CN107287121A - A kind of lactic acid bacteria freeze drying protective agent and preparation method thereof and application method - Google Patents
A kind of lactic acid bacteria freeze drying protective agent and preparation method thereof and application method Download PDFInfo
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- CN107287121A CN107287121A CN201710683736.7A CN201710683736A CN107287121A CN 107287121 A CN107287121 A CN 107287121A CN 201710683736 A CN201710683736 A CN 201710683736A CN 107287121 A CN107287121 A CN 107287121A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
Abstract
A kind of lactic acid bacteria freeze drying protective agent and preparation method thereof and application method; its active ingredient is made up of trehalose, skimmed milk power, compound addO-on therapy and seepage stability component; compound addO-on therapy is that glycerine and sorbierite are mixed, and seepage stability component is mixed by L cysteines, vitamin C and sodium acetate.The present invention is by adding seepage stability component and compound addO-on therapy; when both are added in freeze drying protectant; cell interior can be penetrated into; the bound water molecule in system; generation aquation; make the viscosity increase of system, and then slow down the ice crystal growth rate of system in freeze-drying process, reduce the ratio that water changes into ice；Meanwhile, protective agent enters cell interior, improves cell interior pressure, reduces cell dehydration degree and speed caused by extracellular icing, it is suppressed that the growth of intracellular ice crystal, so as to reduce the injury to cell.
Description
Technical field
The present invention relates to microorganism fungus kind fermentation arts, specifically a kind of lactic acid bacteria freeze drying protective agent and its preparation
Method and application method.
Background technology
Lactic acid bacteria has different physiological roles, including treatment lactose intolerance；Promote the battalion such as protein, monose and calcium, magnesium
Support the absorption of material；Secrete vitamin B；Improve human body intestinal canal function, suppress the breeding of spoilage organisms, reducing blood lipid, hypotensive；It is immune
Regulation and the effect of antitumor, pre- anti-cancer etc..With developing rapidly for China's dairy products industry, market is to lactic acid bacteria product
Demand increases.The quality of lactic acid bacteria fermenting agent is paid close attention to by researcher.The preservation of lactic acid bacteria and the system of lactic acid bacteria fermenting agent
Standby is that lactic acid bacteria various functions are utilized.
Current lactic acid bacteria fermenting agent can be prepared using methods such as vacuum drying, spray drying, freeze-dryings.Many researchers
It was found that freeze-drying has the advantages that other method is incomparable, it is to prepare and preserve biomaterial most efficient method, and
To being increasingly widely applied.Lactobacillus cell can be damaged in freeze-drying process by certain damage, including mechanical damage, solute
Wound, cell membrane damage, the damage of cell metabolism adjustment effect, so as to cause cell death.
Research is found, appropriate protective agent is added before lyophilized, thus it is possible to vary physics, chemical ring when thalline is freeze-dried
Border, mitigates or prevents the infringement of freeze-drying or rehydration to cell, original various physio-biochemical characteristics and life are kept as far as possible
Thing activity, so as to improve the survival rate of cell.
The content of the invention
It is of the invention it is an object of the invention to provide a kind of lactic acid bacteria freeze drying protective agent and preparation method thereof and application method
Method freeze-drying effect is good, and in the freeze-drying of lactic acid bacteria, can greatly improve the survival rate of thalline and in storage
Viable detection during Tibetan.
The present invention be realize technical scheme that above-mentioned technical purpose used for：A kind of lactic acid bacteria freeze drying protective agent, according to
Mass fraction, its active ingredient by 10-18 parts of trehalose, 10-18 parts of skimmed milk power, 2-6 parts of compound addO-on therapy and
1.2-6 parts of seepage stability component composition, wherein, compound addO-on therapy is glycerine and sorbierite with 1-6:1 weight ratio mixing,
Seepage stability component is by Cys, vitamin C and sodium acetate with 1-6:1-2:1-2 weight is than mixing.
A preferred embodiment of the present invention is, the polyethylene in the compound addO-on therapy also containing glycerin weight 10-12%
Pyrrolidones.
A preferred embodiment of the present invention is, the mannitol in the compound addO-on therapy also containing glycerin weight 8-12%.
A preferred embodiment of the present invention is, the sulphur in the seepage stability component also containing Cys weight 4-8%
Sour manganese and Cys weight 10-15% asparatate.
