CN106434463A - Preparation method of lactobacillus rhamnosus lyophilized powder - Google Patents
Preparation method of lactobacillus rhamnosus lyophilized powder Download PDFInfo
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Abstract
The invention discloses a preparation method of lactobacillus rhamnosus lyophilized powder. The preparation method comprises the following steps: (1) preparing strains for fermentation: 1.1) streak culture; 1.2) primary purification culture; 1.3) secondary purification culture; 1.4) bacterial sludge preparation and storage; 2) strain fermentation: 2.1) cryopreserved bacterial sludge resuscitation; 2.2) primary strain culture; 2.3) secondary strain culture; 2.4) tertiary strain culture; 2.5) quaternary strain culture; 3) lyophilized powder preparation: 3.1) fermentation liquid centrifugation; 3.2) freeze-drying; 3.3) lyophilized powder collection; crushing and packaging according to the quality requirements. According to the preparation method disclosed by the invention, the purposes of rhamnose lactobacillus multiplication culture, large viable number of the lyophilized powder, cell yield improvement and production cost reduction are achieved.
Description
Technical field
The invention belongs to biological technical field is and in particular to a kind of preparation method of lactobacillus rhamnosuss lyophilized powder.
Background technology
Lactobacillus rhamnosuss are subordinated to Lactobacillus, are Gram-positive facultative anaerobe, plasmid-free；Can not be using breast
Sugar, but can metabolism monosaccharide；Can grow very well under anaerobic conditions, have CO2In the presence of also can grow.
Lactobacillus rhamnosuss are one of Body normal floras, and intestinal Stickiness is high, and colonization ability is strong, is that human research is the widest
One of general probiotic bacteria, has balance and improves gastrointestinal function, strengthen human autoimmune's ability, promote bacillus bifiduss and thermophilic
Lactobacillus lactiss growth and effect, prevention and help are treated diarrhoea, preventing respiratory tract infection, are discharged toxin, pre- anti-caries, prevented
Quick grade acts on.Based on multiple functions to human body for the lactobacillus rhamnosuss, increasing enterprise utilizes lactobacillus rhamnosuss lyophilizing
Powder is raw material production biological product, then unit product viable count just directly affects effect of product.Therefore, produce in lyophilized powder
Improve viable count just particularly important in preparation process.
Content of the invention
The invention provides a kind of preparation method of lactobacillus rhamnosuss lyophilized powder, reach lactobacillus rhamnosuss propagation training
Support, lyophilized powder number of viable is big, thalline yield improves, the purpose of production cost reduction.
The technical solution used in the present invention is：
A kind of preparation method of lactobacillus rhamnosuss lyophilized powder, comprises the steps：
1) strain of preparation fermentation
1.1) streak culture：After lactobacillus rhamnosuss mycopowder is diluted with sterilized water, sectional streak in MC culture medium, 37 DEG C
Incubator constant temperature culture 48～72 hours；
1.2) one-level purification culture：Single bacterium colony on picking MC culture medium plate, is seeded to equipped with MRS fluid medium
In test tube, sealing, 37 DEG C of incubator constant temperature quiescent culture 17～24 hours, obtain one-level purification bacteria suspension；
1.3) two grades of purification cultures：By 3～5% inoculum concentrations, by cultured one-level bacterial suspension inoculation to equipped with MRS liquid
In the triangular flask of culture medium, sealing, 37 DEG C of incubator constant temperature quiescent culture 17～24 hours, obtain two grades of purification bacteria suspensions；
1.4) bacterium mud preparation and preservation：Two grades of purification bacteria suspensions are centrifuged, 12000rpm centrifugation obtains bacterium in 10 minutes
Mud, bacterium mud is dissolved in MRS fluid medium and the mixed liquor of 50% glycerite composition, obtains bacterium mud mixed liquor, by bacterium mud
Mixed liquor is divided in sterilized cryopreservation tube, seal film phonograph seal, frozen in -40～-80 DEG C of cryogenic refrigerators, obtain rhamnose breast
Bacillus bacterium mud cryopreservation tube, standby；
2) strain fermentation
2.1) frozen bacterium mud recovery：Take the lactobacillus rhamnosuss bacterium mud cryopreservation tube being stored in cryogenic refrigerator, be immediately placed in 37
Carry out strain recovery in DEG C water-bath, all melt to frozen liquid in pipe；
2.2) strain one-level culture：By the bacterium mud recovered mixed liquor 1～2ml direct inoculation to equipped with basal medium
In triangular flask, triangular flask seals, 37 DEG C of incubator constant temperature quiescent culture 17～24 hours；
2.3) two grades of cultures of strain：According to 2～6% inoculum concentrations, by the bacterial suspension inoculation of one-level culture end to equipped with base
