CN110305811A - A kind of lactobacillus plantarum microbial inoculum and its high density fermentation preparation method and application - Google Patents
A kind of lactobacillus plantarum microbial inoculum and its high density fermentation preparation method and application Download PDFInfo
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- CN110305811A CN110305811A CN201910573991.5A CN201910573991A CN110305811A CN 110305811 A CN110305811 A CN 110305811A CN 201910573991 A CN201910573991 A CN 201910573991A CN 110305811 A CN110305811 A CN 110305811A
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- fermentation
- lactobacillus plantarum
- sucrose
- feed supplement
- microbial inoculum
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- 230000004151 fermentation Effects 0.000 title claims abstract description 167
- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 115
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 115
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 114
- 238000002360 preparation method Methods 0.000 title claims abstract description 37
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 36
- 229930006000 Sucrose Natural products 0.000 claims abstract description 62
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 62
- 239000005720 sucrose Substances 0.000 claims abstract description 62
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- 229960005486 vaccine Drugs 0.000 claims abstract description 19
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- 239000002609 medium Substances 0.000 claims description 42
- 241000894006 Bacteria Species 0.000 claims description 33
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 30
- 235000015097 nutrients Nutrition 0.000 claims description 28
- 239000002054 inoculum Substances 0.000 claims description 22
- 238000012545 processing Methods 0.000 claims description 20
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 10
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- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 7
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
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- 108010000912 Egg Proteins Proteins 0.000 description 1
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 1
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- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
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- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
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- 229920002521 macromolecule Polymers 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Birds (AREA)
- Food Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Animal Husbandry (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
Abstract
The invention discloses a kind of lactobacillus plantarum microbial inoculum and its high density fermentation preparation methods, belong to field of biotechnology, and process flow includes the high density fermentation culture of lactobacillus plantarum and the freeze drying process of microbial inoculum.In lactobacillus plantarum high density fermentation culture of the invention, sucrose feed supplement, the sucrose feed profile are as follows: when fermented and cultured to 7-8h, feed supplement is started with the rate of 5-6mL/Lh are carried out during the fermentation；After fermented and cultured is to 11-15h, with 10-18mL/Lh rate feed supplement, after continuing fermentation to 17-18h, with 10-15mL/min rate feed supplement, the 1-2h that continues to ferment terminates；Lactobacillus plantarum microbial inoculum is obtained by the way that freeze drying protectant is added into thallus.The present invention is since using multistage differentiation feed supplement is divided, the viable count that final fermentation obtains is more than 1011Cfu/mL, breaches normal fermentation bacterial strain activity level obtained, and viable count is obviously improved；The survival rate of the lactobacillus plantarum freeze-dried vaccine powder prepared simultaneously using vacuum freeze-drying method of the invention is greater than 90%, and count plate result is greater than 3.0 × 1012cfu/g。
Description
Technical field
The invention belongs to field of biotechnology, be related to a kind of lactobacillus plantarum microbial inoculum and its high density fermentation preparation method with
And application.
Background technique
At the beginning of the seventies, the countries such as America and Europe begin to development of new probiotics, it is desirable to which substitution is anti-to a certain extent
Raw element, and play the effect for adjusting body immunity.In recent years, probiotics are recognized with consumer groups, China continuous
Deepen and understand, lactobacillus micro-ecological preparation with its recuperating gastrointestinal tract and improve immunity in terms of significant advantage, increasingly by
To the extensive concern of people.
Beneficial flora of the lactobacillus plantarum as gastrointestinal tract has and maintains intestinal flora balance, improves immunity of organisms
Enteron aisle is colonized in play its prebiotic effect, nothing by stomach with multiple functions, lactobacillus plantarums such as promotion absorption of nutrient ingredients
By in food fermentation, or in fields such as health cares, suffers from and be widely applied.
High Density Cultivation technology, that is, high-density fermentation technology.In general, High Density Cultivation, which refers to, applies centainly
Culture technique or device improve the fermentation density of thallus, so that the more traditional training method of cell density is increased significantly, to reach
To volume of culture is reduced, the production cycle can also be shortened, the final specific production rate for improving specific product, reduce equipment investment from
And production cost is reduced, improve competitiveness in the market.Fermentation liquid is through thalline were collected by centrifugation, a certain proportion of protective agent of addition
Afterwards, freeze-dried vaccine powder is obtained by drying process.
Such as patent of invention " a kind of method for producing lactic acid and its special plant cream bar of Publication No. CN101302488A
Bacterium ", disclose that the invention discloses a kind of method for producing D-ALPHA-Hydroxypropionic acid and its special plant bacterium lacticums.It is provided by the present invention
Lactobacillus plantarum is lactobacillus plantarum (Lactobacillus plantarum) SMB-2CGMCCNo.2566.At 35-39 DEG C
Under conditions of D-ALPHA-Hydroxypropionic acid can be obtained within stationary culture lactobacillus plantarum 48 hours.The concentration of total lactic acid is 18% in fermentation liquid,
The optical purity of D-ALPHA-Hydroxypropionic acid is 84-86%, fermentation production rate 3.75g/Lh.And the lactobacillus plantarum is easy to be deposited to fermentation
Pot bottom is grown, and is conducive to bacterium-liquid in industrial production and is isolated and purified, and the industrial cost of lactic acid can be reduced, tool
There is important industrial application value.Its fermentation condition be 35-39 DEG C stationary culture 48 hours.The fermented and cultured mode is in batches
Fed-batch fermentation culture, every glucose for adding 60mL 1000g/L for 12 hours are primary.But the feed process is more extensive, does not examine
Strain is considered in the performance of growth different phase, is easily caused sugar source excessive concentration and is generated the growth inhibition effect to strain.
Patent of invention " lactobacillus plantarum and its High Density Cultivation and the freeze-dried vaccine powder of Publication No. CN102864096A
The method of preparation ", technical solution is: it is characterized by: the lactobacillus plantarum (Lactobacillus plantarum
It P8) is the probiotics being isolated from Wulate Middle Banner, Baya ur, NeiMengGu city traditional zymotic dairy product (yoghurt), tool
There are simulated gastric fluid and artificial digestion liquid tolerance, Bile salt resistance, enteron aisle agglutination and the common pathogenic entero becteria of inhibition etc. special
Property；The bacteria strain is on November 18th, 2011 in China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation
Center preservation, deposit number are CGMCC No.5468.High density fermentation liquid, zymotic fluid viable count is up to 1010Cfu/ml or more is obtained
To lactobacillus plantarum (Lactobacillus plantarum P8) freeze-dried vaccine powder.The viable count of bacterium powder is up to 4 × 1011cfu/g
More than.
To sum up, in the prior art, to provide bacterial strain vigor and stability, in high-density culture process, often through sieve
High vigor strain is selected, the modes such as batch feeding, feed component is often complex, and feed supplement amount is larger, while zymotechnique is more
It is extensive, it sets out not according to the growth performance of strain different phase, improves fermenting microbe vigor.
