CN104431355B - A kind of preparation method of lactic acid bacterium particle capsule - Google Patents
A kind of preparation method of lactic acid bacterium particle capsule Download PDFInfo
- Publication number
- CN104431355B CN104431355B CN201410678385.7A CN201410678385A CN104431355B CN 104431355 B CN104431355 B CN 104431355B CN 201410678385 A CN201410678385 A CN 201410678385A CN 104431355 B CN104431355 B CN 104431355B
- Authority
- CN
- China
- Prior art keywords
- bacterium
- lactic acid
- grain
- wet
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention discloses a kind of preparation method of lactic acid bacterium particle capsule, including bacterial strain preparation：The bacterial strain domestication in the MRS fluid nutrient mediums containing mannitol by lactobacillus inoculum, the Best Times of results are the mid-early stage of growth stationary phase, the lactobacillus suspension for terminating culture is collected by centrifugation bacterium mud, bacterium mud is washed with PBS afterwards, the wet bacterium mud for obtaining lactic acid bacteria is collected by centrifugation again, antioxidant is added after adding sterilized water stirring, the bacterium core of lactic acid bacteria granulation is obtained；The granulation of bacterium core：Gained bacterium core is coated the wet bacterium powder for obtaining that gently pressure dissipates using ground floor coating agent；Second layer coating is carried out with second layer coating agent after first time granulation is carried out to wet bacterium powder, bacterium grain is dried to obtain lactic acid bacterium particle after second granulation；Capsule is packed.The present invention improves the tolerance of thalline using mannitol domestication, reduces the bacterium loss of activity in post processing；The lactic acid bacteria harvested in the mid-early stage of growth stationary phase can farthest preserve strain activity after microbial inoculum is prepared into.
Description
Technical field
The present invention relates to a kind of preparation method of lactic acid bacterium particle capsule, belong to lactic acid bacteria technique field.
Background technology
Lactic acid bacteria or its culture etc. have been developed that to be healthy food, medicine, food additives, feed addictive, herbage
The materials such as additives for ensiling, and, recent research indicate that after lactic acid bacteria living reaches enteron aisle, the enteron aisle of end user can be improved
Function；The activity for improving the product and the production cost for reducing the product have be applied to it is excellent in terms of the industries such as specific products
Point.
But, the high activity of lactic acid bacteria is influenceed by bacterial strain, incubation time, protective agent, preparation method etc., lactic acid bacteria
The production cost of microbial inoculum is influenceed by tolerance, apparatus for preparation, the energy consumption etc. of lactic acid bacteria, it is difficult to keeping lactic acid bacteria high activity
While maintenance goods low cost.Therefore, often high activity is obtained using the preparation technology of vacuum freeze drying all the time
Lactic acid bacteria, but there is high cost.In addition, the bacteria cake prepared under the conditions of vacuum freeze drying easily forms block, this meeting
The imperfect of bacteria cake coating is caused, this will cause lactic bacteria activity reduction and increase using difficulty.
Such as patent document 1:State Intellectual Property Office of the People's Republic of China awards in the invention of bulletin on the 21st of September in 2011
A kind of power lactobacillus micro-capsule of patent and its production and use (A of patent No. ZL 101496555), Authorization Notice No. is
CN101496555B, publication number 101496555.It is the manufacture of preservation lactic acid bacteria high activity in being prepared as the freezing of lactic acid bacteria agent
Method, discloses and protective agent skimmed milk power, trehalose, glycerine, sodium glutamate and maltodextrin is added in microbial inoculum, makes lactic acid bacteria
Survival rate improve.However, these protective agents need, by multiple addition steps, to which increase the difficulty of operation and the machine of microbiological contamination
Meeting.
Patent document 2:The invention mandate that State Intellectual Property Office of the People's Republic of China announced on April 23rd, 2014
Patent a kind of compound coating lactic acid bacteria product and preparation method (A of patent No. ZL 102987055), Authorization Notice No. is
CN102987055B.Also disclose and the method that method prepares lactic acid bacteria agent is coated with by compound, disclose the lactic acid bacteria in fermentation
By adding Arabic gum, sodium alginate, atoleine, calcium chloride etc. to be made lactic acid bacteria capsule in liquid, lactic acid bacteria capsule
Zymotic fluid enter again cornstarch of passing through, the microencapsulation of microcrystalline cellulose and through cornstarch, hydroxypropyl methyl cellulose, pectin,
The coating of carragheen, improves the tolerance of lactic acid bacteria.However, the coating agent that compound coating method is selected during preparing microbial inoculum
It is liquid, this can cause microbial inoculum to dry the increase of number of times and the increase of water consumption, this will increase the wind of lactic bacteria activity reduction
Danger and the waste of water resource, and, the price of coating agent is higher, and this will improve the production cost of lactic acid bacteria agent, on the other hand,
Compound coating method to be prepared and select fluid bed the granulation instrument of microbial inoculum more, this microbial inoculum yield can be caused low and bacterium grain indissoluble phenomenon.
Patent document 3:The invention mandate that State Intellectual Property Office of the People's Republic of China announced on June 20th, 2012
A kind of lactobacillus vaginal capsule of patent and preparation method thereof (A of patent No. ZL 101690734), Authorization Notice No. is
CN101690734B.The method that lactic acid bacteria agent is prepared by preparing lactic acid bacteria capsule is also disclosed, is disclosed in fermentation
By adding the excipient of calcium carbonate and medical starch in lactobacillus suspension, lactic acid bacteria is made through 37 DEG C of freeze-day with constant temperature or freeze-drying
Powder, then lactic acid bacteria medicinal powder is loaded into capsule with conventional method.However, the lactic acid bacteria powder in lactic acid bacteria capsule material is in preparation process
In easily it is hardened, and, lactic acid bacteria is vulnerable to the infringement of oxygen, drying condition or freezing conditions in preparation process, and this can be caused
The low phenomenon with bacterium grain indissoluble in capsule of strain activity in microbial inoculum.
In sum as can be seen that prior art do not solve keep lactic acid bacteria high activity while maintenance goods it is low into
This technical barrier.
The content of the invention
It is an object of the invention to solve deficiency of the prior art, there is provided a kind of by lactic acid bacteria strains feature and microbial inoculum system
Preparation Method is combined, simple production process, the preparation method of the lactic acid bacterium particle capsule of low production cost.