In the triangular flask of basal culture medium, basal medium dress liquid amasss and accounts for the 40% of triangular flask volume, and triangular flask seals, 37 DEG C of incubators
Constant temperature quiescent culture 17～24 hours；
2.4) strain third stage culture：According to 2～6% inoculum concentrations, by the bacterial suspension inoculation of two grades of culture end to equipped with base
In the fermentation tank of basal culture medium, basal medium dress liquid amasss and accounts for the 60% of fermenter volume, opens fermentation tank stirring paddle, rotating speed
For 100rpm, ventilation is 0,37 DEG C of constant temperature culture 8～15 hours；
2.5) strain level Four fermentation：According to 2～6% inoculum concentrations, the bacterial suspension inoculation that third stage culture is terminated is to equipped with excellent
Change in the fermentation tank of culture medium, Optimal Medium dress liquid amasss and accounts for the 60% of fermenter volume, opens fermentation tank stirring paddle, rotating speed
For 100rpm, ventilation is 0,37 DEG C of constant temperature culture, after fermentation starts 3 hours, is tieed up by stream plus food stage NaOH solution
The permanent pH holding bacterium solution is 5.50～6.00, and cultivation cycle 8～15 hours, until monitor bacterium solution OD600Value stops increasing, immediately
Stop fermentation；
3) preparation of lyophilized powder
3.1) fermentation liquid centrifugation：After fermentation ends, fermentation tank pot temperature is turned down, when in tank, broth temperature is less than 20
DEG C when, prepare be centrifuged using centrifugation apparatus, centrifugation terminate after, collect centrifugation apparatus rotary drum in bacterium mud；
3.2) lyophilization：Bacterium mud is collected after finishing, according to bacterium mud：Freeze drying protectant=1:1～2 volume ratio, to bacterium
In mud add freeze drying protectant, and stir, mix homogeneously, be dispensed into freezer dryer pallet, then by freezer dryer pallet
Put into the lyophilizing carrying out mycopowder in freezer dryer, keep vacuum 0～1.0Pa, continue lyophilizing 57～72 hours；
3.3) collect lyophilized powder, pack after being pulverized by prescription.
Described preparation method, step 1.4) volume ratio of described MRS fluid medium and 50% glycerite is 1:1,
Before the mixed liquor of MRS fluid medium and 50% glycerite composition and centrifugation, the volume ratio of bacteria suspension is 1:10.
Described preparation method, step 2) in basal medium preparation, formula：Yeast protein peptone 6.0～10.0g/L,
Glucose 19.0～25.0g/L, yeast extract 2.0～4.0g/L, sodium acetate 4.5～6.0g/L, magnesium sulfate 0.4～0.6g/
L, Tween 80 0.5～0.75g/L, oligomeric isomaltose 1.0～2.5g/L, potassium dihydrogen phosphate 1.5～3.0g/L, balance of nothing
Bacterium water, pH value 6.20～6.80；According to formula proportion weighing, heating for dissolving, if the pH value of culture medium is less than 6.20, use
1mol/L food stage NaOH solution adjusts Medium's PH Value to sterilize at 6.20～6.80,115 DEG C 30min.
Described preparation method, step 2) in Optimal Medium preparation, formula：Yeast protein peptone 11.0～15.0g/L,
Glucose 25.0～30.0g/L, yeast extract 6.0～8.0g/L, sodium acetate 2.0～3.0g/L, magnesium sulfate 0.10～
0.15g/L, Tween 80 0.5～0.75g/L, oligomeric isomaltose 1.0～2.5g/L, potassium dihydrogen phosphate 1.5～3.0g/L, remaining
Measure as sterilized water, pH value 6.20～6.80；According to formula proportion weighing, heating for dissolving, if the pH value of culture medium is less than 6.20,
1mol/L food stage NaOH solution is then used to adjust Medium's PH Value to sterilize at 6.20～6.80,115 DEG C 30min.
Described preparation method, step 3.1) described centrifugation apparatus adopt tube centrifuge, before centrifugation, rotary drum steamed
Vapour sterilizing 30min, rotating speed is 10000～15000rpm.
Described preparation method, step 3.2) in frozen-dried protective agent prescription be：Defatted milk powder 60.0～80.0g/L, Fructus Hordei Germinatus
Dextrin 30.0～45.0g/L, trehalose 100.0～300.0g/L, Tween 80 0.5～0.75g/L, glycerol 8.0～15.0g/L,
Sodium glutamate 0.8～1.0g/L, balance of sterilized water, pH value is 6.80～7.50.
Described preparation method, the preparation method of described freeze drying protectant is：
Accurately weigh defatted milk powder by formula proportion, take part sterilized water in formula to be used for being completely dissolved defatted milk powder, in
Sterilize at 115 DEG C 30min, obtains solution A；
Other components weigh according to formula proportion, mixing, with the aseptic water dissolution of remainder in formula, at 115 DEG C
Sterilizing 30min, obtains solution B；
Sterilizing terminate place to room temperature, in aseptic by solution A and solution B mix homogeneously obtain pH value be 6.80～
7.50 freeze drying protectant, the freeze drying protectant preparing cold preservation is standby in -4 DEG C of refrigerators.
Described preparation method, if the pH value of freeze drying protectant is less than 6.80, is adjusted with 1mol/L food stage NaOH solution
PH value is to 6.80～7.50.