Summary of the invention
The object of the present invention is to provide a kind of lactobacillus plantarum microbial inoculum and its high density fermentation preparation method and application, with
Problems of the prior art are solved, the freezing tolerance of its high density fermentation level, enhancing lactobacillus plantarum is improved, improves and produce
Product viable count.
In order to achieve the above objectives, the technical solution adopted by the present invention is that: a kind of high density fermentation of lactobacillus plantarum microbial inoculum
Preparation method, steps are as follows: by lactobacillus plantarum after seed culture, accessing fermentation medium, concussion according to inoculum concentration 3-5%
It cultivates, feed supplement is carried out by addition sucrose in fermentation process, the sucrose concentration of the feed supplement is 800-1000g/L, the sucrose
Feed profile are as follows: when fermented and cultured to 7-8h, feed supplement is started with the rate of 5-6mL/Lh；After fermented and cultured is to 11-15h,
When sucrose residual quantity is 0.04-0.10%, with 10-18mL/Lh rate feed supplement, after continuing fermentation to 17-19h, sucrose residual
When amount is 0.10-0.15%, with 10-15mL/min rate feed supplement, continuing fermentation 1-2.5h terminates, by gained fermentation liquid through being centrifuged
Thallus is obtained, freeze drying protectant mixing, and freeze-dried acquisition lactobacillus plantarum microbial inoculum are added in Xiang Suoshu thallus.
Obviously risen using the thallus content that the fermentation method obtains compared with common fermentation processes, OD600 value can reach at this time
30 or more.By count plate, the viable count measured after fermentation reaches 1.0 × 1011Cfu/mL or more.
Preferably, the sucrose feed profile are as follows: fermented and cultured 7-8h, when sucrose residual quantity is 0.04-0.06%, with 5-
The rate of 6mL/Lh starts feed supplement, after fermented and cultured to 11-12h, when sucrose residual quantity is 0.04-0.1%, with 10-15mL/
Lh rate feed supplement, after fermentation to 14-15h, when sucrose residual quantity is 0.05-0.10%, with the continuation of 15-18mL/Lh rate
Fed-batch cultivation, continues fermentation to after 18-19h, when sucrose residual quantity is 0.10-0.15%, with 10-15mL/min rate feed supplement,
Continuing fermentation 1.5-2.5h terminates.
Obviously risen using the thallus content that the fermentation method obtains compared with common fermentation processes, OD600 value can reach at this time
35 or more.By count plate, the viable count measured after fermentation reaches 2.0 × 1011Cfu/mL or more；
Preferably, during the fermented and cultured, fermented and cultured temperature is 35-37 DEG C, speed of agitator 90-100r/min,
Maintain fermentation pH6.1-6.3.
Preferably, the freeze drying protectant composition are as follows: skimmed milk power 750-800g/L, lactose 450-500g/L, glycerine
20-50g/L, 1-20g/L sodium glutamate, VC 5-10g/L, surplus are that concentration is 0.1M, the PBS solution that pH is 6.2.
Preferably, the fermentation medium composition are as follows: peptone 8-10g/L, sucrose 15-20g/L, yeast powder 15-20g/
L, MgSO4﹒ 7H2O 0.2-0.5g/L, MnSO40.1-0.2g/L, K2HPO4The cane molasses of 1-3g/L, 0.5-1g/L, acetic acid
Sodium 3-5g/L, Tween 80 1.0-2.0mL/L, defoaming agent 0.3-0.5mL/L, surplus are water.
Preferably, the high density fermentation preparation method of a kind of lactobacillus plantarum microbial inoculum, the specific steps are as follows:
(1) seed culture: lactobacillus plantarum is accessed and stands activation training in seed fluid nutrient mediums of saccharomycete in 35-37 DEG C of incubator
21-24h is supported, primary seed solution is obtained；The primary seed solution is inoculated in seed fluid nutrient mediums of saccharomycete with inoculum concentration 3-5%,
35-37 DEG C of stationary culture 21-24h, obtains secondary seed solution；The secondary seed solution is connect with inoculum concentration 3-5%
Enter in seed fluid nutrient mediums of saccharomycete, 35-37 DEG C of stationary culture 21-24h obtains three-level seed liquor；
(2) fermented and cultured: the three-level seed liquor is accessed in fermentation medium with inoculum concentration 3-5%, fermented and cultured temperature
Degree is 35-37 DEG C, speed of agitator 90-100r/min, adjusts constant pH 6.1-6.3 using 50% ammonium hydroxide, and be passed through nitrogen
Maintaining fermentation pressure tank is 0.04-0.06MPa；Sucrose feed supplement is taken in fermentation process, the sucrose concentration of the feed supplement is 800-
1000g/L, the sucrose feed profile are as follows: as fermented and cultured 7-8h, when sucrose residual quantity is 0.04-0.06%, with 5-6mL/
The rate of Lh starts feed supplement；After fermented and cultured is to 11-15h, when sucrose residual quantity is 0.04-0.10%, with 10-18mL/
Lh rate feed supplement, after continuing fermentation to 17-19h, when sucrose residual quantity is 0.10-0.15%, with 10-15mL/min rate benefit
Material, continuing fermentation 1-2.5h terminates；
(3) prepared by microbial inoculum: the fermentation liquid of obtained lactobacillus plantarum being centrifuged through 5000-10000g, obtains bacterium mud；It will freeze
Dry protective agent and bacterium mud are mixed according to the ratio of mass ratio 2-4:1, after pre-cooled processing, Yu Wendu -55 DEG C--60 DEG C,
Vacuum degree 1-7Pa handles 10-12h, obtains lactobacillus plantarum freeze-dried vaccine powder after dry 18-30h.
The viable count of the lactobacillus plantarum freeze-dried vaccine powder is greater than 3.0 × 1012Cfu/g, survival rate are not less than 98.0%.
Preferably, in the step (1), the seed fluid nutrient mediums of saccharomycete is specially MRS fluid nutrient medium；The level-one,
Second level, three-level seed fluid nutrient mediums of saccharomycete volume ratio are as follows: 5:45:1500.
Preferably, in the step (2), the fermented and cultured carries out in the fermenter, the fermentation medium liquid amount
It is 3:5 with fermenter volume ratio.
Preferably, in the step (3), the pre- cool condition is that -80 DEG C of precoolings handle 12h.
Preferably, in the step (4), the freeze drying protectant and bacterium mud mass ratio are 3:1.
Preferably, the lactobacillus plantarum deposit number is CGMCC No.9513.
Another object of the present invention is to provide the lactobacillus plantarum microbial inoculum prepared by above-specified high density fermentation preparation.
Another object of the present invention is to provide the purposes of the lactobacillus plantarum microbial inoculum.
Preferably, the purposes is purposes of the lactobacillus plantarum microbial inoculum in chicken feed.
Preferably, additive amount of the lactobacillus plantarum microbial inoculum in Broiler chicks basal diet is 0.05-0.3%, microbial inoculum
Vigor is 2 × 108CFU/g。
Preferably, the Broiler chicks are 14 age in days Broiler chicks；The feeding cycle is 15-42 age in days.