To achieve these goals, the technical solution of use is the present invention：A kind of preparation of lactic acid bacterium particle capsule
Method, step includes：
First, the preparation of bacterial strain：By lactobacillus inoculum carried out in the MRS fluid nutrient mediums containing mannitol strain culturing with
Its dehydration tolerance is improved, the Best Times that bacterial strain is harvested are the mid-early stage of growth stationary phase, will terminate the lactic acid bacteria of culture
Centrifugation in liquid input centrifuge, collection is deposited in the bacterium mud of bottom, then, is carried out during PBS is put into bacterium mud
Thalline is washed, and is reused centrifuge and is separated and collect and is deposited in the bacterium mud of bottom to obtain the wet bacterium mud of lactic acid bacteria, plus
Enter sterilized water, be stirring evenly and then adding into antioxidant, continue to be stirred until homogeneous to obtain the bacterium core of lactic acid bacteria granulation；
2nd, the granulation of bacterium core：Ground floor coating is carried out using ground floor coating agent to step one gained bacterium core, is gently pressed
I.e. scattered wet bacterium powder, ground floor coating agent is sterile solid cornstarch and sodium cellulose glycolate；Using comminutor to wet bacterium
Powder carries out first time granulation, and second layer coating is then carried out to bacterium grain with second layer coating agent, and second layer coating agent is aseptic solid
Any one solid mixt for being formed in state cornstarch or sterile solid cornstarch and lactose or 1B salt, again
Second granulation is carried out to the bacterium grain of secondary coating using comminutor, the bacterium grain after secondary granulation is dried and is obtained lactic acid bacteria
Particle；
3rd, capsule packaging：The lactic acid bacterium particle that will be obtained in step 2 loads capsule, and packaging is obtained final product.
Further, in the step one, by -20 DEG C of lactobacillus inoculums of preservation to the MRS liquid training containing mannitol
In supporting base, after strain culturing 3~5 times, the lactic acid bacteria strains after being tamed, be stored in -20 DEG C it is standby；Containing sweet
The MRS fluid nutrient mediums for revealing alcohol refer to any one for being adjusted in glucose, sucrose, lactose by the glucose of basal liquid medium
The mixture that sugar is constituted with mannitol, it is 1 with the mass ratio of mannitol：1~1：3；Basal liquid medium is by following
Into being grouped into：The g/L of glucose 20, the g/L of peptone 10, dusty yeast 5g/L, the g/L of beef extract 10, diammonium hydrogen citrate
2 g/L、 MgSO4﹒ 7H2O 0.58 g/L、 MnSO4﹒ H2O 0.25 g/L、 CH3COONa ﹒ 3H2O 5 g/L、 K2HPO4 2 g/
L, 10 g CaCO3 10 g/L、Tween-80 10ml/L。
Further, in the step one, the lactobacillus inoculum after -20 DEG C of domestications of preservation is trained to basal liquid
Support in base, 8 h are cultivated in shaking, and speed is 70rpm/min, obtains one-level shake-flask seed liquid；By one-level shake-flask seed liquid by 8%
Inoculum concentration is inoculated into basal liquid medium or new any one culture medium with MRS fluid nutrient mediums, shaking 8 h of culture, speed
It is 70rpm/min to spend, and then, the growth curve of bacterial strain is determined at interval of 30min, for determining that bacterial strain reaches growth stationary phase
The time of mid-early stage, culture obtains second-level shake flask seed liquor after terminating；Second-level shake flask seed liquor is inoculated into by 8% inoculum concentration
In basal medium or new any one culture medium with MRS fluid nutrient mediums, before shaking was cultivated into strain growth stationary phase
Phase；Newly it is made up of following compositions with MRS fluid nutrient mediums:The g/L of peptone 5, the g/L of Dried Corn Steep Liquor Powder 5, the g/L of dusty yeast 5,
The g/L of brown sugar 120 or sugar content are the sorghum juice of 120 g/L, the g/L of diammonium hydrogen citrate 2, the g/L of sodium acetate 5, Sodium Pyruvate
The g/L of 0.18 g/L, L- semi-gloss cystamine acid hydrochloride 0.05, the g/L of ferroheme 0.5, the g/L of farnoquinone 2, calcium carbonate 10
g/L、K2HPO4 2 g/L、MgSO4·7H2O 0.58 g/L、MnSO4·H2O 0.25 g/L、Tween-80 1ml/L ；Its pH
It is 6.4 to be worth, and is obtained within 15 minutes in 121 °C of sterilizings.
Further, PBS refers to described in the step one：2.4g containing Nacl in every 300 ml aqueous solution,
KCL 0.06g 、Na2HPO4·12H2O 0.462g、KH2PO4 0.06g。
Further, the sterilized water for being added in wet bacterium mud in the step one, water volume is 3.5 with wet thallus mass ratio：1
~4：1, the antioxidant is 2~3 kinds in vitamin C, vitamin E and sodium glutamate, and the addition of antioxidant is
The 4%~8% of wet thallus quality.
Further, the ground floor coating in the step 2 refers to：By sterile solid cornstarch according in step one
Wet bacterium mud mass ratio 6：1~8：1 ratio is added in the bacterium core prepared by step one, is stirred, and mixing speed is
50 rpm/min~70 rpm/min, then add sodium cellulose glycolate, and its addition is wet bacterium mud in step one
Quality 0.5 %~1.5%, continue to be stirred until homogeneous, mixing speed be 50 rpm/min~70 rpm/min,
Obtain the i.e. scattered wet bacterium powder of light pressure；Pelletize for the first time and refer to：Wet bacterium powder is put into comminutor, adjustment granularity is 65~100
Mesh, obtains the bacterium grain of lactic acid bacteria；It is coated for second and refers to：By sterile solid cornstarch or sterile solid cornstarch and lactose,
The solid mixt that any one material in 1B salt is formed is added to the bacterium grain that for the first time prepared by granulation by a certain percentage
In, solid-state cornstarch addition is 3~5 times of the wet bacterium mud quality in step one, lactose and 1B salt addition point
The 0.5%~1.5% of wet bacterium mud quality that Wei be in step one, then, is stirred with agitator, and mixing speed is 10 rpm
/ min~30 rpm/min；Second granulation of bacterium grain：Bacterium grain input comminutor after second is coated, adjusts grain
It is 50~80 mesh to spend, and prepares the bacterium grain of lactic acid bacteria.