The above-mentioned protectant preparation method of lactic acid bacteria freeze drying, weighs each component, then by sea respectively according to above-mentioned ratio
Algae sugar, skimmed milk power, compound addO-on therapy are dissolved in 52-74 part of water, and the 20min that sterilizes under conditions of 105 DEG C, are finally added
Enter load weighted seepage stability component, be well mixed and product is made.
The method for preparing freeze dried lactobcillus fermenting preparation using above-mentioned lactic acid bacteria freeze drying protective agent is to activate lactic acid bacteria, train
Support, then centrifugal enrichment obtains zymotic fluid, added into zymotic fluid after the freeze drying protectant of the above-mentioned preparation isometric with it,
It is well mixed under aseptic condition, and 10-15h is stood to strengthen the frost resistance of somatic cells in 0-5 DEG C of gnotobasis, finally
By the lyophilized i.e. obtained product of above-mentioned mixed liquor.
The mixed liquor is lyophilized to be referred to, by the mixed liquor Jing Guo stand at low temperature in -50 DEG C, the bar that vacuum is 0.2mbar
Vacuum freeze drying is solid-state under part.
The lactic acid bacteria activation, culture refer to that the inoculating lactic acid bacterium in MRS agar mediums recycles liquid MRS cultures
Base is cultivated in 37 DEG C of constant incubators and centrifugation thalline is received after 16 h, and gained thalline is washed twice with the PBS that pH is 7.4 and sent out
Zymotic fluid.
In the present invention, polysaccharide, polyvinylpyrrolidone, sugar alcohol etc. being combined into as the freeze drying protectant of lactic acid bacteria
Point, wherein research thinks that the protecting effect of trehalose is particularly evident.Trehalose is a kind of irreducibility disaccharide of stabilization, due to tool
There are multiple hydroxyls, can be tied with phage surface free radical, it is to avoid thalline exposes in media as well, can also be formed with protein
Hydrogen bond is to replace water, it is ensured that the stability of protein；On the other hand easy bound water molecule in the solution, occurs aquation, subtracts
Lack the content of free water and added the stickiness of solution, so as to slow down the growth course of nucleus, made the ice crystal to be formed relatively fine,
To reach the purpose of protection cell；
Skimmed milk power is one of conventional freeze drying protectant of most of Dairy Enterprises, because it is capable of the composition of stabilizing cell membrane,
Be conducive to rehydration and form one layer of cells diaphragm, the skimmed milk power supplemented with extra freeze drying protectant can be improved in it
Protective effect；
One of creation point of the present invention is, glycerine and sorbierite and mannitol is compound as addO-on therapy, due to glycerine
There are multiple hydroxyls with sorbierite and mannitol, hydrone and the phosphorus in somatic cells membrane phospholipid are may replace in freeze-drying process
Acid groups or with bacterium protein polar group formation hydrogen bond, so as to protect the complete of cell membrane and albumen and structure and function
Property；
Another creation point of the present invention is, is added using the mixing of Cys, vitamin C and sodium acetate as seepage stability component
Enter into freeze drying protectant, these three compositions can penetrate into cell interior, the bound water molecule in system, occur hydration and make
With, make the viscosity increase of system, and then slow down the ice crystal growth rate of system in freeze-drying process, reduce water and change into ice
Ratio；Meanwhile, protective agent enters cell interior, improves cell interior pressure, reduces cell caused by extracellular icing and takes off
Water degree and speed, it is suppressed that the growth of intracellular ice crystal, so as to reduce the injury to cell；And the manganese sulfate additionally added and
Asparatate and original Cys, can play a part of extending bacterium powder storage stability.
Compared with prior art, the invention has the advantages that：
1）The present invention is compound as addO-on therapy using glycerine and sorbierite and mannitol, due to glycerine and sorbierite and sweet dew
Alcohol has multiple hydroxyls, may replace in freeze-drying process phosphate group in hydrone and somatic cells membrane phospholipid or with thalline egg
White matter polar group formation hydrogen bond, so as to protect the integrality of cell membrane and albumen and structure and function, improves lactic acid bacteria
Survival rate；
2）The present invention is added to freeze drying protectant using the mixing of Cys, vitamin C and sodium acetate as seepage stability component
In, these three compositions can penetrate into cell interior, the bound water molecule in system, occur aquation, make the viscosity of system
Increase, and then slow down the ice crystal growth rate of system in freeze-drying process, reduce the ratio that water changes into ice；Meanwhile, protection
Agent enters cell interior, improves cell interior pressure, reduces cell dehydration degree and speed caused by extracellular icing, suppression
The growth of intracellular ice crystal is made, so as to reduce the injury to cell；And the manganese sulfate and asparatate that additionally add and
Original Cys, can play a part of extending bacterium powder storage stability；
3）Prepared during freeze dried lactobcillus fermenting preparation, handled using stand at low temperature, Neng Gou great using the freeze drying protectant of the present invention
Amplitude strengthens the frost resistance of lactic acid bacteria somatic cells, so as to strengthen survival rate of the lactic acid bacteria in freeze-drying process, is obtained after freezing
Leavening normal temperature under can preserve 30 days, Viable detection reaches more than 75%, and performance is stable in preservation term.