Described preparation method, step 3.2) in when mycopowder lyophilizing is carried out using freezer dryer, the temperature of freezer dryer
Degree and freeze-drying time press section setting, specifically, the 1st section：Lyophilizing duration 3h, temperature is set as -25 DEG C；2nd section：Lyophilizing
Duration 40～55h, temperature is set as -25 DEG C；3rd section：Lyophilizing duration 4h, temperature is set as -10 DEG C；4th section：During lyophilizing
Long 4h, temperature is set as 10 DEG C；5th section：Lyophilizing duration 2h, temperature sets 25 DEG C；6th section：Lyophilizing duration 4h, temperature sets
It is set to 30 DEG C.
The invention has the advantages that：
1st, the present invention is to realize the enrichment culture of lactobacillus rhamnosuss, be enriched with the thalline of high viable count for producing as food
The purpose of the lyophilized powder of product additive, has been respectively adopted different more suitable for the culture of lactobacillus rhamnosuss purification and strain fermentation
In basal medium and the Optimal Medium of thalline different growth phases, the component raw material that culture medium adopts is food stage, and
With the addition of somatomedin oligomeric isomaltose in the medium.
2nd, fermented using the centrifugation frozen strain of bacterium mud, controlled in sweat by stream plus food stage NaOH solution
PH is constant, takes classification amplification culture mode to promote thalline quantity quickly to increase, obtains substantial amounts of thalline in the short time, and
Strain is stable, and variation, degeneration are few.
3rd, lactobacillus rhamnosuss bacterium mud is separated and collected using tube centrifuge, bacterium mud yield can reach more than 1.5%, and
The rotary drum of tube centrifuge can use steam sterilization, effectively prevent microbiological contamination in centrifugal process.
4th, producing active bacteria formulation using Freeze Drying Technique has the advantages that many projecting：1. due to being dried at low temperature, micro-
Biology will not lose biological activity；2., during cold drying, the growth of microorganism and the effect of enzyme almost cannot be carried out, energy
Best keep being frozen the original character of dry；3. after being dried, volume, shape are basically unchanged；4. being frozen dry productss is in sponge
Shape, not drying shrinkage, thus big with water engaging surface during dissolving, original character can be recovered rapidly；5. 95%-99% in material can be removed
Moisture, extend product storage life.6. adopt thermograde to reduce and go up, reduce the damage to thalline.
5th, it is to avoid causing the damage of bacterium cell membrane in freezing dry process, add freeze drying protectant, in freeze drying protectant
Middle defatted milk powder mainly plays protective layer effect in cell surface；Glycerol and sodium glutamate can penetrate cell interior, can improve jelly
Speed and permeability of the membrane that during dry, ice crystal generates；Trehalose forms a kind of glassy state in freezing dry process, in glass
Under glass state, viscosity is high, thus coefficient of molecular diffusion is very low, makes macromolecular substances retard motion, and the permeability of cell wall reduces,
Thus playing a protective role；Maltodextrin is conducive to improving the bacterium during the thalline survival rate after lyophilization and storage
Body survival rate, can also improve the instant capacity of mycopowder simultaneously；Tween 80 can prevent protein denaturation as surfactant, during freezing
The formation at ice-water interface can cause protein surface induced denaturation, and surfactant can make protein solution reduction in surface tension,
Reduce the driving force that ice-water interfacial protein matter absorbs and assembles.In compounding protective agent system, every kind of protective agent composition is dry in freezing
All play respective effect during dry, there is synergism each other simultaneously again.
6th, the defatted milk powder in freeze drying protectant takes independent sterilization method, adequately protect its active ingredient not by peracid,
Cross alkali or high temperature, preferably can play protective effect in mycopowder freeze-drying process, improve freeze drying viable microorganism rate；And it is obtained
Lyophilized powder color shallower, beneficial to appetite stimulator.
7th, the technology of the present invention prepares lactobacillus rhamnosuss lyophilized powder, and viable count is up to 1011More than CFU/g.
Brief description
Fig. 1 is the survival rate comparison diagram that lyophilized powder is obtained using different formulations freeze drying protectant.
Fig. 2 is the preparation technology flow chart of lactobacillus rhamnosuss lyophilized powder.
Specific embodiment
A kind of preparation method of lactobacillus rhamnosuss lyophilized powder, as shown in Fig. 2 comprise the steps：
1) prepare fermentation strain
1.1) streak culture：After lactobacillus rhamnosuss mycopowder is diluted with sterilized water, four rides in MC culture medium, line
After end, MC culture medium plate is inverted, 37 DEG C of incubator constant temperature culture 48～72 hours；
1.2) one-level purification culture：On picking plate, the larger single bacterium colony of molten calcium circle is seeded to equipped with the training of 5ml MRS liquid
In the test tube of foster base, test tube seals, and 37 DEG C of incubator constant temperature quiescent culture 17～24 hours obtain one-level purification bacteria suspension；
1.3) two grades of purification cultures：By 3～5% inoculum concentrations, by cultured one-level bacterial suspension inoculation to equipped with MRS liquid
In the triangular flask of culture medium, culture medium dress liquid amasss and accounts for the 40% of triangular flask volume, and triangular flask seals, and 37 DEG C of incubator constant temperature are quiet
Put culture 17～24 hours, obtain two grades of purification bacteria suspensions；
1.4) bacterium mud preparation and preservation：Two grades of purification bacteria suspensions that two grades of purification cultures are obtained are centrifuged,
12000rpm obtains bacterium mud after being centrifuged 10 minutes, and bacterium mud is dissolved in mixing of MRS fluid medium and 50% glycerite composition
Close in liquid, obtain bacterium mud mixed liquor；Bacterium mud mixed liquor is divided in sterilized cryopreservation tube, seal film phonograph seal, frozen in -40
In～-80 DEG C of cryogenic refrigerators, obtain lactobacillus rhamnosuss bacterium mud cryopreservation tube, standby；Described MRS fluid medium is molten with 50% glycerol
The volume ratio of liquid is 1:The first two grade of purification bacteria suspension of the mixed liquor of 1, MRS fluid medium and 50% glycerite composition and centrifugation
Volume ratio be 1:10.