Further, the drying of bacterium grain refers to：By pelletized for the second time in step 2 obtain bacterium grain input drier
Inside it is dried, controls 40 DEG C of temperature, time 24h turns speed for 0 rpm/min~20 rpm/min.
Further, the lactic acid bacteria is lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus salivarius, VREF and breast
Any one in sour enterococcus.
The beneficial effects of the invention are as follows：The tolerance of thalline is improved using mannitol domestication, lactobacillus suspension is reduced rear
The bacterium loss of activity of handling process.Lactobacillus suspension is that microbial inoculum activity prepared by this period exists in the mid-early stage of growth stationary phase
Other times section is higher than in heatproof, acidproof and bile tolerance tolerance test, prepared by the lactic acid bacteria that this time period harvests
Strain activity can be farthest preserved after into microbial inoculum.Lactic acid bacteria adds antioxidant before microbial inoculum is prepared into, and this will make breast
Sour bacterium reduces the damage that oxygen is caused to it in follow-up preparation technology, and this is beneficial to bacterial strain and retains in subsequent technique
High activity and reduce the risk of microbiological contamination.Coating agent is solid, reduces the consumption of water.
Specific embodiment
With reference to specific embodiment, the present invention is further elaborated.In following embodiments, unless otherwise specified,
It is conventional method；The reagent and biomaterial, unless otherwise specified, commercially obtain；The percentage composition or
Concentration, is mass percent unless otherwise specified.
Culture medium used is as follows in following embodiments：
Basal liquid medium：The g/L of glucose 20, the g/L of peptone 10, dusty yeast 5g/L, the g/ of beef extract 10
L, diammonium hydrogen citrate 2 g/L, MgSO4﹒ 7H2O 0.58 g/L、 MnSO4﹒ H2O 0.25 g/L、 CH3COONa ﹒ 3H2O 5
g/L、 K2HPO42 g/L, 10 g CaCO310 g/L、Tween-80 10ml/L；PH value is 6.4,121 °C and sterilizes 15 points
Clock.
Newly match somebody with somebody MRS fluid nutrient mediums:The g/L of peptone 5, the g/L of Dried Corn Steep Liquor Powder 5, the g/L of dusty yeast 5, brown sugar 120
G/L or sugar content are the sorghum juice of 120g/L, the g/L of diammonium hydrogen citrate 2, the g/L of sodium acetate 5, the g/L of Sodium Pyruvate 0.18,
The g/L of L- semi-gloss cystamines acid hydrochloride 0.05, the g/L of ferroheme 0.5, the g/L of farnoquinone 2, calcium carbonate 10 g/L, K2HPO4
2 g/L、MgSO4·7H2O 0.58 g/L、MnSO4·H2O 0.25 g/L、Tween-80 1ml/L ；Its pH value is 6.4,
It is obtained within 15 minutes in 121 °C of sterilizings.
The component and content of every liter of MRS solid mediums be:1.5% agar powder is added in above-mentioned basal liquid medium
(mass percent), pH value is 6.4,121 °C and sterilizes 15 minutes.
PBS：2.4g containing Nacl, KCL 0.06g, Na in every 300 ml aqueous solution2HPO4·12H2O
0.462g、KH2PO4 0.06g。
Embodiment 1
The lactobacillus paracasei of -20 DEG C of preservations is inoculated into basal liquid medium, and by the glucose of the culture medium
The mixture of sucrose and mannitol composition is adjusted to, its mass ratio is 1：1, after bacterial strain is with this medium culture 5 times, by this bacterium
It is standby to treat that strain is stored in -20 DEG C of preservations；
The lactobacillus paracasei that -20 DEG C are stored in after domestication is inoculated into basal liquid medium, shaking 8 h of culture,
Speed is 70rpm/min, obtains one-level shake-flask seed liquid；Primary seed solution is inoculated into newly with MRS liquid by 8% inoculum concentration
In culture medium, then shaking 8 h of culture, the growth curve of bacterial strain are determined at interval of 30min, determine that bacterial strain reaches growth stabilization
The time of the mid-early stage of phase is 25 h, now, stops culture, obtains second-level shake flask seed liquor；Secondary seed solution is connect by 8%
The amount of kind is inoculated into newly with MRS fluid nutrient mediums, and 25 h are cultivated in shaking；
The bacterial strain of upper step culture is put into centrifuge to be centrifuged, collection is deposited in the bacterium mud of bottom, then, by PBS
Solution carries out thalline washing in putting into bacterium mud, is centrifuged and collects the bacterium mud for being deposited in bottom again；The wet bacterium mud that will be obtained adds
Enter sterilized water, the ratio of water volume and wet bacterium mud quality is 4：1 (V：W), it is sufficiently stirred for be well mixed, then, then by 4 %
Mass ratio (W：W 3 kinds of antioxidants in vitamin C, vitamin E and sodium glutamate) are added, is sufficiently stirred for mixing
It is even；
It is 6 that sterile solid cornstarch is pressed into it with the mass ratio of the wet bacterium mud of preparation in upper step：1（W：W）Ratio add
Enter in the lactic acid bacterium mud containing antioxidant for preparing, stirred with agitator, mixing speed is 50 rpm/min, then is added
Enter sodium cellulose glycolate, its addition is 0.5 % (W of the quality of the wet bacterium mud for preparing：W), stirred with agitator,
Mixing speed is 50 rpm/min, obtains the i.e. scattered wet bacterium powder of light pressure；
In the wet bacterium powder input comminutor that will be prepared, adjustment granularity is 65 mesh, obtains the bacterium grain of the lactic acid bacteria of 65 mesh；By nothing
Bacterium solid-state cornstarch is added in bacterium grain, and sterile solid cornstarch addition is 3 times of the quality of the wet bacterium mud for preparing
(W：W), then, mixture is stirred with agitator, and mixing speed is 10 rpm/min；
In the bacterium grain input comminutor that will be prepared, adjustment granularity is 50 mesh, obtains the bacterium grain of the lactic acid bacteria of 50 mesh；Will system
It is dried in standby bacterium grain input drier, controls temperature for 40 DEG C, turn speed for 5 rpm/min, is obtained after 24 h
The lactic acid bacterium particle of 50 mesh.
The capsule packaging of bacterium grain：Dried bacterium grain is loaded into capsule by a conventional method, packaging is obtained final product.