Embodiment
To be easy to understand technological means, creation characteristic, reached purpose and the beneficial effect of the invention realized, under
Face combines embodiment, and the present invention is expanded on further.
Embodiment 1
A kind of lactic acid bacteria freeze drying protective agent, according to mass fraction, trehalose, 18 part of defatted milk of its active ingredient by 18 parts
The seepage stability component composition of powder, 6 parts of compound addO-on therapy and 6 parts, wherein, be combined addO-on therapy be glycerine and sorbierite with
1:1 weight is than mixing, and seepage stability component is by Cys, vitamin C and sodium acetate with 1: 2:2 weight is than mixed
Close；
The above-mentioned protectant preparation method of lactic acid bacteria freeze drying, each component is weighed according to above-mentioned ratio respectively, then by trehalose,
Skimmed milk power, compound addO-on therapy are dissolved in 52 parts of water, and the 20min that sterilizes under conditions of 105 DEG C, are eventually adding and are weighed
Seepage stability component, be well mixed i.e. be made product；
The method for preparing freeze dried lactobcillus fermenting preparation using above-mentioned lactic acid bacteria freeze drying protective agent is to activate lactic acid bacteria, cultivate, and
Centrifugal enrichment obtains zymotic fluid afterwards, is added into zymotic fluid after the freeze drying protectant of the above-mentioned preparation isometric with it, sterile
Under the conditions of be well mixed, and in 0 DEG C of gnotobasis stand 10h to strengthen the frost resistance of somatic cells, finally will it is above-mentioned mix
Close the lyophilized i.e. obtained product of liquid.
Above for the present invention basic embodiment, can more than on the basis of further improved, optimized and limited：
A kind of preferred scheme of the present embodiment is that the extra polyethylene containing glycerin weight 10% is gone back in the compound addO-on therapy
Pyrrolidones；
Another preferred scheme of the present embodiment is that additionally can also contain the sweet of glycerin weight 8% in the compound addO-on therapy
Reveal alcohol；
Another preferred scheme of the present embodiment is, the sulfuric acid in the seepage stability component also containing Cys weight 4%
The asparatate of manganese and Cys weight 10%；
Mixed liquor described in the present embodiment is lyophilized to be referred to, is in -50 DEG C, vacuum by the mixed liquor Jing Guo stand at low temperature
Vacuum freeze drying is solid-state under conditions of 0.2mbar；
The activation of lactic acid bacteria described in the present embodiment, culture refer to that the inoculating lactic acid bacterium in MRS agar mediums recycles liquid
MRS culture mediums are cultivated in 37 DEG C of constant incubators receives centrifugation thalline after 16 h, gained thalline is washed twice with pH for 7.4 PBS
Obtain zymotic fluid.
Embodiment 2
A kind of lactic acid bacteria freeze drying protective agent, according to mass fraction, trehalose, 10 part of defatted milk of its active ingredient by 10 parts
Powder, 2 parts of compound addO-on therapy and 1.2 parts of seepage stability component composition, wherein, it is glycerine and sorbierite to be combined addO-on therapy
With 6:1 weight is than mixing, and seepage stability component is by Cys, vitamin C and sodium acetate with 6:1:1.5 weight is than mixed
Close；
The above-mentioned protectant preparation method of lactic acid bacteria freeze drying, each component is weighed according to above-mentioned ratio respectively, then by trehalose,
Skimmed milk power, compound addO-on therapy are dissolved in 74 parts of water, and the 20min that sterilizes under conditions of 105 DEG C, are eventually adding and are weighed
Seepage stability component, be well mixed i.e. be made product；
The method for preparing freeze dried lactobcillus fermenting preparation using above-mentioned lactic acid bacteria freeze drying protective agent is to activate lactic acid bacteria, cultivate, and
Centrifugal enrichment obtains zymotic fluid afterwards, is added into zymotic fluid after the freeze drying protectant of the above-mentioned preparation isometric with it, sterile
Under the conditions of be well mixed, and in 5 DEG C of gnotobasis stand 15h to strengthen the frost resistance of somatic cells, finally will it is above-mentioned mix
Close the lyophilized i.e. obtained product of liquid.