Lactobacillus plantarum of the present invention, culture presevation number are as follows: CGMCC No.9513, the strain is in Publication No.
It is disclosed in the patent of invention " a kind of method of lactobacillus plantarum and its High Density Cultivation " of CN104357348A.
The utility model has the advantages that
1, significant bacterial strain vigor
The conventional final viable count of lactobacillus plantarum batch fermentation only only reaches to 109Cfu/mL or so, even if passing through routine
Batch feeding after final zymotic fluid viable count be also difficult to break through 1010Cfu/mL is horizontal, and the present invention passes through difference stage by stage
After changing feed supplement, final viable count is more than 1011Cfu/mL breaches normal fermentation bacterial strain activity level obtained, viable count
It is obviously improved.
Using the three stages differentiation feed supplement of " low-high-low ", mainly according to the lag phase of this lactobacillus plantarum, logarithmic phase
The growth characteristics of (forward and backward), stationary phase, into after logarithm early period, growing microorganism rate is accelerated, and the carbon source needed at this time maintains speed
Degree is increase accordingly, and feed supplement demand increases, and needs to be continuously improved feed rate, after reaching late log phase, growing microorganism reduced rate,
Carbon source demand is reduced, and feed rate slows down, so both can demand with effective guarantee thalli growth to carbon source, and in turn avoid carbon
Source builds up caused growth inhibitory effect, can utmostly promote this lactobacillus plantarum during the fermentation numerous
It grows.
Thallus content obviously rises compared with common fermentation processes, and OD600 value can reach 30 or more at this time.By count plate,
The viable count measured after fermentation reaches 1.0 × 1011Cfu/mL or more.
Using the quadravalence segment difference alienation feed supplement of feed rate " low-in-high-low ", main prolonging according to this lactobacillus plantarum
Demurrage, logarithmic phase (it is preceding-in-in after-it is rear), the growth characteristics of stationary phase, enter logarithmic phase especially for lactobacillus plantarum, will
The strain fermentation period be refined as logarithm before, in, in after and late log phase.Its mid-log phase and the thallus of logarithm middle and later periods hair
There are significant differences for ferment, it is a discovery of the invention that in logarithm early period and mid-log phase, demand phase of the lactobacillus plantarum for carbon source
Closely, and in the logarithm middle and later periods, as cell density increases, growing microorganism rate is accelerated, at this time demand of the thallus for carbon source
It significantly increases, the carbon source needed maintains speed to increase accordingly, and feed supplement demand increases, and needs to be continuously improved feed rate, arrival pair
After the number later period, growing microorganism reduced rate, carbon source demand is reduced, and feed rate slows down, then equally reduces carbon source and increase, to promote
Into thallus for the effective rate of utilization of carbon source.Thallus content obviously rises compared with common fermentation processes, and OD600 value can reach 35 at this time
More than.By count plate, the viable count measured after fermentation is not less than 2.0 × 1011cfu/mL。
The conventional final viable count of lactobacillus plantarum batch fermentation only only reaches to 109Cfu/mL or so, even if passing through routine
Batch feeding after final zymotic fluid viable count be also difficult to break through 1010Cfu/mL is horizontal, and the present invention passes through three stage differences
After changing feed supplement, final viable count is more than 1011Cfu/mL breaches normal fermentation bacterial strain activity level obtained, viable count
It is obviously improved；And the present invention is by the way that after the alienation feed supplement of quadravalence segment difference, final fermentation level is 2 times of three stages difference feed supplement.
Furthermore in the present invention, the use of molasses can be further able to enrichment vitamin and mineral in fermentation medium, effectively
Promote the growth of thallus.
2, freeze drying protectant
Protective agent combines in the present invention, to thallus protectiveness, homogeneity, inoxidizability, anti-cryoprotection and final bacterium
It is suitable for that physics and chemistry aspect is preferred that powder, which stores: skimmed milk power, lactose, glycerine, sodium glutamate, VC are preferably square as protective agent
Case, and freeze-drying formula is obtained by optimization constituent content；Wherein: skimmed milk power is macromolecular compound, has protection to microorganism
Effect, and promote its distillation to form heat-resisting skeleton and block heat transfer and heat radiation, it is protected for heat labile lactobacillus plantarum
Shield effect is obvious；Lactose is small molecule compound, can promote to form uniform suspension, plays moisture relaxation effect.Glycerine can
Prevent strain ice crystal in refrigerating process from forming the injury to thallus；It is appropriate that the close effect of sodium glutamate and water retains bacterium powder
Moisture meets the Minimum requirements that bacterial strain sustains life.VC can significantly improve the reserve temperature of bacterium powder as preferred antioxidant
And the time, increase its stability.
3, yield
The final viable count that ferments in the present invention can achieve 1.0 × 1011Normal fermentation phase in CFU/mL or more, with comparative example
Than in the situation similar in fermentation time and cost of material, viable count of the present invention can significantly improve 30 times, even and if comparative example
Middle optimization routine culture medium fermentation is compared, and viable count is improved also greater than 10 times, and yield and economical gain are significant.
Specific embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
Under the premise of invention spirit and scope, to the various changes or change of material component and dosage progress in these embodiments
It belongs to the scope of protection of the present invention.Below in conjunction with specific embodiment, invention is further explained.
1 high density fermentation culture lactobacillus plantarum of embodiment
(1) seed culture of lactobacillus plantarum: the lactobacillus plantarum strain CGMCC No.9513 that will be stored in glycerol tube
Access in 5mL MRS fluid nutrient medium 37 DEG C of standing activation cultures for 24 hours, as first order seed；Again by activated first order seed
Liquid is inoculated in 45mL MRS fluid nutrient medium with 3% inoculum concentration, and 37 DEG C of stationary cultures are secondary seed for 24 hours；Finally will
Activated secondary seed solution is in 3% inoculum concentration access 1500mL MRS fluid nutrient medium, 37 DEG C of stationary cultures are for 24 hours
Three-level seed liquor.The formula of the MRS fluid nutrient medium are as follows: 20g/L glucose, 10g/L soy peptone, the leaching of 5g/L yeast
Powder, 10g/L beef extract, 5g/L anhydrous sodium acetate, 2g/L dipotassium hydrogen phosphate, 2g/L sodium citrate, 0.2g/L MgSO4·7H2O,
0.054g/L MnSO4,1g/L Tween-80, distilled water dissolution are prepared.
(2) fermentation tank culture: fermenter volume 50L loads fermentation medium 30L, and sterilization temperature is 121 DEG C,
20min；The three-level seed liquor that culture obtains all is pumped into fermentor with 5% inoculum concentration, fermented and cultured temperature is 35 DEG C, is stirred
Revolving speed 100r/min is mixed, pH is automatically maintained at pH 6.2 using 50% ammonium hydroxide, and is passed through nitrogen and fermentation pressure tank is maintained to exist
0.05MPa；Sucrose feed supplement is taken in fermentation process, the sucrose concentration of the feed supplement is 1000g/L, the sucrose feed profile
Are as follows: fermented and cultured 7h when sucrose residual quantity is 0.05%, starts feed supplement with the rate of 5mL/Lh, after fermented and cultured to 13h,
When sucrose residual quantity is 0.06%, with 15-16mL/Lh rate feed supplement, after continuing fermentation to 18h, sucrose residual quantity is
When 0.12%, with 14mL/min rate feed supplement, continuing fermentation 2h terminates.