Note：Step 1) operation all must aseptic operating platform and aseptic in execute；
2) strain fermentation
2.1) frozen bacterium mud recovery：Take the lactobacillus rhamnosuss bacterium mud cryopreservation tube being stored in cryogenic refrigerator, be immediately placed in 37
Carry out strain recovery, 30～45s in DEG C water-bath, all melt to frozen liquid in pipe；
2.2) strain one-level culture：By the bacterium mud recovered mixed liquor 1～2ml direct inoculation to equipped with the culture of 10ml basis
In the 50ml triangular flask of base, triangular flask seals, 37 DEG C of incubator constant temperature quiescent culture 17～24 hours；
2.3) two grades of cultures of strain：According to 2～6% inoculum concentrations, by the bacterial suspension inoculation of one-level culture end to equipped with base
In the triangular flask of basal culture medium, basal medium dress liquid amasss and accounts for the 40% of triangular flask volume, and triangular flask seals, 37 DEG C of incubators
Constant temperature quiescent culture 17～24 hours；
2.4) strain third stage culture：According to 2～6% inoculum concentrations, by the bacterial suspension inoculation of two grades of culture end to equipped with base
In the fermentation tank of basal culture medium, basal medium dress liquid amasss and accounts for the 60% of fermenter volume, opens fermentation tank stirring paddle, rotating speed
For 100rpm, ventilation is 0,37 DEG C of constant temperature culture 8～15 hours；
2.5) strain level Four fermentation：According to 2～6% inoculum concentrations, the bacterial suspension inoculation that third stage culture is terminated is to equipped with excellent
Change in the fermentation tank of culture medium, Optimal Medium dress liquid amasss and accounts for the 60% of fermenter volume, opens fermentation tank stirring paddle, rotating speed
For 100rpm, ventilation is 0,37 DEG C of constant temperature culture, after fermentation starts 3 hours, is tieed up by stream plus food stage NaOH solution
The permanent pH holding bacterium solution is 5.50～6.00, and cultivation cycle 8～15 hours, until monitor bacterium solution OD600Value stops increasing, immediately
Stop fermentation；
Wherein basal medium formulation and preparation method：Yeast protein peptone 6.0～10.0g/L, glucose 19.0～
25.0g/L, yeast extract 2.0～4.0g/L, sodium acetate 4.5～6.0g/L, magnesium sulfate 0.4～0.6g/L, Tween 80 0.5
～0.75g/L, oligomeric isomaltose 1.0～2.5g/L, potassium dihydrogen phosphate 1.5～3.0g/L, balance of sterilized water, pH value 6.20
～6.80；According to formula proportion weighing, heating for dissolving, if the pH value of culture medium is less than 6.20, use 1mol/L food stage
NaOH solution adjusts Medium's PH Value to sterilize at 6.20～6.80,115 DEG C 30min；
Optimization culture based formulas and preparation method：Yeast protein peptone 11.0～15.0g/L, glucose 25.0～30.0g/L,
Yeast extract 6.0～8.0g/L, sodium acetate 2.0～3.0g/L, magnesium sulfate 0.10～0.15g/L, Tween 80 0.5～
0.75g/L, oligomeric isomaltose 1.0～2.5g/L, potassium dihydrogen phosphate 1.5～3.0g/L, balance of sterilized water, pH value 6.20～
6.80；According to formula proportion weighing, heating for dissolving, if the pH value of culture medium is less than 6.20, use 1mol/L food stage NaOH
Solution adjusts Medium's PH Value to sterilize at 6.20～6.80,115 DEG C 30min；
Note：Nutrient media components raw material is food stage.