Above for the present invention basic embodiment, can more than on the basis of further improved, optimized and limited：
A kind of preferred scheme of the present embodiment is that the extra polyethylene containing glycerin weight 12% is gone back in the compound addO-on therapy
Pyrrolidones；
Another preferred scheme of the present embodiment is that additionally can also contain glycerin weight 12% in the compound addO-on therapy
Mannitol；
Another preferred scheme of the present embodiment is, the sulfuric acid in the seepage stability component also containing Cys weight 8%
The asparatate of manganese and Cys weight 15%；
Mixed liquor described in the present embodiment is lyophilized to be referred to, is in -50 DEG C, vacuum by the mixed liquor Jing Guo stand at low temperature
Vacuum freeze drying is into solid-state under conditions of 0.2mbar；
The activation of lactic acid bacteria described in the present embodiment, culture refer to that the inoculating lactic acid bacterium in MRS agar mediums recycles liquid
MRS culture mediums are cultivated in 37 DEG C of constant incubators receives centrifugation thalline after 16 h, gained thalline is washed twice with pH for 7.4 PBS
Obtain zymotic fluid.
Embodiment 3
A kind of lactic acid bacteria freeze drying protective agent, according to mass fraction, trehalose, 14 part of defatted milk of its active ingredient by 14 parts
Powder, 4 parts of compound addO-on therapy and 3.6 parts of seepage stability component composition, wherein, it is glycerine and sorbierite to be combined addO-on therapy
With 3.5:1 weight is than mixing, and seepage stability component is by Cys, vitamin C and sodium acetate with 3.5:1.5:1 weight
Than mixing；
The above-mentioned protectant preparation method of lactic acid bacteria freeze drying, each component is weighed according to above-mentioned ratio respectively, then by trehalose,
Skimmed milk power, compound addO-on therapy are dissolved in 63 parts of water, and the 20min that sterilizes under conditions of 105 DEG C, are eventually adding and are weighed
Seepage stability component, be well mixed i.e. be made product；
The method for preparing freeze dried lactobcillus fermenting preparation using above-mentioned lactic acid bacteria freeze drying protective agent is to activate lactic acid bacteria, cultivate, and
Centrifugal enrichment obtains zymotic fluid afterwards, is added into zymotic fluid after the freeze drying protectant of the above-mentioned preparation isometric with it, sterile
Under the conditions of be well mixed, and in 3 DEG C of gnotobasis stand 13h to strengthen the frost resistance of somatic cells, finally will it is above-mentioned mix
Close the lyophilized i.e. obtained product of liquid.
Above for the present invention basic embodiment, can more than on the basis of further improved, optimized and limited：
A kind of preferred scheme of the present embodiment is that the extra polyethylene containing glycerin weight 11% is gone back in the compound addO-on therapy
Pyrrolidones；
Another preferred scheme of the present embodiment is that additionally can also contain glycerin weight 10% in the compound addO-on therapy
Mannitol；
Another preferred scheme of the present embodiment is, the sulfuric acid in the seepage stability component also containing Cys weight 6%
The asparatate of manganese and Cys weight 12.5%；
Mixed liquor described in the present embodiment is lyophilized to be referred to, is in -50 DEG C, vacuum by the mixed liquor Jing Guo stand at low temperature
Vacuum freeze drying is into solid-state under conditions of 0.2mbar；
The activation of lactic acid bacteria described in the present embodiment, culture refer to that the inoculating lactic acid bacterium in MRS agar mediums recycles liquid
MRS culture mediums are cultivated in 37 DEG C of constant incubators receives centrifugation thalline after 16 h, gained thalline is washed twice with pH for 7.4 PBS
Obtain zymotic fluid.