Thallus content obviously rises compared with common fermentation processes, and OD600 value reaches 31 at this time.By count plate, after fermentation
The viable count of measurement is 1.1 × 1011cfu/mL；
Wherein, fermentation medium forms are as follows: peptone 8g/L, sucrose 15g/L, yeast powder 15g/L, MgSO4﹒ 7H2O
0.2g/L, MnSO40.1g/L, K2HPO4The cane molasses of 1g/L, 0.5g/L, sodium acetate 3g/L, Tween 80 1.0mL/L disappear
Infusion 0.3mL/L, surplus are water.
(3) the fermentation liquid 5000g of obtained lactobacillus plantarum the separation of fermentation liquid: is centrifugated bacterium mud.
(4) vacuum freeze drying of bacterium mud: freeze drying protectant and bacterium mud weight in wet base are mixed according to the ratio of mass ratio 3:1
30min makes to suspend after uniformly mixing, and cold freeze at -80 DEG C carries out precooling processing 12h in case, then at -55 DEG C of temperature or so,
Vacuum degree 1-7Pa, the dry viable count that obtains afterwards for 24 hours is 3.1 × 1012The lactobacillus plantarum freeze-dried vaccine powder of cfu/g.
The component and content of freeze drying protectant are as follows: skimmed milk power 750g/L, lactose 450g/L, glycerine 20g/L, 1g/L paddy
Propylhomoserin sodium, VC 5g/L, are 0.1M with concentration, and the PBS liquid that pH is 6.2 dissolves.
(5) survival rate of the lactobacillus plantarum freeze-dried vaccine powder prepared reaches 98.9%.
2 high density fermentation culture lactobacillus plantarum of embodiment
(1) seed culture of lactobacillus plantarum: the lactobacillus plantarum strain CGMCC No.9513 that will be stored in glycerol tube
Access in 5mL MRS fluid nutrient medium 37 DEG C of standing activation cultures for 24 hours, as first order seed；Again by activated first order seed
Liquid is inoculated in 45mL MRS fluid nutrient medium with 3% inoculum concentration, and 37 DEG C of stationary cultures are secondary seed for 24 hours；Finally will
Activated secondary seed solution is in 3% inoculum concentration access 1500mL MRS fluid nutrient medium, 37 DEG C of stationary cultures are for 24 hours
Three-level seed liquor.The formula of the MRS fluid nutrient medium are as follows: 20g/L glucose, 10g/L soy peptone, the leaching of 5g/L yeast
Powder, 10g/L beef extract, 5g/L anhydrous sodium acetate, 2g/L dipotassium hydrogen phosphate, 2g/L sodium citrate, 0.2g/L MgSO4·7H2O,
0.054g/L MnSO4,1g/L Tween-80, distilled water dissolution are prepared.
(2) fermentation tank culture: fermenter volume 50L loads fermentation medium 30L, and sterilization temperature is 121 DEG C,
20min；The three-level seed liquor that culture obtains all is pumped into fermentor with 5% inoculum concentration, fermented and cultured temperature is 35 DEG C, is stirred
Revolving speed 100r/min is mixed, pH is automatically maintained at pH 6.2 using 50% ammonium hydroxide, and is passed through nitrogen and fermentation pressure tank is maintained to exist
0.05MPa；Sucrose feed supplement is taken in fermentation process, the sucrose concentration of the feed supplement is 1000g/L, the sucrose feed profile
Are as follows: fermented and cultured 7-8h starts feed supplement with the rate of 5mL/Lh, after fermented and cultured to 12h, with 18mL/Lh rate feed supplement,
After continuing fermentation to 17h, with 10mL/min rate feed supplement, continuing fermentation 2h terminates.
Thallus content obviously rises compared with common fermentation processes, and OD600 value can reach 30 or more at this time.By count plate,
The viable count measured after fermentation is 1.2 × 1011cfu/mL；
Wherein, fermentation medium forms are as follows: peptone 10g/L, sucrose 20g/L, yeast powder 20g/L, MgSO4﹒
7H2O0.5g/L, MnSO40.2g/L, K2HPO4The cane molasses of 3g/L, 1g/L, sodium acetate 5g/L, Tween 80 2.0mL/L,
Defoaming agent 0.5mL/L, surplus are water.
(3) the fermentation liquid 7000g of obtained lactobacillus plantarum the separation of fermentation liquid: is centrifugated bacterium mud.
(4) vacuum freeze drying of bacterium mud: freeze drying protectant and bacterium mud weight in wet base are mixed according to the ratio of mass ratio 3:1
30min makes to suspend after uniformly mixing, and cold freeze at -80 DEG C carries out precooling processing 12h in case, then at -55 DEG C of temperature or so,
Viable count is obtained greater than 3.2 × 10 after vacuum degree 1-7Pa, dry 18-30h12The lactobacillus plantarum freeze-dried vaccine powder of cfu/g.
The component and content of freeze drying protectant are as follows: skimmed milk power 800g/L, lactose 500g/L, glycerine 50g/L, glutamic acid
Sodium 20g/L, VC 10g/L are that the PBS liquid that 0.1M pH is 6.2 dissolves with concentration.
(5) survival rate of the lactobacillus plantarum freeze-dried vaccine powder prepared is 98.5%.
3 high density fermentation culture lactobacillus plantarum of embodiment
1, the seed culture of lactobacillus plantarum: the lactobacillus plantarum strain CGMCC No.9513 that will be stored in glycerol tube
Access in 5mL MRS fluid nutrient medium 37 DEG C of standing activation cultures for 24 hours, as first order seed；Again by activated first order seed
Liquid is inoculated in 45mL MRS fluid nutrient medium with 3% inoculum concentration, and 37 DEG C of stationary cultures are secondary seed for 24 hours；Finally will
Activated secondary seed solution is in 3% inoculum concentration access 1500mL MRS fluid nutrient medium, 37 DEG C of stationary cultures are for 24 hours
Three-level seed liquor.The formula of the MRS fluid nutrient medium are as follows: 20g/L glucose, 10g/L soy peptone, the leaching of 5g/L yeast
Powder, 10g/L beef extract, 5g/L anhydrous sodium acetate, 2g/L dipotassium hydrogen phosphate, 2g/L sodium citrate, 0.2g/L MgSO4·7H2O,
0.054g/L MnSO4,1g/L Tween-80, distilled water dissolution are prepared.