Embodiment 2
Difference from Example 1 is：
The second layer of bacterium grain is coated：The solid mixt that sterile solid cornstarch and lactose are formed is proportionally added into
In the bacterium grain prepared in pelletizing for the first time, sterile solid cornstarch addition is 3 times of (W of the quality of wet bacterium mud：W), lactose
Addition is 0.5% (W of the quality of wet bacterium mud：W), then, mixture is stirred with agitator, and mixing speed is 10
rpm / min。
Embodiment 3
Difference from Example 1 is：
The second layer of bacterium grain is coated：The solid mixt that sterile solid cornstarch and 1B salt are formed is in proportion
It is added in the bacterium grain prepared in granulation for the first time, wherein, sterile solid cornstarch addition is 3 times of wet bacterium mud quality
(W：W), 1B salt addition is respectively 0.5% (W of wet bacterium mud quality：W), then, mixture agitator stirs equal
Even, mixing speed is 10 rpm/min；
Second granulation of bacterium grain：In bacterium grain input comminutor prepared by upper step, adjustment granularity is 50 mesh, obtains 50
Purpose lactic acid bacteria bacterium grain.
Embodiment 4
Difference from Example 1 is：
Primary seed solution is inoculated into newly with MRS fluid nutrient mediums by 8% inoculum concentration, 8 h are cultivated in shaking, then, often
Interval 30min determines the growth curve of bacterial strain, determines that the time that bacterial strain reaches the latter stage of growth stationary phase is 30 h, now, stops
Only cultivate, obtain second-level shake flask seed liquor；Secondary seed solution is inoculated into by 8% inoculum concentration and newly match somebody with somebody in MRS fluid nutrient mediums,
30 h of shaking culture.
Embodiment 5
Difference from Example 1 is：
Primary seed solution is inoculated into newly with MRS fluid nutrient mediums by 8% inoculum concentration, 8 h are cultivated in shaking, then, often
Interval 30min determines the growth curve of bacterial strain, determines that the time that bacterial strain reaches the latter stage of growth stationary phase is 30 h, now, stops
Only cultivate, obtain second-level shake flask seed liquor；Secondary seed solution is inoculated into by 8% inoculum concentration and newly match somebody with somebody in MRS fluid nutrient mediums,
30 h of shaking culture.
The second layer of bacterium grain is coated：The solid mixt that sterile solid cornstarch is formed with lactose is added by a certain percentage
Enter to first time in the bacterium grain prepared in pelletizing, sterile solid cornstarch addition is 3 times of (W of wet bacterium mud quality：W), breast
Sugared addition is respectively 0.5% (W of wet bacterium mud quality：W).
Embodiment 6
Difference with embodiment 1 is：
Primary seed solution is inoculated into newly with MRS fluid nutrient mediums by 8% inoculum concentration, 8 h are cultivated in shaking, then, often
Interval 30min determines the growth curve of bacterial strain, determines that the time that bacterial strain reaches the latter stage of growth stationary phase is 30 h, now, stops
Only cultivate, obtain second-level shake flask seed liquor；Secondary seed solution is inoculated into by 8% inoculum concentration and newly match somebody with somebody in MRS fluid nutrient mediums,
30 h of shaking culture；
The second layer of bacterium grain is coated：The solid mixt that sterile solid cornstarch and 1B salt are formed is in proportion
It is added in the bacterium grain that for the first time prepared by granulation, sterile solid cornstarch addition is 3 times of (W of wet bacterium mud quality：W), L-
Lysine salt addition is respectively 0.5% (W of wet bacterium mud quality：W)；
Second granulation of bacterium grain：In bacterium grain input comminutor prepared by upper step, adjustment granularity is 50 mesh, obtains 50
The bacterium grain of purpose lactic acid bacteria.
Embodiment 7
The Lactobacillus plantarum of -20 DEG C of preservations is inoculated into basal liquid medium, and the glucose of the culture medium is adjusted
It is whole for sucrose and mannitol composition mixture, its mass ratio be 1：3, after bacterial strain is with this medium culture 5 times, by this bacterial strain
It is stored in -20 DEG C of preservations standby to treat；
The Lactobacillus plantarum that -20 DEG C are stored in after domestication is inoculated into basal liquid medium, static mixer culture 8
H, obtains one-level shake-flask seed liquid；One-level shake-flask seed liquid is inoculated into newly with MRS fluid nutrient mediums by 8% inoculum concentration, it is quiet
Only after the h of stir culture 8, the growth curve of bacterial strain is determined at interval of 30min, determine that bacterial strain reaches the mid-early stage of growth stationary phase
Time be 18h, now, stop culture, obtain second-level shake flask seed liquor；Second-level shake flask seed liquor is inoculated with by 8% inoculum concentration
To newly with MRS fluid nutrient mediums, the h of static mixer culture 18；
The bacterial strain of upper step culture is put into centrifuge to be centrifuged, collection is deposited in the bacterium mud of bottom, then, by PBS
Solution carries out thalline washing in putting into bacterium mud, is centrifuged and collects the bacterium mud for being deposited in bottom again；The wet bacterium mud that will be obtained, plus
Enter sterilized water, the ratio of water volume and wet bacterium mud quality is 4：1 (V：W), it is sufficiently stirred for be well mixed, then, then by 6 %
Mass ratio (W：W vitamin C, 3 kinds of antioxidants of vitamin E and sodium glutamate) are added, is sufficiently stirred for well mixed；
The ground floor of bacterium mud is coated：Sterile solid cornstarch is pressed into itself and wet bacterium mud mass ratio 7：1（W：W）Ratio add
Enter in the lactic acid bacterium mud containing antioxidant, stirred with agitator, mixing speed is 60 rpm/min, adds hydroxyl first
Base sodium cellulosate, its addition is 1.5 % (W of wet bacterium mud quality：W), stirred with agitator, mixing speed is 60
Rpm/min, obtains the i.e. scattered wet bacterium powder of light pressure；
Pelletize for the first time：In wet bacterium powder input comminutor prepared by upper step, adjustment granularity is 65 mesh, obtains the breast of 65 mesh
The bacterium grain of sour bacterium；
The second layer of bacterium grain is coated：Sterile solid cornstarch is proportionally added into bacterium grain prepared by upper step, it is aseptic
Solid-state cornstarch addition is 5 times of (W of wet bacterium mud quality：W), then, mixture is stirred with agitator, stirring speed
It is 10 rpm/min to spend；
Second granulation of bacterium grain：In bacterium grain input comminutor prepared by upper step, adjustment granularity is 50 mesh, obtains 50
The bacterium grain of purpose lactic acid bacteria；
The drying of bacterium grain：It is dried in bacterium grain input drier prepared by upper step, controls temperature for 40 DEG C, turns speed
It is 5 rpm/min to spend, and the lactic acid bacterium particle of 50 mesh is obtained after 24 h；
The capsule packaging of bacterium grain：The dried bacterium grain of upper step is loaded into capsule by a conventional method, packaging is obtained final product.