In order to verify the effect of freeze drying protectant of the present invention, spy makees following experiment and is compared checking：
Experiment material 1：Lactic acid bacteria freeze drying protective agent prepared by above example 3 is used as protective agent A；
Experiment material 2：Conventional freeze drying protectant is as protective agent B, and its composition is：12 portions of skimmed milk powers, 12 portions of sucrose, 1.5
Part sodium glutamate, 0.3 part of glycerine, 0.2 part of vitamin C；
Experiment material 3：The residual components that freeze drying protectant in embodiment 3 is removed after compound addO-on therapy are used as protective agent C；
Experiment material 4：The residual components that freeze drying protectant in embodiment 3 is removed after seepage stability component are used as protective agent D；
Experiment culture medium：MRS culture mediums, MRS agar mediums；
Experiment key instrument：German CHRIST vacuum freeze driers, the type biochemical cultivation cases of SPX-250BS- II；SW-CJ-
The single one side clean work station of IFD types, GI54DW type autoclaves, high speed freezing centrifuge；
First, lactic acid bacteria activation and cultural method：
The activation culture of lactic acid bacteria：By preservation of bacteria strain lactic acid bacteria in glycerine cryopreservation tube（Lactobacillus casei）With transfer needle in MRS
Rule in agar medium flat board, 37 DEG C of incubated 16 h choose single bacterium and fallen within and 16 h are cultivated in fluid nutrient medium, and liquid passes two
It is used for subsequent experimental after generation；
Lactobacillus casei after activation is inoculated into liquid MRS culture mediums with 2% inoculum concentration and is enlarged culture, in 37 DEG C of perseverances
Cultivated in warm incubator and centrifugation thalline is received after 16 h, gained thalline is washed twice with pH7.4 PBS, for follow-up test；
The MRS Liquid Culture based formulas cultivated used in Lactobacillus casei is as follows：The g/L of peptone 10.0, the g/L of beef extract 8.0
, the g/L of yeast extract 4.0, the g/L of glucose 20.0, Tween 80 1mL/L, the g/L of dipotassium hydrogen phosphate 2.0, sodium acetate trihydrate 5.0
G/L, the g/L of Triammonium citrate 2.0, the g/L of epsom salt 0.2, manganese sulfate 0.05 g/L, pH 6.2 ± 0.2；
MRS agar mediums are that the g/L of agar 15 is added on the basis of MRS fluid nutrient mediums.
2nd, experimental method is as follows：
(1) it is protectant to prepare
Above-mentioned protective agent A, protective agent B, protective agent C and protective agent D are dissolved by identical matched proportion density distilled water, 105 DEG C
Sterilize standby after 20 min；
(2) lactic acid bacterial count before freezing
Using bacterium colony colony counting method, i.e., under aseptic technique, take l mL bacterium solution, plus 9 mL sterile saline
It is made 1:10 uniform dilution, then 10 times of incremental dilutions are carried out successively, to appropriate dilution factor 10-6,10-7, l0-8, point
Do not take each μ L of diluted concentration 100 in solid culture ware, thalline is uniformly dispersed with painting glass rod, be inverted, put 37 DEG C of constant humidity of people
48 h are cultivated in incubator, the colony counts formed in each plate are recorded, according to extension rate, every milliliter of original sample are calculated
Contained total number of bacterial colonies in product, to represent viable count, each dilution factor do 3 it is parallel, be repeated 3 times；
(3) collection of thalline and protectant addition
Take 30 mL bacterium solutions to centrifuge 20 min in the rpm of centrifuge tube 4000 for having sterilized and having numbered, remove supernatant, add 30 mL
Twice of the PBS of sterilizing；Sterilized 30 mL protective agents A, protective agent B, protective agent C and guarantor are separately added into each pipe
Protect agent D to be resuspended, re-suspension liquid is sub-packed in 5 mL cillin bottles with l mL, and reference numeral；
(4) lactic acid bacteria freeze drying and survival rate are calculated
By the freeze drying protectant bacteria suspension dispensed after 12 h are stored during temperature is 4 DEG C of environment, uncap, be put into vacuum refrigeration
Drying machine, lyophilisation condition is：Vacuum 0.2mbar, -50 DEG C of cryogenic temperature, the h of freeze-drying time 24；Lid is compressed, taken out,
Obtain lactic acid bacteria freeze drying bacterium powder.
Lyophilized bacterium powder is reverted into lyophilized front volume with sterile saline, lyophilized rear breast is carried out with bacterium colony colony counting method
Sour bacterium counts.