2, fermentation tank culture: fermenter volume 50L loads fermentation medium 30L, and sterilization temperature is 121 DEG C,
20min；The three-level seed liquor that culture obtains all is pumped into fermentor with 5% inoculum concentration, fermented and cultured temperature is 35 DEG C, is stirred
Revolving speed 100r/min is mixed, pH is automatically maintained at pH 6.2 using 50% ammonium hydroxide, and is passed through nitrogen and fermentation pressure tank is maintained to exist
0.05MPa；Sucrose feed supplement is taken in fermentation process, the sucrose concentration of the feed supplement is 800g/L, the sucrose feed profile are as follows:
Fermented and cultured when sucrose residual quantity is 0.06% at this time, starts to carry out feed supplement, be started by feed supplement pump with 5mL/Lh's to 7h
Rate flow feeding culture medium, maintaining residual sugar content in fermentation liquid is 0.05%-0.10%, after fermented and cultured is to 11h, with
Cell density increases, and when OD600 reaches 15, residual sugar content is reduced to 0.05% in fermentation liquid at this time, therefore improves feed rate extremely
10mL/Lh continues fed-batch cultivation, and maintaining residual sugar content in fermentation liquid is 0.05%-0.10%, ferments about when proceeding to
After 15h, as cell density further increases, when OD600 reaches 20, residual sugar content is reduced to again in fermentation liquid at this time
0.05%, it needs to further increase feed rate at this time to 15mL/Lh and continues fed-batch cultivation, maintain residual sugar in fermentation liquid
Content is 0.05%-0.10%, continues fermentation to after 18h, when OD600 is 26, residual sugar content 0.15%, bacterium in fermentation liquid at this time
Body grows into the transitional period of late log phase and stationary phase, and carbon source consumption is no longer linearly increasing, should turn down feed rate extremely at this time
10mL/min, maintaining residual sugar content in fermentation liquid is 0.05%-0.10%, and continuing fermentation 2h terminates.
The fermentative medium formula are as follows: peptone 10g/L, sucrose 20g/L, yeast powder 20g/L, MgSO4 ﹒
7H2O0.5g/L, MnSO40.1g/L, K2HPO4The cane molasses of 2g/L, 0.5g/L, sodium acetate 5g/L, Tween 80 1mL/L disappear
Infusion 0.3mL/L, distilled water dissolution are prepared.The feed-batch culture based formulas are as follows: 1000g/L sucrose.Final OD600 value reaches about
It is 29.4.By viable plate count, the viable count measured after fermentation is higher than 2.2 × 1011cfu/mL
3, the fermentation liquid 5000g of obtained lactobacillus plantarum the separation of fermentation liquid: is centrifugated bacterium mud.
4, the vacuum freeze drying of bacterium mud: freeze drying protectant and bacterium mud weight in wet base are mixed according to the ratio of mass ratio 3:1
30min makes to suspend after uniformly mixing, and cold freeze at -80 DEG C carries out precooling processing 12h in case, then at -55 DEG C of temperature or so,
Obtaining viable count after vacuum degree 1-7Pa, dry 30h is 3.1 × 1012The lactobacillus plantarum freeze-dried vaccine powder of cfu/g.The freeze-drying is protected
Protect the component and content of agent are as follows: skimmed milk power 750g/L, lactose 500g/L, glycerine 20g/L, 1g/L sodium glutamate, VC5g/L,
It is the PBS liquid dissolution that 0.1M pH is 6.2 with concentration.；
5, the survival rate of the lactobacillus plantarum freeze-dried vaccine powder prepared can reach 99.4%.
4 high density fermentation culture lactobacillus plantarum of embodiment
1, the seed culture of lactobacillus plantarum: the lactobacillus plantarum strain CGMCC No.9513 that will be stored in glycerol tube
Access in 5mL MRS fluid nutrient medium 37 DEG C of standing activation cultures for 24 hours, as first order seed；Again by activated first order seed
Liquid is inoculated in 45mL MRS fluid nutrient medium with 3% inoculum concentration, and 37 DEG C of stationary cultures are secondary seed for 24 hours；Finally will
Activated secondary seed solution is in 3% inoculum concentration access 1500mL MRS fluid nutrient medium, 37 DEG C of stationary cultures are for 24 hours
Three-level seed liquor.The formula of the MRS fluid nutrient medium are as follows: 20g/L glucose, 10g/L soy peptone, the leaching of 5g/L yeast
Powder, 10g/L beef extract, 5g/L anhydrous sodium acetate, 2g/L dipotassium hydrogen phosphate, 2g/L sodium citrate, 0.2g/L MgSO4·7H2O,
0.054g/L MnSO4,1g/L Tween-80, distilled water dissolution are prepared.
2, fermentation tank culture: fermenter volume 50L loads fermentation medium 30L, and sterilization temperature is 121 DEG C,
20min；The three-level seed liquor that culture obtains all is pumped into fermentor with 5% inoculum concentration, fermented and cultured temperature is 35 DEG C, is stirred
Revolving speed 100r/min is mixed, pH is automatically maintained at pH 6.2 using 50% ammonium hydroxide, and is passed through nitrogen and fermentation pressure tank is maintained to exist
0.05MPa；Sucrose feed supplement is taken in fermentation process, the sucrose concentration of the feed supplement is 900g/L, the sucrose feed profile are as follows:
Fermented and cultured about 8h when sucrose residual quantity is 0.05% at this time, starts to carry out feed supplement, be started by feed supplement pump with 6mL/Lh's
Rate flow feeding culture medium, maintaining residual sugar content in fermentation liquid is 0.05%-0.10%, after fermented and cultured is to 11h, with
Cell density increases, and when OD600 reaches 18, residual sugar content is reduced to 0.05% in fermentation liquid at this time, therefore improves feed rate extremely
15mL/Lh continues fed-batch cultivation, and maintaining residual sugar content in fermentation liquid is 0.05%-0.10%, ferments about when proceeding to
After 15h, as cell density further increases, when OD600 reaches 25, residual sugar content is reduced to again in fermentation liquid at this time
0.05%, it needs to further increase feed rate at this time to 18mL/Lh and continues fed-batch cultivation, maintain residual sugar in fermentation liquid
Content is 0.05%-0.10%, continues fermentation to after 18h, when OD600 is 28, residual sugar content 0.15%, bacterium in fermentation liquid at this time
Body grows into the transitional period of late log phase and stationary phase, and carbon source consumption is no longer linearly increasing, should turn down feed rate extremely at this time
15mL/min, maintaining residual sugar content in fermentation liquid is 0.05%-0.10%, and continuing fermentation 2h terminates.
Wherein fermentative medium formula are as follows: peptone 9g/L, sucrose 15g/L, yeast powder 15g/L, MgSO4﹒ 7H2O 0.5g/
L, MnSO40.1g/L, K2HPO4The cane molasses of 2g/L, 0.5g/L, sodium acetate 5g/L, Tween 80 1mL/L, defoaming agent
0.3mL/L, distilled water dissolution are prepared.The feed-batch culture based formulas are as follows: 900g/L sucrose.Final OD600 value reaches about
31.8.By viable plate count, the viable count measured after fermentation is higher than 2.1 × 1011cfu/mL
3, the fermentation liquid 5000g of obtained lactobacillus plantarum the separation of fermentation liquid: is centrifugated bacterium mud.