3) preparation of lyophilized powder
3.1) fermentation liquid centrifugation：After level Four fermentation ends, fermentation tank pot temperature is turned down, when in tank, broth temperature is low
Can prepare to be centrifuged when 20 DEG C, centrifugation apparatus adopt tube centrifuge, carry out steam sterilization 30min to rotary drum before centrifugation, turn
Speed is 10000～15000rpm, after centrifugation terminates, collects the bacterium mud in rotary drum；
3.2) freeze drying protectant preparation：
Frozen-dried protective agent prescription：Defatted milk powder 60.0～80.0g/L, maltodextrin 30.0～45.0g/L, trehalose
100.0～300.0g/L, Tween 80 0.5～0.75g/L, glycerol 8.0～15.0g/L, sodium glutamate 0.8～1.0g/L, surplus
For sterilized water, pH value is 6.80～7.50；
Freeze drying protectant preparation method：Accurately weigh defatted milk powder by formula proportion, take the sterilized water of 2/3 volume in formula
For being completely dissolved defatted milk powder, 115 DEG C of sterilizing 30min, obtain solution A；Other components weigh according to formula proportion, mixing, use
The aseptic water dissolution of remaining 1/3 volume in formula, 115 DEG C of sterilizing 30min, obtain solution B；It is sterilized separately end to place to room temperature
Afterwards, in aseptic, solution A and solution B mix homogeneously are obtained freeze drying protectant, if the pH value of freeze drying protectant is less than 6.80,
1mol/L food stage NaOH solution is then used to adjust pH value to 6.80～7.50, by the freeze drying protectant preparing cold preservation in -4 DEG C of refrigerators
Interior standby；
3.3) lyophilization：Bacterium mud is collected after finishing, according to bacterium mud：Freeze drying protectant=1:1～2 volume ratio, to bacterium
In mud add freeze drying protectant, and stir, mix homogeneously, be dispensed into freeze dryer pallet, then freeze dryer pallet put into lyophilizing
Carry out the lyophilizing of mycopowder in machine, freezer dryer temperature arranges (being shown in Table 1) by section in time, vacuum 0～1.0Pa, continue
Lyophilizing 57～72 hours；
Table 1 freezer dryer temperature setting table
| Section | 1 | 2 | 3 | 4 | 5 | 6 |
| Run time/h | 3 | 40～55 | 4 | 4 | 2 | 4 |
| End temp/DEG C | -25 | -25 | -10 | 10 | 25 | 30 |
13) collect lyophilized powder, pack after being pulverized by prescription.
Note：2) 3) operation is both needed to carry out in 100,000 grades of cleaning shops.
A kind of preparation method of lactobacillus rhamnosuss lyophilized powder of embodiment 1
Comprise the steps：
1) preparation of fermentation strain
1.1) streak culture：After lactobacillus rhamnosuss mycopowder is diluted with sterilized water, four rides in MC culture medium, line
After end, plate is inverted, 37 DEG C of incubator constant temperature culture 60 hours；
1.2) one-level purification culture：On picking plate, the larger single bacterium colony of molten calcium circle is seeded to equipped with the training of 5ml MRS liquid
In the test tube of foster base, test tube seals, 37 DEG C of incubator constant temperature quiescent culture 20 hours；
1.3) two grades of purification cultures：By 3% inoculum concentration, by cultured one-level bacterial suspension inoculation to equipped with 100ml MRS
In the 250ml triangular flask of fluid medium, triangular flask seals, 37 DEG C of incubator constant temperature quiescent culture 20 hours；
1.4) bacterium mud preparation and preservation：The 100ml bacteria suspension that the culture of two grades of purification is obtained is centrifuged, 12000rpm from
The heart obtained bacterium mud after 10 minutes, bacterium mud is dissolved in 10ml mixed liquor (mixed liquor by 5ml MRS fluid medium with
5ml50% glycerite mix homogeneously is formulated) in, bacterium mud mixed liquor is divided in sterilized 1ml cryopreservation tube, sealing
Film phonograph seal, frozen in -80 DEG C of cryogenic refrigerators, standby；
Note：1.1) 1.4) operation all must aseptic operating platform and aseptic in execute；
2) strain fermentation
2.1) frozen bacterium mud recovery：Take the lactobacillus rhamnosuss bacterium mud cryopreservation tube being stored in cryogenic refrigerator, be immediately placed in 37
Carry out strain recovery, 30～45s in DEG C water-bath, all melt to frozen liquid in pipe；
2.2) strain one-level culture：By the 1ml having recovered bacterium mud mixed liquor direct inoculation to equipped with 10ml basal medium
50ml triangular flask in, triangular flask seals, 37 DEG C of incubator constant temperature quiescent culture 20 hours；
2.3) two grades of cultures of strain：According to 2% inoculum concentration, by the bacterial suspension inoculation of one-level culture end to equipped with 100ml
In the 250ml triangular flask of basal medium, inoculate 4 bottles by this, triangular flask seals, 37 DEG C of incubator constant temperature quiescent culture 20 are little
When；
2.4) strain third stage culture：According to 2% inoculum concentration, by the bacterial suspension inoculation of two grades of culture end to equipped with 18L base
In the 30L fermentation tank of basal culture medium, open fermentation tank stirring paddle, rotating speed is 100rpm, ventilation is 0,37 DEG C of constant temperature culture 12
Hour；
2.5) strain level Four fermentation：According to 5% inoculum concentration, the bacterial suspension inoculation that third stage culture is terminated is extremely excellent equipped with 300L
Change in the 500L fermentation tank of culture medium, open fermentation tank stirring paddle, rotating speed is 100rpm, ventilation is 0,37 DEG C of constant temperature culture,
After fermentation starts 3 hours, maintaining constant pH by stream plus food stage NaOH solution is 5.60 ± 0.1, cultivates to 11 hours
When, monitor bacterium solution OD600Value stops increasing, and stops fermentation immediately；
Wherein, basal medium composition and preparation：Yeast protein peptone 8.0g/L, glucose 25.0g/L, yeast extract
3.0g/L, sodium acetate 6.0g/L, magnesium sulfate 0.5g/L, Tween 80 0.5g/L, oligomeric isomaltose 1.0g/L, potassium dihydrogen phosphate
3.0g/L, balance of sterilized water, pH value 6.20；According to formula proportion weighing, heating for dissolving, with 1mol/L food stage NaOH solution
Medium's PH Value is adjusted to sterilize at 6.20,115 DEG C 30min；
Optimization culture based formulas：Yeast protein peptone 14.0g/L, glucose 30.0g/L, yeast extract 6.0g/L, acetic acid
Sodium 3.0g/L, magnesium sulfate 0.10g/L, Tween 80 0.5g/L, oligomeric isomaltose 2.0g/L, potassium dihydrogen phosphate 3.0g/L, surplus
For sterilized water, pH value 6.80；According to formula proportion weighing, heating for dissolving, adjust medium pH with 1mol/L food stage NaOH solution
It is worth at 6.80,115 DEG C sterilizing 30min；
Note：2.1) 2.5) operation is both needed to carry out in 100,000 grades of cleaning shops.