Cell survival rate（%）The viable count of 1 mL sample liquids after=lyophilized rehydration（cfu/mL）/ freeze preceding 1 mL sample liquids
Viable count（cfu/mL）×100%.
3rd, experimental result
The cells in sample survival rate for adding protective agent A is 89.5%；
The cells in sample survival rate for adding protective agent B is 72.3%；
The cells in sample survival rate for adding protective agent C is 66.8%；
The cells in sample survival rate for adding protective agent D is 61.7%；
It follows that the freeze drying protectant of the present invention is compared to for protective agent common on the market, its effect is more preferable, and
Because protective agent A cell survival rate is much larger than protective agent C and protective agent D, that is to say, that at the same add compound addO-on therapy and
Seepage stability component can play composite synergistic effect, can further improve cell survival rate.
Principal character, general principle and the advantages of the present invention of the present invention has been shown and described above.Industry technology
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, the present invention can also have various change according to actual conditions
And improvement, these changes and improvements all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by appended
Claims and its equivalent thereof.
Claims (8)
1. a kind of lactic acid bacteria freeze drying protective agent, it is characterised in that：According to mass fraction, its active ingredient is by 10-18 parts of marine alga
Sugar, 10-18 parts of skimmed milk power, 2-6 parts of compound addO-on therapy and 1.2-6 parts of seepage stability component composition, wherein, it is combined
AddO-on therapy is glycerine and sorbierite with 1-6:1 weight than mixing, seepage stability component by Cys, vitamin C and
Sodium acetate is with 1-6:1-2:1-2 weight is than mixing.
2. a kind of lactic acid bacteria freeze drying protective agent according to claim 1, it is characterised in that：In the compound addO-on therapy also
Polyvinylpyrrolidone containing glycerin weight 10-12%.
3. a kind of lactic acid bacteria freeze drying protective agent according to claim 1, it is characterised in that：In the compound addO-on therapy also
Mannitol containing glycerin weight 8-12%.
4. a kind of lactic acid bacteria freeze drying protective agent according to claim 1, it is characterised in that：In the seepage stability component also
The asparatate of manganese sulfate containing Cys weight 4-8% and Cys weight 10-15%.
5. the protectant preparation method of a kind of lactic acid bacteria freeze drying according to claim 1, it is characterised in that：Will according to right
Ask the ratio described in 1 to weigh each component respectively, then trehalose, skimmed milk power, compound addO-on therapy are dissolved in 52-74 parts of water
In, and the 20min that sterilizes under conditions of 105 DEG C, load weighted seepage stability component is eventually adding, is well mixed and production is made
Product.
6. the method that the lactic acid bacteria freeze drying protective agent prepared using claim 5 prepares freeze dried lactobcillus fermenting preparation, its feature exists
In：Lactic acid bacteria is activated, cultivated, then centrifugal enrichment obtains zymotic fluid, the right isometric with it is added into zymotic fluid will
After the freeze drying protectant for asking 5 preparations, be aseptically well mixed, and in 0-5 DEG C of gnotobasis stand 10-15h with
Strengthen the frost resistance of somatic cells, finally by the lyophilized i.e. obtained product of above-mentioned mixed liquor.
7. the method that utilization lactic acid bacteria freeze drying protective agent according to claim 6 prepares freeze dried lactobcillus fermenting preparation, it is special
Levy and be, the mixed liquor is lyophilized to be referred to, by the mixed liquor Jing Guo stand at low temperature in -50 DEG C, the condition that vacuum is 0.2mbar
Lower vacuum freeze drying is solid-state.
8. the method that utilization lactic acid bacteria freeze drying protective agent according to claim 6 prepares freeze dried lactobcillus fermenting preparation, it is special
Levy and be, the lactic acid bacteria activation, culture refer to that the inoculating lactic acid bacterium in MRS agar mediums recycles liquid MRS cultures
Base is cultivated in 37 DEG C of constant incubators and centrifugation thalline is received after 16 h, and gained thalline is washed twice with the PBS that pH is 7.4 and sent out
Zymotic fluid.
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| CN115478017A (en) * | 2022-10-11 | 2022-12-16 | 微康益生菌(苏州)股份有限公司 | Cryoprotectant for improving activity of lactobacillus bulgaricus, freezing method and application |
| CN115478017B (en) * | 2022-10-11 | 2023-10-03 | 微康益生菌(苏州)股份有限公司 | Cryoprotectant for improving activity of lactobacillus bulgaricus, and freezing method and application thereof |
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