skimmed milk power, in 115
Sterilize 30min at DEG C, obtains solution A；
B other components are weighed according to formula rate, mixing is sterilized at 115 DEG C with the purified water dissolution of remainder in formula
30min obtains solution B；
C sterilizing terminates to place to after room temperature, is uniformly mixed solution A and solution B to obtain freeze drying protectant in sterile, will prepare
Good freeze drying protectant is refrigerated in spare in -4 DEG C of refrigerators.
8. preparation method as claimed in claim 7, which is characterized in that purified in step a and purify water consumption in water consumption and b
Mass ratio is 1~2:3.
9. preparation method as described in claim 1, which is characterized in that carry out bacterium powder jelly using freeze drier in step 3.2)
When dry, the temperature and freeze-drying time of freeze drier are arranged by section, specifically, the 1st section: freeze-drying 2~4h of duration, temperature are set
It is set to -40 DEG C；2nd section: freeze-drying 24~36h of duration, temperature are set as -25 DEG C；3rd section: freeze-drying 2~6h of duration, temperature
It is set as -15 DEG C；4th section: freeze-drying 2~6h of duration, temperature are set as -5 DEG C；5th section: freeze-drying 1~3h of duration, temperature are set
It is set to 0 DEG C；6th section: freeze-drying 2~4h of duration, temperature are set as 15 DEG C；7th section: freeze-drying 2~4h of duration, temperature are set as
25℃；8th section: freeze-drying 4~6h of duration, temperature are set as 30 DEG C.
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