3) preparation of lyophilized powder
3.1) fermentation liquid centrifugation：After level Four fermentation ends, fermentation tank pot temperature is turned down, when in tank, broth temperature is low
Prepare centrifugation when 20 DEG C, centrifugation apparatus adopt tube centrifuge, carry out steam sterilization 30min to rotary drum, rotating speed is before centrifugation
12000rpm, after centrifugation terminates, collects bacterium mud 5.55L in rotary drum, and bacterium mud yield is 1.85%；
3.2) prepare 6L freeze drying protectant：Weigh defatted milk powder 360.0g, be dissolved completely in 2974L sterilized water, 115 DEG C
Sterilizing 30min；Weigh maltodextrin 210.0g, trehalose 900.0g, Tween 80 3.0g, glycerol 60.0g, sodium glutamate 6.0g,
It is dissolved completely in after mixing in 1487L sterilized water, 115 DEG C of sterilizing 30min；It is sterilized separately end to place to room temperature, aseptic
Interior by two solution mix homogeneously, then use 1mol/L food stage NaOH solution to adjust pH value to 7.00, the frozen-dried protective that will be obtained
Agent cold preservation is standby in -4 DEG C of refrigerators；
3.3) lyophilization：Bacterium mud is collected after finishing, according to bacterium mud：Freeze drying protectant=1:1 volume ratio, adds lyophilizing
Protective agent, and stir, mix homogeneously, be dispensed into freeze dryer pallet, then freeze dryer pallet put into and in freeze dryer, carry out mycopowder
Lyophilizing, freezer dryer temperature setting is shown in Table 2, vacuum 0～1.0Pa, continues lyophilizing 62 hours；
Table 2 freezer dryer temperature setting table
| Section | 1 | 2 | 3 | 4 | 5 | 6 |
| Run time/h | 3 | 45 | 4 | 4 | 2 | 4 |
| End temp/DEG C | -25 | -25 | -10 | 10 | 25 | 30 |
3.4) collect lyophilized powder, detection viable count is 5.0 × 1011CFU/g；
3.5) pack after being pulverized by prescription.
Note：3.1) 3.5) operation is both needed to carry out in 100,000 grades of cleaning shops.
Embodiment 2 contrast test
Freeze drying protectant formulation selection is tested
Formula A：Defatted milk powder 160g/L, maltodextrin 100g/L, trehalose 100g/L, glutamic acid 10g/L, ascorbic acid
20g/L, glycerol 20g/L；
Formula B：Whey powder 100g/L, maltodextrin 50g/L, ascorbic acid 20g/L, glycerol 50g/L；
Formula C：Fructus hordei germinatus leaching powder 150g/L, glycerol 20g/L, inulin 70g/L；
Formula D (embodiment 1 formula)：Defatted milk powder 60g/L, maltodextrin 35g/L, trehalose 150g/L, tween
800.5g/L, glycerol 10g/L, sodium glutamate 1g/L, pH value 7.00.
Prepare freeze drying protectant respectively according to above-mentioned tetra- kinds of formula of A, B, C, D, be obtained under the conditions of identical lyophilization
Lyophilized powder A, B, C, D, store 12 months under the conditions of lyophilized powder A, B, C, D are positioned over -20 DEG C, store before and after viable bacteria by detection
Number calculates its survival rate, sees Fig. 1.Shown by Fig. 1：The lactobacillus rhamnosuss being obtained using the freeze drying protectant of embodiment 1 formula
Lyophilized powder, after storing 12 months under the conditions of -20 DEG C, thalline survival rate is far above other 3 kinds of freeze drying protectants.