Embodiment 8
Difference from Example 7 is：
The second layer of bacterium grain is coated：The solid mixt that sterile solid cornstarch and lactose are formed is proportionally added into
In bacterium grain prepared by upper step, sterile solid cornstarch addition is 5 times of (W of wet bacterium mud quality：W), Inclusion of Lactose difference
It is 1.0% (W of wet bacterium mud quality：W).
Embodiment 9
Difference with embodiment 7 is：
One-level shake-flask seed liquid is inoculated into by 8% inoculum concentration and newly match somebody with somebody in MRS fluid nutrient mediums, static mixer culture 8
H, then, the growth curve of bacterial strain is determined at interval of 30min, determines that the time that bacterial strain reaches the latter stage of growth stationary phase is 24
H, now, stops culture, obtains second-level shake flask seed liquor；Second-level shake flask seed liquor is inoculated into newly with MRS by 8% inoculum concentration
In fluid nutrient medium, the h of static mixer culture 24.
Embodiment 10
Difference with embodiment 7 is：
One-level shake-flask seed liquid is inoculated into by 8% inoculum concentration and newly match somebody with somebody in MRS fluid nutrient mediums, static mixer culture 8
H, then, the growth curve of bacterial strain is determined at interval of 30min, determines that the time that bacterial strain reaches the latter stage of growth stationary phase is 24
H, now, stops culture, obtains second-level shake flask seed liquor；Second-level shake flask seed liquor is inoculated into newly with MRS by 8% inoculum concentration
In fluid nutrient medium, the h of static mixer culture 24.
The second layer of bacterium grain is coated:The solid mixt that sterile solid cornstarch and lactose are formed is proportionally added into
In bacterium grain prepared by upper step, sterile solid cornstarch addition is 5 times of (W of wet bacterium mud quality：W), Inclusion of Lactose is wet
1.0% (W of bacterium mud quality：W).
Embodiment 11
The VREF of -20 DEG C of preservations is inoculated into basal liquid medium, and the carbon source of the culture medium is adjusted to
The mixture that sucrose is constituted with mannitol, its mass ratio is 1：2, after bacterial strain is with this medium culture 5 times, this bacterial strain is preserved
It is standby to treat in -20 DEG C of preservations；
The VREF that -20 DEG C are stored in after upper step is tamed is inoculated into basal liquid medium, static mixer culture 8
H, obtains one-level shake-flask seed liquid；One-level shake-flask seed liquid is inoculated into newly with MRS fluid nutrient mediums by 8% inoculum concentration, it is quiet
Only the h of stir culture 8, then, the growth curve of bacterial strain is determined at interval of 30min, before determining that bacterial strain was reached in growth stationary phase
The time of phase is 25 h, now, stops culture, obtains second-level shake flask seed liquor；Second-level shake flask seed liquor is pressed 8% inoculum concentration
It is inoculated into and newly match somebody with somebody in MRS fluid nutrient mediums, the h of static mixer culture 25；
The bacterial strain of upper step culture is put into centrifuge to be centrifuged, collection is deposited in the bacterium mud of bottom, then, by PBS
Solution carries out thalline washing in putting into bacterium mud, is centrifuged and collects the bacterium mud for being deposited in bottom again；The wet bacterium mud for preparing is added
Enter sterilized water, water volume is 4 with wet thallus mass ratio：1 (V：W), it is sufficiently stirred for be well mixed, then, then by the matter of 6 %
Amount is than (W：W vitamin C, 3 kinds of antioxidants of vitamin E and sodium glutamate) are added, is sufficiently stirred for well mixed；
The ground floor of bacterium mud is coated：Sterile solid cornstarch is pressed into itself and wet bacterium mud mass ratio 7：1（W：W）Ratio add
Enter in the lactic acid bacterium mud containing antioxidant, stirred with agitator, mixing speed is 50 rpm/min, adds hydroxyl first
Base sodium cellulosate, its addition is 1.5 % (W of wet bacterium mud quality：W), stirred with agitator, mixing speed is 50
Rpm/min, obtains the i.e. scattered wet bacterium powder of light pressure；
Pelletize for the first time：In wet bacterium powder input comminutor prepared by upper step, adjustment granularity is 80 mesh, obtains the breast of 80 mesh
The bacterium grain of sour bacterium；
The second layer of bacterium grain is coated：The solid mixt that sterile solid cornstarch and lactose are formed is proportionally added into
In bacterium grain prepared by upper step, sterile solid cornstarch addition is 4 times of (W of wet bacterium mud quality：W), Inclusion of Lactose is wet
1.5% (W of bacterium mud quality：W), then, mixture is stirred with agitator, and mixing speed is 10 rpm/min；
Second granulation of bacterium grain：In bacterium grain input granulator prepared by upper step, adjustment granularity is 65 mesh, obtains 65
The bacterium grain of purpose lactic acid bacteria；
The drying of bacterium grain：It is dried in bacterium grain input drier prepared by upper step, controls temperature for 40 DEG C, turns speed
It is 5 rpm/min to spend, and the lactic acid bacterium particle of 65 mesh is obtained after 24 h.
The capsule packaging of bacterium grain：The dried bacterium grain of upper step is loaded into capsule by a conventional method, packaging is obtained final product.
Embodiment 12
Difference with embodiment 11 is：
One-level shake-flask seed liquid is inoculated into by 8% inoculum concentration and newly match somebody with somebody in MRS fluid nutrient mediums, static mixer culture 8
H, then, the growth curve of bacterial strain is determined at interval of 30min, determines that the time that bacterial strain reaches the latter stage of growth stationary phase is 30
H, now, stops culture, obtains second-level shake flask seed liquor；Second-level shake flask seed liquor is inoculated into newly with MRS by 8% inoculum concentration
In fluid nutrient medium, the h of static mixer culture 30.