4, the vacuum freeze drying of bacterium mud: freeze drying protectant and bacterium mud weight in wet base are mixed according to the ratio of mass ratio 3:1
30min makes to suspend after uniformly mixing, and cold freeze at -80 DEG C carries out precooling processing 12h in case, then at -55 DEG C of temperature or so,
Obtaining viable count after vacuum degree 1-7Pa, dry 28h is 3.2 × 1012The lactobacillus plantarum freeze-dried vaccine powder of cfu/g.The freeze-drying is protected
Protect the component and content of agent are as follows: skimmed milk power 800g/L, lactose 500g/L, glycerine 50g/L, sodium glutamate 10g/L, VC
10g/L is that the PBS liquid that 0.1M pH is 6.2 dissolves with concentration.
5, the survival rate of the lactobacillus plantarum freeze-dried vaccine powder prepared can reach 99.1%.
What the above-mentioned method prepared referring to embodiment to the culture of lactobacillus plantarum high density liquid and bacterium powder carried out retouches in detail
It states, is illustrative without being restrictive, several embodiments can be enumerated according to limited range, therefore do not departing from this
Change and modification under invention general plotting should belong within protection scope of the present invention.
Comparative example 1: common process high density fermentation culture lactobacillus plantarum
1, the seed culture of lactobacillus plantarum: with embodiment 1；
2, fermentation tank culture: fermenter volume 50L loads fermentation medium 30L, and sterilization temperature is 121 DEG C,
20min；The three-level seed liquor that culture obtains all is pumped into fermentor with 5% inoculum concentration, fermented and cultured temperature is 35 DEG C, is stirred
Revolving speed 100r/min is mixed, pH is automatically maintained at pH 6.2 using 50% ammonium hydroxide, and is passed through nitrogen and fermentation pressure tank is maintained to exist
0.05MPa；In fermented and cultured about 8h, pH goes up not down in culture solution, it was demonstrated that fermentation terminates, OD600 measured value is at this time
13.5 or so, viable count 3.0 × 109cfu/mL.Fermentation medium is formed with embodiment 1.
3, the separation of fermentation liquid: with embodiment 1；
4, the vacuum freeze drying of bacterium mud: freeze drying protectant and bacterium mud weight in wet base are mixed according to the ratio of mass ratio 3:1
30min makes to suspend after uniformly mixing, and cold freeze at -80 DEG C carries out precooling processing 12h in case, then at -55 DEG C of temperature or so,
Obtaining viable count after vacuum degree 1-7Pa, dry 30h is 2.1 × 1011The lactobacillus plantarum freeze-dried vaccine powder of cfu/g.The freeze-drying is protected
Protect the component and content of agent are as follows: skimmed milk power 750g/L, lactose 500g/L, VC 5g/L are dissolved with distilled water.
5, the survival rate of the lactobacillus plantarum freeze-dried vaccine powder prepared can reach 80.8%.
2 high density fermentation culture lactobacillus plantarum of comparative example
1, the seed culture of lactobacillus plantarum: with embodiment 1；
2, fermentation tank culture: fermenter volume 50L loads fermentation medium 30L, and sterilization temperature is 121 DEG C,
20min；The three-level seed liquor that culture obtains all is pumped into fermentor with 5% inoculum concentration, fermented and cultured temperature is 35 DEG C, is stirred
Revolving speed 100r/min is mixed, pH is automatically maintained at pH 6.2 using 50% ammonium hydroxide, and is passed through nitrogen and fermentation pressure tank is maintained to exist
0.05MPa；In fermented and cultured about 8h, sucrose residual quantity is 0.06%, should start to carry out feed supplement at this time, is started by feed supplement pump
With the rate flow feeding culture medium of 6mL/Lh, after fermented and cultured is to 13h, as cell density increases, OD600 reaches 17
When, feed rate is adjusted at this time to 18mL/min and continues fed-batch cultivation, and continuing to ferment to 20h terminates.The fermented and cultured
Based formulas are as follows: fermentation medium is formed with embodiment 1.The feed-batch culture based formulas are as follows: 900g/L sucrose.Final OD600 value
Reach about 23.8.By viable plate count, the viable count measured after fermentation is higher than 7.4 × 1010cfu/mL
3, the separation of fermentation liquid: with embodiment 1；
4, the vacuum freeze drying of bacterium mud: freeze drying protectant and bacterium mud weight in wet base are mixed according to the ratio of mass ratio 3:1
30min makes to suspend after uniformly mixing, and cold freeze at -80 DEG C carries out precooling processing 12h in case, then at -55 DEG C of temperature or so,
Obtaining viable count after vacuum degree 1-7Pa, dry 28h is 3.4 × 1012The lactobacillus plantarum freeze-dried vaccine powder of cfu/g.The freeze-drying is protected
Protect the component and content of agent are as follows: skimmed milk power 800g/L, lactose 500g/L, glycerine 50g/L, sodium glutamate 10g/L, VC
10g/L is dissolved with distilled water and is prepared.
5, the survival rate of the lactobacillus plantarum freeze-dried vaccine powder prepared can reach 90.9%.
3 optimization routine culture method high density fermentation culture lactobacillus plantarum of comparative example
(1) activation of strain:
The lactobacillus plantarum CGMCC No.9513 of freezen protective is inoculated in MRS fluid nutrient medium (MRS culture medium: egg
White peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, trisodium citrate 2g are spat
Temperature -801g, magnesium sulfate 200mg, manganese sulfate 54mg, 1000mL, pH6.5,121 DEG C of 15min sterilizings of distilled water are spare), 37 DEG C of trainings
Activated spawn is obtained after supporting 15-20 hours, such secondary culture 2 times；
(2) preparation of Optimal Medium:
Sucrose 15-30kg, yeast powder 15-30kg, peptone 7-15kg, composite buffering salting liquid 1-3mL, complex inorganic salt catalyst
Solution 0.1-0.3mL, distilled water 1000L.Sufficiently dissolution, pH value are not adjusted, and sterilize 20min at 121 DEG C.
Each component content ratio of the composite buffering salting liquid is as follows: anhydrous sodium acetate 45kg, dipotassium hydrogen phosphate 70kg,
Potassium dihydrogen phosphate 77kg, sodium bicarbonate 37kg, distilled water 1000L；
Each component content ratio of the composite inorganic salting liquid is as follows: MgSO4.7H2O 2kg, MnSO4·5H2O 2
3kg, anhydrous calcium chloride 1.5kg, distilled water 1000L.
(3) preparation of seed fermentation liquid:
Above-mentioned activated strain is inoculated in Optimal Medium with the percentage by volume for accounting for Optimal Medium 3%, 30
DEG C culture 12h；
(4) inoculation fermentation
Seed culture fluid is inoculated in Optimal Medium with the percentage by volume for accounting for Optimal Medium 6%, 30-37 DEG C of perseverance
Temperature culture, spontaneous fermentation in 0-2 hours, automatic control Feeding ammonia water (concentration 25%) holding pH5.5-5.8,6-8 are small within 2-6 hours
When spontaneous fermentation, until fermentation terminate, obtain lactobacillus plantarum high-density culture fluid, viable count reaches 8.1x109CFU/mL。
The survival rate of 1 microbial inoculum of experimental example measures
By the lactobacillus plantarum microbial inoculum of 1-4 of the embodiment of the present invention and comparative example 1-2 according to actual storage, traffic condition,
The survival rate of bacterial strain is measured with 25 DEG C of room temperature.
Survival rate (%) overview that 1 thallus freeze-dried powder of table saves under normal temperature condition (25 DEG C)
As shown in Table 1, the lactobacillus plantarum freeze-dried powder prepared using freeze drying protectant provided by the present application and technique is 25
It is stored 12 months under the conditions of DEG C, survival rate is still up to 80.0% or more, and uses comparative example freeze drying protectant and method system
At identical conditions, 1 freeze-dried powder of comparative example has fallen to standby freeze dried powder in the time of preservation 6 months, survival rate
10% hereinafter, 2 freeze-dried powder of comparative example save 12 months later periods survival rate be 74.0%, illustrate protective agent provided by the present application and
The freeze-dried powder storage stability of method preparation is more preferable.
Influence of 2 microbial inoculum of experimental example to meat chicken production performance and immune function
1 materials and methods
1.1 experimental material
1.1.1 experimental animal
14 age in days AA white meat-type chickens, 120 plumage.
1.1.2 reagent
The lactobacillus plantarum microbial inoculum 3.2 × 10 prepared using the embodiment of the present invention 412Cfu/g, with dextrin dilution 2 ×
108CFU/g manufactures experimently unit: Tianjin Shengji Group Co.Ltd.The dextrin derives from starch.
1.2 experimental method
1.2.1 grouping
Healthy chick on the 14th similar in weight is randomly divided into 4 groups, every group of 3 repetitions, each 10 plumage of repetition is remembered respectively
For 0.3% lactobacillus plantarum preparation processing group T1, 0.1% lactobacillus plantarum preparation processing group T2, 0.05% lactobacillus plantarum preparation
Processing group T3With blank control group T0.The lactobacillus plantarum preparation in 14 age in days test group daily rations, blank control group feed basic day
Grain.Feeding cycle is 15-42 age in days.
The test grouping of table 1
| Processing group | Daily ration |
| T0 | Basal diet |
| T1 | + 0.3% lactobacillus plantarum preparation processing group of basal diet |
| T2 | + 0.1% lactobacillus plantarum preparation processing group of basal diet |
| T3 | + 0.05% lactobacillus plantarum preparation processing group of basal diet |
It is immune according to conventional Broiler chicks immune programme to henhouse and breeding facility strict sterilization before test.It is fixed during test
Amount feeds intake, and is freely eaten and drinks water.
1.2.2 daily ration is tested
Table 2 tests daily ration and forms (air bells dry basis) %:
Note: premix ingredient: multidimensional 0.03；Micro 0.3, choline 0.1, empty portions supply 100% with zeolite powder
1.2.2 Testing index
1.2.2.1 growth performance measures
Test measures each repeating groups chicken weight and feed consumption rate daily, calculates daily gain, day feed consumption and feed-weight ratio.
Day feed consumption: record each group broiler chicken feed consumption rate daily counts each group feed consumption total amount respectively, calculates daily material consumption.
Feed-weight ratio: according to feed consumption total amount and weight gain total amount, each group feedstuff-meat ratio is calculated；
Average daily gain: according to initial weight, end weight and age in days, each group daily gain is calculated.
1.2.2.2 Biochemical Indices In Serum measures
In 28,42 age in days early mornings, each repeating groups take 3 chickens at random, and Culling heart blood prepares blood serum sample, measure in serum
The content of total protein (TP), albumin (ALB) and globulin (GLOB).Biochemical Indices In Serum measurement according to kit specification into
Row.
1.2.2.3 Development of Immune Organs index determining
In 28,42 age in days early mornings, each repeating groups take 3 chickens at random, analyse after Culling heart blood, take thymus gland, spleen, method
Family name's capsule, weighing, Computation immunity shoot formation.
2 results
Influence of the 2.1 lactobacillus plantarum preparation processing groups to broiler growth performance
Seen from table 3, diet adds lactobacillus plantarum preparation processing group prepared by the present invention, microbial inoculum bacterium bacterium with higher
Strain vigor, increases significantly for the growth performance of broiler chicken, and different microbial inoculum additive amounts are young to the meat of different stages of growth
Chicken growth performance has significant impact.
The influence of daily gain: different disposal group chicken average daily gain has more apparent difference, and test group tests full period
Average daily gain is extremely significant to be higher than control group (P < 0.01), and in 22-28d and 36-42d, average daily gain is significantly higher than control
Group (P < 0.05), it is then extremely significant lower than control group (P < 0.01) in 29-35d.
The influence of average daily gain: test group and control group average daily gain only in 29-35d it is extremely significant lower than pair
According to group (P < 0.01), the substantially less than control group (P < 0.05) in 36-42d；It is not significant (P > 0.05) to test full period difference.
The influence of feed-weight ratio: compared with the control group, each growth phase of test group chicken and test full period feed-weight ratio compared with
Low, wherein heteropolar significant (P < 0.01) in 22-28d the and 36-42d time difference, test full period difference reaches the level of signifiance (P < 0.05).
The influence difference of the broiler growth performance indicator of various dose test group different stages of growth is not significant, test
The a little higher than blank control group of full period test group average daily gain, and feed-weight ratio is lower than control group.
Influence of 3. different disposal of table to broiler growth performance
Note: colleague's data subscript difference lowercase letter indication difference is significant (P < 0.05), and different capitalizations indicate difference
Extremely significant (P < 0.01), non-marking-up mother person indicate that difference is not significant (P > 0.05).Similarly hereinafter.
Influence of the 2.2 lactobacillus plantarum preparation processing groups to Broiler chicks Biochemical Indices In Serum
By table 4 as it can be seen that when 28-42 age in days of age in days, various dose test group the Total albumen content and blank control group phase
Than significant difference (P < 0.05)；The Broiler chicks serum biochemistry of various dose test group and blank control group to different stages of growth
The influence difference of index is not significant, and in addition to 42d albumin content, the serum albumin content of various dose test group chicken is equal
There is different degrees of raising compared with blank control group.
Influence of 4. different disposal of table to Broiler chicks Biochemical Indices In Serum
Influence of the 2.3 lactobacillus plantarum preparation processing groups to Broiler chicks Development of Immune Organs
It is learnt by table 5, each test group and control group Broiler chicks 28d and 42d thymus gland, spleen and bursal index are compared with control group
Difference that there are no significant.But on the whole, in addition to 42d index and spleen index, various dose lactobacillus plantarum preparation processing group chicken
Immune Organs Index have different degrees of raising compared with control group, illustrate the present invention prepare lactobacillus plantarum preparation have promote meat
The trend of chick Development of Immune Organs.
Influence of 5. different disposal of table to Broiler chicks Development of Immune Organs
3 discuss
The result shows that: after high density fermentation culture, viable count is more than lactobacillus plantarum preparation prepared by the present invention
1011Cfu/mL breaches normal fermentation bacterial strain activity level obtained, and viable count is obviously improved, and microbial inoculum of the present invention has
Standby significant application activity, production performance and immune function for broiler chicken have significant impact, by by microbial inoculum of the present invention
It is added in feed, in the test a little higher than blank control group of full period test group average daily gain, and feed-weight ratio is lower than blank control
Group；In addition to 42d albumin content, the serum albumin content of test group chicken has different degrees of raising compared with positive controls；
In addition to 42d index and spleen index, the Immune Organs Index of test group chicken has different degrees of raising compared with blank control group, shows to try
The lactobacillus plantarum preparation tested has significant application effect.
Prepared by the method lactobacillus plantarum formulation application is in broiler fodder, hence it is evident that improves the growth of broiler chicken
Can, health status is improved, average daily gain, feed-weight ratio and average daily gain etc. are improved in varying degrees, and can obviously be promoted
Into the development of immune organ.
Claims (10)
1. a kind of high density fermentation preparation method of lactobacillus plantarum microbial inoculum, it is characterised in that: steps are as follows: by lactobacillus plantarum
After seed culture, fermentation medium is accessed according to inoculum concentration 3-5%, shake culture carries out sucrose feed supplement, institute in fermentation process
The sucrose concentration for stating feed supplement is 800-1000g/L, the sucrose feed profile are as follows: when fermented and cultured to 7-8h, sucrose residual quantity
When for 0.04-0.06%, feed supplement is started with the rate of 5-6mL/Lh；After fermented and cultured is to 11-15h, sucrose residual quantity is
When 0.04-0.10%, with 10-18mL/Lh rate feed supplement, after continuing fermentation to 17-19h, sucrose residual quantity is 0.10-
When 0.15%, with 10-15mL/min rate feed supplement, continuing fermentation 1-2.5h terminates, and gained fermentation liquid is obtained thallus through centrifugation,
Freeze drying protectant mixing, and freeze-dried acquisition lactobacillus plantarum microbial inoculum are added into the thallus.
2. a kind of high density fermentation preparation method of lactobacillus plantarum microbial inoculum as described in claim 1, it is characterised in that: the sugarcane
Sugared feed profile are as follows: fermented and cultured 7-8h starts to mend when sucrose residual quantity is 0.04-0.06% with the rate of 5-6mL/Lh
Expect, after fermented and cultured to 11-12h, when sucrose residual quantity is 0.04-0.1%, with 10-15mL/Lh rate feed supplement, fermentation is extremely
After 14-15h, when sucrose residual quantity is 0.05-0.10%, fed-batch cultivation is continued with 15-18mL/Lh rate, continues fermentation extremely
After 18-19h, when sucrose residual quantity is 0.10-0.15%, with 10-15mL/min rate feed supplement, continue the 1.5-2.5h knot that ferments
Beam.
3. a kind of high density fermentation preparation method of lactobacillus plantarum microbial inoculum as described in claim 1, it is characterised in that: the jelly
Dry protective agent composition are as follows: skimmed milk power 750-800g/L, lactose 450-500g/L, glycerine 20-50g/L, sodium glutamate 1-
20g/L, VC 5-10g/L, surplus are that concentration is 0.1M, the PBS solution that pH is 6.2.
4. a kind of high density fermentation preparation method of lactobacillus plantarum microbial inoculum as described in claim 1, it is characterised in that: the hair
Ferment culture medium composition are as follows: peptone 8-10g/L, sucrose 15-20g/L, yeast powder 15-20g/L, MgSO4﹒ 7H20 0.2-0.5g/
L, MnSO40.1-0.2g/L, K2HPO4The cane molasses of 1-3g/L, 0.5-1g/L, sodium acetate 3-5g/L, Tween 80 1.0-
2.0mL/L, defoaming agent 0.3-0.5mL/L, surplus are water.
5. a kind of high density fermentation preparation method of lactobacillus plantarum microbial inoculum described in claim 1, it is characterised in that: the fermentation
In incubation, fermented and cultured temperature is 35-37 DEG C, speed of agitator 90-100r/min, maintains fermentation pH6.1-6.3.
6. a kind of high density fermentation preparation method of lactobacillus plantarum microbial inoculum as claimed in claim 3, it is characterised in that: specific step
It is rapid as follows:
(1) seed culture: lactobacillus plantarum is accessed in seed fluid nutrient mediums of saccharomycete and stands activation culture in 35-37 DEG C of incubator
21-24h obtains primary seed solution；The primary seed solution is inoculated in seed fluid nutrient mediums of saccharomycete with inoculum concentration 3-5%, 35-
37 DEG C of stationary culture 21-24h, obtain secondary seed solution；By the secondary seed solution with inoculum concentration 3-5% access seed liquid training
It supports in base, 35-37 DEG C of stationary culture 21-24h obtains three-level seed liquor；
(2) fermented and cultured: by the three-level seed liquor in inoculum concentration 3-5% access fermentation medium, fermented and cultured temperature is
35-37 DEG C, speed of agitator 90-100r/min, constant pH 6.1-6.3 is adjusted using 50% ammonium hydroxide, and be passed through nitrogen maintenance
Fermentation pressure tank is 0.04-0.06MPa；Sucrose feed supplement is taken in fermentation process, the sucrose concentration of the feed supplement is 800-
1000g/L, the sucrose feed profile are as follows: as fermented and cultured 7-8h, when sucrose residual quantity is 0.04-0.06%, with 5-6mL/
The rate of Lh starts feed supplement；After fermented and cultured is to 11-15h, when sucrose residual quantity is 0.04-0.10%, with 10-18mL/
Lh rate feed supplement, after continuing fermentation to 17-19h, when sucrose residual quantity is 0.10-0.15%, with 10-15mL/min rate benefit
Material, continuing fermentation 1-2.5h terminates；
(3) prepared by microbial inoculum: the fermentation liquid of obtained lactobacillus plantarum being centrifuged through 5000-10000g, obtains bacterium mud；Freeze-drying is protected
Agent and bacterium mud is protected to be mixed according to the ratio of mass ratio 2-4:1, after pre-cooled processing, Yu Wendu -55 DEG C--60 DEG C, vacuum
1-7Pa is spent, 10-12h is handled, obtains lactobacillus plantarum freeze-dried vaccine powder after dry 18-30h.
7. a kind of high density fermentation preparation method of lactobacillus plantarum microbial inoculum as claimed in claim 6, it is characterised in that: the step
Suddenly in (1), the seed fluid nutrient mediums of saccharomycete is specially MRS fluid nutrient medium；The level-one, second level, three-level seed Liquid Culture
Base volume ratio are as follows: 5:45:1500.
8. a kind of high density fermentation preparation method of lactobacillus plantarum microbial inoculum as claimed in claim 6, it is characterised in that: the step
Suddenly in (4), the freeze drying protectant and bacterium mud mass ratio are 3:1.
9. the lactobacillus plantarum microbial inoculum that the high density fermentation preparation method as described in claim 1-8 is any prepares.
10. purposes of the lactobacillus plantarum microbial inoculum as claimed in claim 9 in chicken feed.
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