Claims (9)
1. a kind of preparation method of lactobacillus rhamnosuss lyophilized powder is it is characterised in that comprise the steps：
1) strain of preparation fermentation
1.1) streak culture：After lactobacillus rhamnosuss mycopowder is diluted with sterilized water, sectional streak in MC culture medium, 37 DEG C of cultures
Case constant temperature culture 48～72 hours；
1.2) one-level purification culture：Single bacterium colony on picking MC culture medium plate, is seeded to the test tube equipped with MRS fluid medium
In, sealing, 37 DEG C of incubator constant temperature quiescent culture 17～24 hours, obtain one-level purification bacteria suspension；
1.3) two grades of purification cultures：By 3～5% inoculum concentrations, by cultured one-level bacterial suspension inoculation to equipped with MRS liquid culture
In the triangular flask of base, sealing, 37 DEG C of incubator constant temperature quiescent culture 17～24 hours, obtain two grades of purification bacteria suspensions；
1.4) bacterium mud preparation and preservation：Two grades of purification bacteria suspensions are centrifuged, 12000rpm centrifugation obtains bacterium mud in 10 minutes, will
Bacterium mud is dissolved in MRS fluid medium and the mixed liquor of 50% glycerite composition, obtains bacterium mud mixed liquor, by bacterium mud mixed liquor
Be divided in sterilized cryopreservation tube, seal film phonograph seal, frozen in -40～-80 DEG C of cryogenic refrigerators, obtain lactobacillus rhamnosuss bacterium
Mud cryopreservation tube, standby；
2) strain fermentation
2.1) frozen bacterium mud recovery：Take the lactobacillus rhamnosuss bacterium mud cryopreservation tube being stored in cryogenic refrigerator, be immediately placed in 37 DEG C of water
Carry out strain recovery in bath, all melt to frozen liquid in pipe；
2.2) strain one-level culture：By the bacterium mud recovered mixed liquor 1～2ml direct inoculation to the triangle equipped with basal medium
In bottle, triangular flask seals, 37 DEG C of incubator constant temperature quiescent culture 17～24 hours；
2.3) two grades of cultures of strain：According to 2～6% inoculum concentrations, the bacterial suspension inoculation that one-level culture is terminated is trained to equipped with basis
In the triangular flask of foster base, basal medium dress liquid amasss and accounts for the 40% of triangular flask volume, and triangular flask seals, 37 DEG C of incubator constant temperature
Quiescent culture 17～24 hours；
2.4) strain third stage culture：According to 2～6% inoculum concentrations, the bacterial suspension inoculation that two grades of cultures are terminated is trained to equipped with basis
In the fermentation tank of foster base, basal medium dress liquid amasss and accounts for the 60% of fermenter volume, opens fermentation tank stirring paddle, rotating speed is
100rpm, ventilation is 0,37 DEG C of constant temperature culture 8～15 hours；
2.5) strain level Four fermentation：According to 2～6% inoculum concentrations, the bacterial suspension inoculation that third stage culture is terminated is trained to equipped with optimizing
In the fermentation tank of foster base, Optimal Medium dress liquid amasss and accounts for the 60% of fermenter volume, opens fermentation tank stirring paddle, rotating speed is
100rpm, ventilation is 0,37 DEG C of constant temperature culture, after fermentation starts 3 hours, is maintained by stream plus food stage NaOH solution
The permanent pH of bacterium solution is 5.50～6.00, cultivation cycle 8～15 hours, until monitoring bacterium solution OD600Value stops increasing, and stops immediately
Only ferment；
3) preparation of lyophilized powder
3.1) fermentation liquid centrifugation：After fermentation ends, fermentation tank pot temperature is turned down, when in tank, broth temperature is less than 20 DEG C
When, prepare to be centrifuged using centrifugation apparatus, after centrifugation terminates, collect the bacterium mud in centrifugation apparatus rotary drum；
3.2) lyophilization：Bacterium mud is collected after finishing, according to bacterium mud：Freeze drying protectant=1:1～2 volume ratio, in bacterium mud
Add freeze drying protectant, and stir, mix homogeneously, be dispensed into freezer dryer pallet, then freezer dryer pallet put into
Carry out the lyophilizing of mycopowder in freezer dryer, keep vacuum 0～1.0Pa, continue lyophilizing 57～72 hours；
3.3) collect lyophilized powder, pack after being pulverized by prescription.
2. preparation method as claimed in claim 1 is it is characterised in that step 1.4) described MRS fluid medium is sweet with 50%
The volume ratio of oil solution is 1:The body of bacteria suspension before the mixed liquor of 1, MRS fluid medium and 50% glycerite composition and centrifugation
Long-pending ratio is 1:10.
3. preparation method as claimed in claim 1 is it is characterised in that step 2) in basal medium preparation, formula：Yeast
Peptone 6.0～10.0g/L, glucose 19.0～25.0g/L, yeast extract 2.0～4.0g/L, sodium acetate 4.5～6.0g/
L, magnesium sulfate 0.4～0.6g/L, Tween 80 0.5～0.75g/L, oligomeric isomaltose 1.0～2.5g/L, potassium dihydrogen phosphate 1.5
～3.0g/L, balance of sterilized water, pH value 6.20～6.80；According to formula proportion weighing, heating for dissolving, if the pH of culture medium
Value is less than 6.20, then use 1mol/L food stage NaOH solution to adjust Medium's PH Value to sterilize at 6.20～6.80,115 DEG C 30min.