Embodiment 13
The resistance to acidity test of lactic acid bacterium particle capsule
(1) microbial inoculum：The lactic acid bacterium particle capsule of 1~embodiment of embodiment 12.
(2) sour environment:It is 2,2.5 and 3 that basal liquid medium 5M HCl adjust pH value.
(3) resistance to acidity test:Weigh respectively 1g lactic acid bacterium particle capsules be added to 9 mL pH value be 2,2.5 and 3 basis
In fluid nutrient medium, then 37 DEG C of incubation 2h, take out the sodium phosphate buffer (pH7.0) that sample is added to 0.4 M of 9mL,
Mix, take 0.5 mL mixed liquors and coat conventional MRS solid mediums, clump count on flat board is carried out after 37 DEG C of 24 h of culture
Count, survival rate is the ratio of the viable count and untreated viable count for determining, 3 repetitions of each pH measuring setting.
The viable count average survived in lactic acid bacterium particle capsule under the different acidic conditions of table 1
As seen from Table 1, in acid condition, bacterial strain stationary phase mid-early stage prepare particles capsule (example 1,2,3,
7th, 8, viable count 11) is greater than the particles capsule (example 4,5,6,9,10,12) that bacterial strain is prepared in the latter stage of stationary phase.
Embodiment 14
Lactic acid bacterium particle capsule bile tolerance is determined
(1) microbial inoculum：The lactic acid bacterium particle capsule of 1~embodiment of embodiment 12.
(2) cholate environment:The Pig cholate of different content is added in basal liquid medium, its final concentration is respectively 0.3%
With 0.6%, and it is 5.8 to adjust pH value with 5M HCl.
(3) bile tolerance is determined:Weigh respectively 1g lactic acid bacterium particle capsules be added to 9 mL gallbladder salinity be 0.3% He
In 0.6% basal liquid medium, then 37 DEG C of incubation 2h, take out the sodium phosphate buffer that sample is added to 0.4 M of 9mL
Liquid (pH7.0), is mixed, and takes 0.5 mL mixed liquors and coats conventional MRS solid mediums, and flat board is carried out after 37 DEG C of 24 h of culture
The counting of upper clump count, survival rate is the ratio of the viable count and untreated viable count for determining, 3 weights of each measuring setting
It is multiple.
The viable count average survived in lactic acid bacterium particle capsule under the conditions of the various concentrations Pig cholate of table 2
As seen from Table 2, under different pig gall salt concentration conditions, the bacterium grain capsule that bacterial strain is prepared in the mid-early stage of stationary phase
The viable count of (example 1,2,3,7,8,11) be greater than bacterial strain stationary phase latter stage prepare bacterium grain capsule (example 4,5,6,9,
10、12)。
Embodiment 15
Lactic acid bacterium particle capsule high temperature resistant is determined
(1) microbial inoculum：The lactic acid bacterium particle capsule of 1~embodiment of embodiment 12.
(2) hot environment:62 DEG C, 80 DEG C and 95 DEG C three thermogrades are set.
(3) high temperature resistant is determined:1g lactic acid bacterias bacterium grain capsule is weighed respectively to put into iron pan, and iron pan is respectively placed in 62
DEG C, in the baking oven of 80 DEG C and 95 DEG C, standing time is 2min, then, takes out the basal liquid medium that sample is added to 5mL
In, mix, take 0.5 mL mixed liquors and coat conventional MRS solid mediums, carry out clump count on flat board after 37 DEG C of 24 h of culture
Counting, survival rate be determine viable count and untreated viable count ratio, 3 repetitions of each measuring setting.
The viable count average survived in lactic acid bacterium particle capsule under the condition of different temperatures of table 3
As seen from Table 3, under different hot conditions, bacterial strain stationary phase mid-early stage prepare bacterium grain capsule (example 1,
2nd, 3,7,8, viable count 11) is greater than bacterium grain capsule (example 4,5,6,9,10,12) that bacterial strain is prepared in the latter stage of stationary phase.
Embodiment 16
Measure of the lactic acid bacterium particle capsule to fresh clover ensiling
(1) microbial inoculum：Embodiment 3, embodiment 7 and reality in the lactic acid bacterium particle capsule of 1~embodiment of selection embodiment 12
The microbial inoculum that example 11 is the present embodiment is applied, 10 is prepared with the water less than 40 DEG C before use10The lactic acid bacteria solution of cfu/mL concentration.
(2) ensiling material：Alfalfa plant in first flower bud phase.
(3) packet and dosage are set：Experiment clover is divided into 10 groups in the way of mono- group of 300g, 3 repetitions of every group of setting, often
Group clover is cut into the lysate of the clover addition lactic acid bacterium particle capsule that 5~7cm is long, after every group of cutting and turns uniformly, so
Afterwards, it is fitted into the vial of 500mL and is compacted, the concentration of lactic acid bacteria is set to 109Cfu/mL and 1010Two concentration ladders of cfu/mL
Degree, one to three group of experiment is respectively 109The microbial inoculum solution of the embodiment 3, embodiment 7 and embodiment 11 of cfu/mL, experiment four to
Six groups are respectively 1010The microbial inoculum solution of the embodiment 3, embodiment 7 and embodiment 11 of cfu/mL, seven groups of experiment is 109cfu/mL
Embodiment 3 and embodiment 7 composite bacteria agent solution, experiment eight groups be 109The embodiment 3 of cfu/mL and being combined for embodiment 11
Microbial inoculum solution, nine groups of experiment is 109The embodiment 7 of cfu/mL and the composite bacteria agent solution of embodiment 11, testing ten groups is
109The composite bacteria agent solution of the embodiment 3, embodiment 7 and embodiment 11 of cfu/mL.
(4) quality determination of Alfalfa Silage：After 30 days, 20 g samples are uniformly accurately weighed;180 mL distilled water are added, is stirred
Mix uniform, plus preservative film;24 h are extracted at 4 DEG C, it is leaching liquor to filter gained liquid twice through four layers of gauze and qualitative filter paper.Leaching
Extract is used for determining alfalfa ensilage material pH value, and pH value is determined using thunder magnetic PHS-25 type pH instrument.Water soluble carbohydrates are used
Anthrone-Sulphuric acid Colorimetry, crude protein uses Kjeldahl nitrogen determination (FOSS companies full-automatic Kjeldahl determination device).