4. preparation method as claimed in claim 1 is it is characterised in that step 2) in Optimal Medium preparation, formula：Yeast
Peptone 11.0～15.0g/L, glucose 25.0～30.0g/L, yeast extract 6.0～8.0g/L, sodium acetate 2.0～
3.0g/L, magnesium sulfate 0.10～0.15g/L, Tween 80 0.5～0.75g/L, oligomeric isomaltose 1.0～2.5g/L, di(2-ethylhexyl)phosphate
Hydrogen potassium 1.5～3.0g/L, balance of sterilized water, pH value 6.20～6.80；According to formula proportion weighing, heating for dissolving, if training
The pH value of foster base is less than 6.20, then use 1mol/L food stage NaOH solution to adjust Medium's PH Value to go out at 6.20～6.80,115 DEG C
Bacterium 30min.
5. preparation method as claimed in claim 1 is it is characterised in that step 3.1) described centrifugation apparatus adopt tubular type to be centrifuged
Machine, carries out steam sterilization 30min to rotary drum before centrifugation, and rotating speed is 10000～15000rpm.
6. preparation method as claimed in claim 1 is it is characterised in that step 3.2) in frozen-dried protective agent prescription be：Defatted milk
Powder 60.0～80.0g/L, maltodextrin 30.0～45.0g/L, trehalose 100.0～300.0g/L, Tween 80 0.5～
0.75g/L, glycerol 8.0～15.0g/L, sodium glutamate 0.8～1.0g/L, balance of sterilized water, pH value is 6.80～7.50.
7. preparation method as claimed in claim 6 is it is characterised in that the preparation method of described freeze drying protectant is：
Accurately weigh defatted milk powder by formula proportion, take part sterilized water in formula to be used for being completely dissolved defatted milk powder, in 115 DEG C
Lower sterilizing 30min, obtains solution A；
Other components weigh according to formula proportion, mixing, with the aseptic water dissolution of remainder in formula, sterilize at 115 DEG C
30min, obtains solution B；
Sterilizing terminates to place to room temperature, and in aseptic, solution A and solution B mix homogeneously being obtained pH value is 6.80～7.50
Freeze drying protectant, the freeze drying protectant preparing cold preservation is standby in -4 DEG C of refrigerators.
If 8. preparation method as claimed in claim 7, it is characterised in that the pH value of freeze drying protectant is less than 6.80, is used
1mol/L food stage NaOH solution adjusts pH value to 6.80～7.50.
9. preparation method as claimed in claim 1 is it is characterised in that step 3.2) in mycopowder jelly is carried out using freezer dryer
When dry, the temperature of freezer dryer and freeze-drying time press section setting, specifically, the 1st section：Lyophilizing duration 3h, temperature sets
For -25 DEG C；2nd section：Lyophilizing duration 40～55h, temperature is set as -25 DEG C；3rd section：Lyophilizing duration 4h, temperature sets
For -10 DEG C；4th section：Lyophilizing duration 4h, temperature is set as 10 DEG C；5th section：Lyophilizing duration 2h, temperature sets 25 DEG C；6th
Section：Lyophilizing duration 4h, temperature is set as 30 DEG C.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701642A (en) * | 2017-03-01 | 2017-05-24 | 汉臣氏（沈阳）儿童制品有限公司 | Preparation method of lactobacillus acidophilus freeze-dried powder |
| CN106983134A (en) * | 2017-04-10 | 2017-07-28 | 北华大学 | The preparation method of northern Chinese caterpillar Fungus bacterium freeze-dried powder |
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| CN111961631A (en) * | 2020-09-04 | 2020-11-20 | 国科智盛(沁阳市)生物科技有限公司 | Fermentation production process of lactobacillus fermentum |
| CN113025514A (en) * | 2019-12-25 | 2021-06-25 | 百岳特生物技术（上海）有限公司 | Particle structure with high-concentration live bacteria and preparation method thereof |
| CN115093993A (en) * | 2022-06-08 | 2022-09-23 | 微康益生菌(苏州)股份有限公司 | High-temperature stable lactobacillus rhamnosus microbial inoculum and preparation method and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101580809A (en) * | 2009-06-16 | 2009-11-18 | 华南理工大学 | Method for preparing direct vat set Lactobacillus casei subsp. rhamnosus freeze-dried powder |
| JP2013192490A (en) * | 2012-03-19 | 2013-09-30 | Research Institute Of Environment Agriculture & Fisheries Osaka Prefecture | Method for producing dried powder of microorganism |
-
2016
- 2016-10-12 CN CN201610891354.9A patent/CN106434463B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101580809A (en) * | 2009-06-16 | 2009-11-18 | 华南理工大学 | Method for preparing direct vat set Lactobacillus casei subsp. rhamnosus freeze-dried powder |
| JP2013192490A (en) * | 2012-03-19 | 2013-09-30 | Research Institute Of Environment Agriculture & Fisheries Osaka Prefecture | Method for producing dried powder of microorganism |
Non-Patent Citations (2)
| Title |
|---|
| 刘璐 等: "低聚异麦芽糖对干酪中鼠李糖乳杆菌促生长的研究", 《中国乳品工业》 * |
| 回晶 等: "鼠李糖乳杆菌培养基的优化", 《辽宁大学学报》 * |
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