The average of the lactic acid bacterium particle capsule silage effect value of table 4
As seen from Table 4, the silage effect that the lactic acid bacterium particle capsule of preparation can reach to clover.
Described above is not limitation of the present invention, and the present invention is also not limited to the example above, the art
Change, remodeling, addition or replacement that technical staff is made in essential scope of the invention, should also belong to protection of the invention
Scope.
Claims (8)
1. a kind of preparation method of lactic acid bacterium particle capsule, it is characterised in that step includes：
First, the preparation of bacterial strain：Lactobacillus inoculum is carried out strain culturing to improve in the MRS fluid nutrient mediums containing mannitol
Its dehydration tolerance, the Best Times that bacterial strain is harvested are the mid-early stage of growth stationary phase, and the lactobacillus suspension that will terminate culture is thrown
Enter centrifugation in centrifuge, collection is deposited in the bacterium mud of bottom, then, thalline is carried out during PBS is put into bacterium mud
Washing, reuses centrifuge and separates and collect and be deposited in the bacterium mud of bottom to obtain the wet bacterium mud of lactic acid bacteria, adds nothing
Bacterium water, is stirring evenly and then adding into antioxidant, continues to be stirred until homogeneous to obtain the bacterium core of lactic acid bacteria granulation；
2nd, the granulation of bacterium core：Ground floor coating is carried out using ground floor coating agent to step one gained bacterium core, light pressure is obtained and is dissipated
Wet bacterium powder, ground floor coating agent be sterile solid cornstarch and sodium cellulose glycolate；Wet bacterium powder is entered using comminutor
Row is pelletized for the first time, then carries out second layer coating to bacterium grain with second layer coating agent, and second layer coating agent is beautiful sterile solid
Any one solid mixt for being formed in rice starch or sterile solid cornstarch and lactose or 1B salt, reuses
Comminutor carries out second granulation to the bacterium grain of secondary coating, the bacterium grain after secondary granulation is dried and obtains lactic acid bacteria
Grain；
3rd, capsule packaging：The lactic acid bacterium particle that will be obtained in step 2 loads capsule, and packaging is obtained final product.
2. the preparation method of lactic acid bacterium particle capsule according to claim 1, it is characterised in that：In the step one, will-
20 DEG C of lactobacillus inoculums of preservation in the MRS fluid nutrient mediums containing mannitol, after strain culturing 3~5 times, after being tamed
Lactic acid bacteria strains, be stored in -20 DEG C it is standby；MRS fluid nutrient mediums containing mannitol refer to basal liquid medium
Glucose be adjusted to the mixture of any one sugar in glucose, sucrose, lactose and mannitol composition, itself and mannitol
Mass ratio is 1：1~1：3；Basal liquid medium is made up of following compositions：The g/L of glucose 20, the g/L of peptone 10, yeast
Powder 5g/L, the g/L of beef extract 10, diammonium hydrogen citrate 2 g/L, MgSO4﹒ 7H2O 0.58g/L、MnSO4﹒ H2O 0.25 g/L、
CH3COONa ﹒ 3H2O 5 g/L、K2HPO42 g/L, CaCO3 10 g/L、Tween-80 10ml/L。
3. the preparation method of lactic acid bacterium particle capsule according to claim 2, it is characterised in that：In the step one, will-
Lactobacillus inoculum after 20 DEG C of domestications of preservation in basal liquid medium, shaking culture 8h, speed is 70rpm, obtains one
Level shake-flask seed liquid；One-level shake-flask seed liquid is inoculated into basal liquid medium or new with the training of MRS liquid by 8% inoculum concentration
Support in any one culture medium of base, 8h is cultivated in shaking, and speed is 70rpm, then, the growth for determining bacterial strain at interval of 30min is bent
Line, for determining that bacterial strain reaches the time of the mid-early stage of growth stationary phase, culture obtains second-level shake flask seed liquor after terminating；By two
During level shake-flask seed liquid is inoculated into basal medium or new any one culture medium with MRS fluid nutrient mediums by 8% inoculum concentration,
Shaking was cultivated to the mid-early stage of strain growth stationary phase；Newly it is made up of following compositions with MRS fluid nutrient mediums：The g/L of peptone 5,
The g/L of Dried Corn Steep Liquor Powder 5, the g/L of dusty yeast 5, the g/L of brown sugar 120 or sorghum juice, diammonium hydrogen citrate that sugar content is 120 g/L
2 g/L, the g/L of sodium acetate 5, the g/L of Sodium Pyruvate 0.18, the g/L of L-cysteine hydrochloride 0.05, the g/L of ferroheme 0.5, dimension
Raw element K22 g/L, calcium carbonate 10 g/L, K2HPO4 2 g/L、MgSO4·7H2O 0.58 g/L、MnSO4·H2O 0.25 g/
L、Tween-80 1ml/L；Its pH value is 6.4, is obtained within 15 minutes in 121 DEG C of sterilizings.
4. the preparation method of lactic acid bacterium particle capsule according to claim 1, it is characterised in that：Described in the step one
PBS refers to：2.4g containing NaCl, KCl 0.06g, Na in per the 300ml aqueous solution2HPO4·12H2O 0.462g、KH2PO4
0.06g。
5. the preparation method of lactic acid bacterium particle capsule according to claim 1, it is characterised in that：The antioxidant is dimension
2~3 kinds in raw element C, vitamin E and sodium glutamate, the addition of antioxidant is the 4%~8% of wet thallus quality.
6. the preparation method of lactic acid bacterium particle capsule according to claim 1, it is characterised in that：In the step 2
One layer of coating refer to：By sterile solid cornstarch according to its with step one in wet bacterium mud mass ratio 6：1~8：1 ratio
It is added in the bacterium core prepared by step one, stirs, mixing speed is 50rpm~70rpm, then adds hydroxylmethyl cellulose
Plain sodium, its addition is the 0.5%~1.5% of the quality of wet bacterium mud in step one, continues to be stirred until homogeneous, and mixing speed is
50rpm~70rpm, obtains the i.e. scattered wet bacterium powder of light pressure；Pelletize for the first time and refer to：Wet bacterium powder is put into comminutor, grain is adjusted
It is 65~100 mesh to spend, and obtains the bacterium grain of lactic acid bacteria；It is coated for second and refers to：By sterile solid cornstarch or sterile solid corn
The solid mixt that any one material in starch and lactose, 1B salt is formed is added to granulation for the first time by a certain percentage
In the bacterium grain of preparation, solid-state cornstarch addition is 3~5 times of the wet bacterium mud quality in step one, lactose and 1B
Salt addition is respectively 0.5%~1.5% of the wet bacterium mud quality in step one, then, is stirred with agitator, mixing speed
It is 10rpm~30rpm；Second granulation of bacterium grain：Will second be coated after bacterium grain input comminutor, adjustment granularity is 50~
80 mesh, prepare the bacterium grain of lactic acid bacteria.
7. the preparation method of lactic acid bacterium particle capsule according to claim 1, it is characterised in that：The drying of bacterium grain is
Refer to：To for the second time be pelletized in step 2 be dried in the bacterium grain input drier for obtaining, and control 40 DEG C of temperature, and time 24h turns over
Speed is mixed for 0rpm~20rpm.
8. the preparation method of lactic acid bacterium particle capsule according to claim 1, it is characterised in that：The lactic acid bacteria is secondary dry
Any one in Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus salivarius, VREF and lactoenterococcus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410678385.7A CN104431355B (en) | 2014-11-24 | 2014-11-24 | A kind of preparation method of lactic acid bacterium particle capsule |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410678385.7A CN104431355B (en) | 2014-11-24 | 2014-11-24 | A kind of preparation method of lactic acid bacterium particle capsule |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104431355A CN104431355A (en) | 2015-03-25 |
| CN104431355B true CN104431355B (en) | 2017-05-31 |
Family
ID=52878703
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410678385.7A Active CN104431355B (en) | 2014-11-24 | 2014-11-24 | A kind of preparation method of lactic acid bacterium particle capsule |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104431355B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112602829A (en) * | 2020-12-17 | 2021-04-06 | 湖北华扬科技发展有限公司 | High-stability enterococcus faecium preparation and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101323850A (en) * | 2008-07-28 | 2008-12-17 | 天津科技大学 | Lactobacillus helveticus microcapsule, preparation and use thereof |
| CN101715876A (en) * | 2009-04-13 | 2010-06-02 | 四川渴望生物科技有限公司 | Preparation method of high-efficiency compound microorganism soybean meal leaven |
| CN102987055A (en) * | 2012-10-17 | 2013-03-27 | 李雪平 | Compound encapsulated lactic acid bacteria product and preparation method thereof |
| CN103497050A (en) * | 2013-09-26 | 2014-01-08 | 天津科技大学 | Method for preparing salt tolerant phosphate solubilizing seed coating |
| CN103749973A (en) * | 2011-10-17 | 2014-04-30 | 陈双露 | Coated microcapsule of lactic acid bacteria and its preparation method |
-
2014
- 2014-11-24 CN CN201410678385.7A patent/CN104431355B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101323850A (en) * | 2008-07-28 | 2008-12-17 | 天津科技大学 | Lactobacillus helveticus microcapsule, preparation and use thereof |
| CN101715876A (en) * | 2009-04-13 | 2010-06-02 | 四川渴望生物科技有限公司 | Preparation method of high-efficiency compound microorganism soybean meal leaven |
| CN103749973A (en) * | 2011-10-17 | 2014-04-30 | 陈双露 | Coated microcapsule of lactic acid bacteria and its preparation method |
| CN102987055A (en) * | 2012-10-17 | 2013-03-27 | 李雪平 | Compound encapsulated lactic acid bacteria product and preparation method thereof |
| CN103497050A (en) * | 2013-09-26 | 2014-01-08 | 天津科技大学 | Method for preparing salt tolerant phosphate solubilizing seed coating |
Non-Patent Citations (1)
| Title |
|---|
| 益生菌微生态制剂及包埋技术研究进展;龚阿琼等;《国外畜牧学(猪与禽)》;20120825;第32卷(第08期);69－72 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104431355A (en) | 2015-03-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ningtyas et al. | The viability of probiotic Lactobacillus rhamnosus (non-encapsulated and encapsulated) in functional reduced-fat cream cheese and its textural properties during storage | |
| US20210261908A1 (en) | Cryoprotective compositions and uses thereof | |
| CN103620021B (en) | New compositions | |
| CN102725397B (en) | Dead lactobacillus biomass for antimicrobial use and a production method therefor | |
| CN100537740C (en) | Metabolic activity microorganism and production method thereof | |
| Bosnea et al. | Microencapsulated cells of Lactobacillus paracasei subsp. paracasei in biopolymer complex coacervates and their function in a yogurt matrix | |
| CN102370057A (en) | Lactobacillus microcapsule and preparation method thereof | |
| CN102283334A (en) | Complex probiotic microcapsule capable of improving intestinal tract micro-ecology of small-sized dogs | |
| CN109717481A (en) | A kind of preparation process of coating probiotics | |
| CN107287136A (en) | A kind of preparation method of the prebiotic solid lactic acid bacteria agent with prebiotic function | |
| CN109007807A (en) | A kind of microcapsules fruit ferment powder and preparation method thereof | |
| CN104271731B (en) | The composition of microbial biomass comprising activation | |
| PL214583B1 (en) | A new strain of Lactobacillus plantarum S, the use of the strain of Lactobacillus plantarum S and preparation for ensiling bulky feed | |
| CN116096856A (en) | Microencapsulated microbial cultures utilizing octenyl succinic anhydride starch-chitosan complex agglomerates | |
| RU2614116C2 (en) | Method of producing probiotic composition | |
| WO2017050773A1 (en) | Method for preparing a probiotic powder using a two-in-one whey-containing nutrient medium | |
| CN104431355B (en) | A kind of preparation method of lactic acid bacterium particle capsule | |
| CN107881133A (en) | A kind of high-activity bifidobacterium powder vacuum drying production technology and application | |
| CN106490307A (en) | A kind of method of antimicrobial composition fermentation Entermorpha | |
| CN104642746A (en) | Preparation method of feeding lactic acid bacteria micro pills | |
| CN107686824A (en) | A kind of preparation method of feeding lactobacillus microcapsules | |
| CN106834164A (en) | For the Chinese yew solid medium and its application method of feed | |
| CN109943505A (en) | A kind of compound probiotic microbial inoculum for harmless treatment excrement slag bed padding | |
| CN109418506A (en) | A kind of stabilization coating technique of the feeding biological vitamin of enteric solubility | |
| US20180153106A1 (en) | Method for Producing Food-Safe Sprouted Seed Products |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |