CN107177522A - One plant height activity forage plant lactobacillus and its cultural method and application - Google Patents
One plant height activity forage plant lactobacillus and its cultural method and application Download PDFInfo
- Publication number
- CN107177522A CN107177522A CN201610132132.9A CN201610132132A CN107177522A CN 107177522 A CN107177522 A CN 107177522A CN 201610132132 A CN201610132132 A CN 201610132132A CN 107177522 A CN107177522 A CN 107177522A
- Authority
- CN
- China
- Prior art keywords
- lactobacillus plantarum
- wei
- cgmcc
- lactobacillus
- culture
- 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.)
- Withdrawn
Links
Classifications
-
- 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
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
-
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
Abstract
The invention belongs to lactic acid bacteria culture technique field, it is related to one plant of forage plant lactobacillus and its cultural method and application.Lactobacillus plantarum WEI 70（Lactobacillus plantarum）CGMCC No.11747 are isolated from the excrement of 60 age in days sodium selenites, can tolerate the Pig cholate of low pH and high concentration, and have stronger fungistatic effect to Escherichia coli, salmonella, streptococcus and staphylococcus aureus, and inhibition zone is up to 30 35 mm.The present invention obtains Lactobacillus plantarum WEI 70 by optimization culture based formulas, cultivation temperature and the most suitable initial pH of fermentation（Lactobacillus plantarum）CGMCC No.11747 high density liquid cultural method, the viable count maximum of zymotic fluid can reach 6.0 × 109CFU/mL, is more than 6 times of the viable count obtained with MRS culture mediums under same condition of culture.By above-mentioned zymotic fluid by high speed tubular type centrifugal collection thalline after, after being mixed by a certain percentage with special-purpose protecting agent, the viable count of lyophilized bacterium powder that vacuum freeze drying is obtained can reach 7.0 × 1011CFU/g, survival rate is up to 93%.
Description
Technical field：
The invention belongs to lactic acid bacteria culture technique field, it is related to the active forage plant lactobacillus of a plant height and its cultural method and answers
With.
Background technology：
Lactobacillus plantarum（Lactobacillus plantarum）It is the Ministry of Agriculture《Catalogue of feed additive varieties》Listed strain,
The normal flora belonged in people and animal intestinal tract, into enteron aisle after can effectively colonize.Lactobacillus plantarum metabolism carbohydrate production
Lactogenesis acid, advantageously reduces intestinal environment pH, suppresses the growth of harmful bacteria.Peroxide can be also produced in Lactobacillus plantarum metabolic process
Change hydrogen, the material such as bacteriocin has certain killing action to pathogenic bacteria, and do not have medicament residue.In view of Lactobacillus plantarum
Above characteristic, increasing producer is selected as one of production strain of probiotics product.
MRS culture mediums being selected traditional lactobacillus-fermented, expensive, the viable count after fermentation is less than 1 × 10 more9
CFU/mL, in the case where the effective dosage of probiotics is fixed, low fermentation level adds cost equal to covert.Low cost
Culture medium, the zymotechnique that can efficiently amplify for promote probiotic products industrialization have it is great guidance and practice anticipate
Justice.
In a word, a plant height active plant lactobacillus is screened, and develops a kind of low cost, high yield, can stay active for long periods
Lactobacillus plantarum probiotics, for development green feed, it is ensured that food security has great importance.
The content of the invention：
（One）Goal of the invention：
First purpose of the present invention is to provide one plant of forage plant lactobacillus（Lactobacillus plantarum）WEI-70
CGMCC No.11747。
The excrement of 60 age in days sodium selenites is gathered, therefrom separates, screen, identify a lactobacillus plantarum
Lactobacillus plantarum, are named as WEI-70, and send Chinese microorganism strain preservation pipe on November 26th, 2015
Reason committee common micro-organisms center, numbering is CGMCC No. 11747, preservation address：BeiChen West Road, Chaoyang District, BeiJing City 1
Institute of microbiology of the Chinese Academy of Sciences of institute postcode:100101 phones：86-10-64807596.
Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 are Gram-positive
Bacterium, nose circle is shaft-like, no gemma, atrichia；Amphimicrobian, optimum growth temperature is 30-40 DEG C, and optimal pH is 6.0-7.0.
20 more than h, viable count accessible 1 × 10 are cultivated in MRS culture mediums9 CFU/mL。
Second object of the present invention is to provide Lactobacillus plantarum（Lactobacillus plantarum）WEI-70
CGMCC No.11747 high density liquid cultural method.
The Lactobacillus plantarum that the present invention is provided（Lactobacillus plantarum）WEI-70 CGMCC No.11747
High density liquid cultural method, be that the bacterium is seeded into special fluid nutrient medium to be cultivated, fluid nutrient medium it is main
Composition is as follows：
The g/L of maltodextrin 20, the g/L of corn starch 32, the g/L of sodium acetate trihydrate 3, the g/L of three hypophosphite monohydrate hydrogen dipotassium 2, lemon
Lemon acid ammonium 2 g/L, the g/L of bitter salt 0.6, the g/L of Manganous sulfate monohydrate 0.1, the g/L of Tween 80 1, pH value is 6.5.
In above-mentioned cultural method, inoculative proportion is 1%；Cultivation temperature is 34-37 DEG C；Stand or stuffiness culture, training
The time of supporting is 16-20 h.
Under 50 L fermentation conditions, using above-mentioned culture medium at 37 DEG C, the rpm of rotating speed 100, airproof condition is issued
The Lactobacillus plantarum obtained after the h of ferment 24（Lactobacillus plantarum）WEI-70 CGMCC No.11747 fermentation
The viable count of liquid can reach 5.0 × 109-6.0×109CFU/mL.And under similarity condition, Lactobacillus plantarum（Lactobacillus
plantarum）Viable count after WEI-70 CGMCC No.11747 are cultivated in MRS culture mediums only has 9.2 × 108 CFU/
ML, the culture medium after optimization makes viable count improve more than 5 times.
Third object of the present invention is to provide Lactobacillus plantarum（Lactobacillus plantarum）WEI-70
CGMCC No.11747 as probiotics preparation method, by strain through first order seed shaking flask and 50 L fermentation tanks enrichment after
Zymotic fluid by high speed tubular type centrifugal collection bacterium mud, be well mixed with bacteria protectant, prior to -70 DEG C pre-freeze 4-5 h, so
After be placed in vacuum freeze drier, freezed under following lyophilized parameter：Vacuum is less than 30 Pa, and condenser temperature is -40
DEG C to -50 DEG C, 41 h are freezed, active microbe powder is obtained after drying, by the bacterium powder corncob crushed after crusher machine with same granularity
Powder is mixed, and obtains probiotics product.
The rotating speed of the high speed tubular type centrifugal collection thalline is 15000-20000 rpm.
The bacteria protectant formula is：Contain in every 1000 mL water：The g of skimmed milk power 100, the g of sodium glutamate 10, sea
Algae sugar 10 g, the g of glycerine 30.
The mass volume ratio of the bacterium mud and bacteria protectant is 1:5.
Forage plant lactobacillus WEI-70 CGMCC No.11747 can be raiseeed as probiotics products application in regulation
In fowl intestinal microecology balance.
Forage plant lactobacillus WEI-70 CGMCC No.11747 can as probiotics products application prevention or
In auxiliary treatment fowl bacterial diarrhoea.
（Two）Meaning：
The invention provides a lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747,
And the fermentation medium of the bacterium is optimized, the effect for realize and reduce cost, improving fermentation viable count.Lactobacillus plantarum
（Lactobacillus plantarum）WEI-70 CGMCC No.11747 carry out the hair of 50 L fermentation tank levels with this technique
Ferment, viable count can reach 6.0 × 109CFU/mL, is more than 6 times of MRS culture mediums.The micro- life made on this basis
State preparation freeze-dried powder viable count reaches as high as 7.0 × 1011 CFU/g。
The Lactobacillus plantarum that the present invention is provided（Lactobacillus plantarum）WEI-70 CGMCC No.11747
With significant probiotic properties, animal can survive after taking by gastric juice and cholate, and in enteron aisle.According to above-mentioned culture side
Probiotics product prepared by method and post-processing approach, as livestock and poultry additive for microbe feedstuff, can suppress harmful intestinal tract
The propagation of bacterium, adjusts animal and bird intestines microecological balance, improves Production of Livestock and Poultry ability, resistance against diseases, finally reduces feeding antibiotic
Use.
Brief description of the drawings
Fig. 1 is Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 are in artificial stomach
Liquid（pH2.0）In survival curve.
Fig. 2 is Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 are in Pig cholate
Solution（The g/L of gallbladder salinity 20）In survival curve.
Fig. 3 is Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 are to artificial stomach
The tolerance of intestinal digestion liquid.Preceding 2 h in figure（Dotted line is boundary）It is survival curve of the bacterial strain in simulated gastric fluid, 22 subsequent h are
Bacterial strain is forwarded to the survival curve after simulated intestinal fluid from simulated gastric fluid.
Fig. 4 is Lactobacillus plantarum（Lactobacillus plantarum）The most suitable fermentations of WEI-70 CGMCC No.11747
The optimization of carbon source.
Fig. 5 is Lactobacillus plantarum（Lactobacillus plantarum）The most suitable fermentations of WEI-70 CGMCC No.11747
The optimization of nitrogen source.
Fig. 6 is Lactobacillus plantarum（Lactobacillus plantarum）The most suitable fermentations of WEI-70 CGMCC No.11747
The optimization of temperature.
Fig. 7 is Lactobacillus plantarum（Lactobacillus plantarum）The most suitable fermentations of WEI-70 CGMCC No.11747
PH optimization.
Fig. 8 is Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 are sent out in 50 L
Growth curve under the conditions of fermentation tank.
Embodiment:
The present invention can be better described in following instantiation, but the application of the present invention is not limited in following instance.
Embodiment one：Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 point
From and identification：
The g of excrement 25 of 60 age in days sodium selenites is weighed, in the triangular flask for being put into 0.85% physiological saline for filling 225 mL sterilizings
（20 sterile glass beads are placed in advance in triangular flask）, 37 DEG C, 180 rpm shake 30 min, draw 1 mL suspensions and do ten times
Incremental gradient dilutes, and chooses 2-3 acceptable diluent degree, draws 1 mL dilutions in sterile petri dish, each dilution factor does two
It is individual parallel；Into the culture dish added with dilution, addition is cooled to 46 DEG C of MRS agar mediums after melting, careful to rotate culture
Ware makes sample be sufficiently mixed with culture medium, 37 DEG C of culture 48-72 h.The bacterium colony line that different shape is selected from culture dish is pure
Change, preservation and carry out 16S rDNA sequencing identification respectively.
Embodiment two：Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 are resistant to
Low pH gastric juice, Pig cholate and artificial gastro-intestinal digestion liquid：
From Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 preservation inclined-plane（MRS
Culture medium）Choose a ring lawn to be connected in the 250 mL triangular flasks equipped with 100 mL liquid MRS culture mediums, in 37 DEG C, quiescent culture
18 h, 10 min, supernatant discarding, with 0.85% brine of sterilizing are centrifuged by bacterium solution under conditions of 6000 rpm
Once, then with same volume physiological saline suspend.To draw 10 mL suspension bacteria liquids and be connected to respectively equipped with 90 mL pH be respectively 1,2,3,
4th, in 250 mL triangular flasks of 5 simulated gastric fluid and the artificial pig gall salting liquids of 90 mL, pig gall salinity is respectively 3 g/L, 5 g/
L, 10 g/L, 15 g/L, 20 g/L, every kind of condition do three it is parallel, while to be not added with the sample of cholate as control.Then 37
DEG C, the min of quiescent culture 180 lives respectively at initial inoculation, 30 min, 60 min, 120 min, 180 min to culture
Bacterium counts, and draws viable count change curve, calculates bacterial strain and is incubated 180 in different pH simulated gastric fluids and various concentrations Pig cholate
Survival rate during min.Bacterium solution is incubated in simulated gastric fluid to be drawn 10 mL bacteria suspensions and is connected to equipped with 90 mL simulated intestinal fluids after 2 h
In 250 mL triangular flasks, equally at 37 DEG C, quiescent culture, respectively at initial inoculation, 0.5 h, 1 h, 1.5 h, 2 h, 6 h, 12
H, 18 h, 24 h carry out count plate to culture, draw viable count change curve.
Interpretation of result：Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 are in pH
180 min are kept in 2.0 simulated gastric fluid, survival rate reaches more than 85%, as a result sees Fig. 1；In the artificial pig that concentration is 20 g/L
180 min are kept in cholate solution, as a result survival rate is shown in Fig. 2 more than 70%.Lactobacillus plantarum（Lactobacillus
plantarum）WEI-70 CGMCC No.11747 keep 24 h in artificial gastro-intestinal digestion liquid, and viable count can maintain 107
CFU/mL, this time meets the intestinal physiology cycle, ensure that thalline plays prebiotic effect, as a result sees Fig. 3.
Embodiment three：Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 suppression
Bacterium activity：
1st, the preparation of culture plate：
From Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 preservation inclined-plane（MRS
Culture medium）Choose a ring lawn to be inoculated in liquid MRS culture mediums, 37 DEG C, cultivate 18 h, take 0.1 mL bacterium solutions to be coated on MRS and put down
Plate, cultivates 48 h by 37 DEG C.
2nd, bacteriostatic activity detects the preparation of flat board：
The LB nutrient solutions of indicator bacteria are diluted after 100 times, 0.1 mL bacterium solutions is drawn and is coated on LB flat boards.37 DEG C, incubated overnight.
The indicator bacteria includes Escherichia coli, salmonella, streptococcus and staphylococcus aureus.
3rd, the measure of bacteriostatic activity：
With aseptic card punch in Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747's
Punch, be then put in fungus block on the LB flat boards for coating indicator bacteria, 37 DEG C on culture plate, cultivate 20 h, observe and measure suppression
Bacterium loop diameter.
4th, interpretation of result：Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 couple
4 kinds of indicator bacterias have stronger inhibitory action, and antibacterial circle diameter size is 30-35 mm.
Example IV：Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 are most suitable
The optimization of fermenting carbon source：
1st, culture medium based on the MRS culture mediums without glucose is prepared, is dispensed into 500 mL triangular flasks, liquid amount 300
ML/ bottles.
2nd, appropriate glucose, sucrose, lactose, oligoisomaltose, maltodextrin, whey powder, molasses are weighed as carbon
Source, is added separately in above-mentioned basal medium, fully after dissolving, adjusts pH to 6.5 with 40% sodium hydroxide solution, moisturizing is extremely
300mL/ bottles, C final concentration is 0.67 M in every bottle.121 DEG C sterilize 20 minutes, and different carbon source culture mediums are made.
3rd, with 5 mL sterile salines by the Lactobacillus plantarum of fresh cultured（Lactobacillus plantarum）
Lawn on WEI-70 CGMCC No.11747 inclined-planes is rinsed completely, is all inoculated into above-mentioned carbon source culture medium, every bottle
Viable count is determined after being inoculated with 0.8 mL bacteria suspensions, 37 DEG C of h of quiescent culture 24.Every kind of carbon source culture medium does three repetitions.
4th, interpretation of result：Carbon source optimizing test result indicates that, Lactobacillus plantarum（Lactobacillus plantarum）
After WEI-70 CGMCC No.11747 are using maltodextrin as the h of carbon source through fermentation 24, maximum viable count can be obtained.Therefore, really
Maltodextrin is determined as fermentation optimum carbon source, as a result sees Fig. 4.
Embodiment five：Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 are most suitable
The optimization of fermentation nitrogen source：
1st, the glucose in MRS culture mediums is substituted with maltodextrin, prepares and be free of organic nitrogen source（Be free of peptone, beef extract
And yeast extract）MRS culture mediums based on culture medium, dispense into 500 mL triangular flasks, mL/ bottles of liquid amount 300.
2nd, appropriate fish peptone, beef extract, yeast extract, corn steep liquor, corn starch, urea, sodium nitrate and sulfuric acid are weighed
Ammonium, is added separately in above-mentioned basal medium, and fully after dissolving, pH to 6.5, moisturizing to 300 are adjusted with 40% sodium hydroxide solution
ML/ bottles, the content of total nitrogen is 2.59 g/L in every bottle.121 DEG C sterilize 20 minutes, and different nitrogen source mediums are made.
3rd, with 5 mL sterile salines by the Lactobacillus plantarum of fresh cultured（Lactobacillus plantarum）
Lawn on WEI-70 CGMCC No.11747 inclined-planes is rinsed completely, is all inoculated into above-mentioned nitrogen source medium, every bottle
Viable count is determined after being inoculated with 0.8 mL bacteria suspensions, 37 DEG C of h of quiescent culture 24.Every kind of nitrogen source medium does three repetitions.
4th, interpretation of result：Nitrogen source Optimal Experimental result shows, Lactobacillus plantarum（Lactobacillus plantarum）
After WEI-70 CGMCC No.11747 are using corn starch as the h of nitrogen source fermentation 24, maximum viable count can be obtained.Therefore, really
Corn starch is determined as fermentation optimum nitrogen source, as a result sees Fig. 5.
Embodiment six：Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 are most suitable
The optimization of fermentation temperature：
, prepare Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 culture mediums, into
Part is as follows：The g/L of corn starch 32, the g/L of maltodextrin 20, the g/L of sodium acetate trihydrate 3, the g/L of three hypophosphite monohydrate hydrogen dipotassium 2,
The g/L of ammonium citrate 2, the g/L of bitter salt 0.6, the g/L of Manganous sulfate monohydrate 0.1, the g/L of Tween 80 1, use 40% hydrogen-oxygen
Change sodium solution and adjust pH to 6.5, dispense into 500 mL triangular flasks, mL/ bottles of liquid amount 300.121 DEG C of 20 min of sterilizing.
2nd, with 5 mL sterile salines by the Lactobacillus plantarum of fresh cultured（Lactobacillus plantarum）
Lawn on WEI-70 CGMCC No.11747 inclined-planes is rinsed completely, is all inoculated into above-mentioned culture medium, every bottle of inoculation
0.8 mL bacteria suspensions, respectively at determining viable count after 31 DEG C, 34 DEG C, 37 DEG C, the 40 DEG C and 43 DEG C h of quiescent culture 24.Often
Plant cultivation temperature and do three repetitions.
3rd, interpretation of result：Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747
The h of quiescent culture 24, can obtain maximum viable count under the conditions of 34 DEG C and 37 DEG C.Accordingly, it is determined that most suitable cultivation temperature is
34-37 DEG C, as a result see Fig. 6.
Embodiment seven：Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 are most suitable
Ferment initial pH optimization：
, prepare Lactobacillus plantarum (Lactobacillus plantarum) WEI-70 CGMCC No.11747 culture mediums, into
Part is as follows：The g/L of corn starch 32, the g/L of maltodextrin 20, the g/L of sodium acetate trihydrate 3, the g/L of three hypophosphite monohydrate hydrogen dipotassium 2,
The g/L of ammonium citrate 2, the g/L of bitter salt 0.6, the g/L of Manganous sulfate monohydrate 0.1, Tween 80 1g/L, packing to 500
In mL triangular flasks, mL/ bottles of liquid amount 300, to adjust pH with glacial acetic acid or 40% NaOH solution respectively be 5.5,6.0,6.5,7.0,
7.5 and 8.0.121 DEG C of 20 min of sterilizing.
2nd, with 5 mL sterile salines by the Lactobacillus plantarum of fresh cultured（Lactobacillus plantarum）
Lawn on WEI-70 CGMCC No.11747 inclined-planes is rinsed completely, is all inoculated into above-mentioned culture medium, every bottle of inoculation
Viable count is determined after 0.8 mL bacteria suspensions, 37 DEG C of h of quiescent culture 24.Every kind of pH does three repetitions.
3rd, interpretation of result：Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747
Under conditions of 6.5,37 DEG C of h of quiescent culture 24 of pH, maximum viable count can be obtained.Accordingly, it is determined that most suitable fermentation pH is
6.5, as a result see Fig. 7.
Embodiment eight：Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 fermentation trainings
Support:
The lawn on the WEI-70 inclined-planes of fresh cultured is rinsed completely with 5 mL sterile salines, 0.8 mL bacterium are drawn
Suspension inoculation is into the 500 mL triangular flasks equipped with 300 mL fermenting culture mediums, in 37 DEG C, the h of quiescent culture 18, with 1%
Inoculum concentration be connected to equipped with 30 L pH for 6.5 dedicated liquid fermentation medium 50 L fermentation tanks in, sent out in 37 DEG C
In ferment culture, fermentation process, rotating speed control cultivates 20 h in 100 rpm, stuffiness, and pH is down to 4.0 or so, fermentation ends, hair
Zymotic fluid viable count is maximum up to 6.0 × 109 CFU/mL。
Interpretation of result：Viable count after 50 L ferment tanks is up to 6.0 × 109CFU/mL, under being the same terms
More than 6 times of fermentation viable count in MRS culture mediums.Lactobacillus plantarum（Lactobacillus plantarum）WEI-70
CGMCC No.1174750 L fermentor grown curve maps are shown in Fig. 8.
Lactobacillus plantarum（Lactobacillus plantarum）WEI-70 CGMCC No.11747 fermenting cultures
Base, composition is as follows：The g/L of corn starch 32, the g/L of maltodextrin 20, the g/L of sodium acetate trihydrate 3, three hypophosphite monohydrate hydrogen dipotassiums 2
G/L, the g/L of ammonium citrate 2, the g/L of bitter salt 0.6, the g/L of Manganous sulfate monohydrate 0.1, Tween 80 1 g/L, pH are
6.5。
Embodiment nine：Lactobacillus plantarum (Lactobacillus plantarum) WEI-70 CGMCC No.11747 ferment
The vacuum freeze drying processing of liquid：
By Lactobacillus plantarum (Lactobacillus plantarum) the WEI-70 CGMCC No.11747 described in embodiment eight
Zymotic fluid passes through high speed tube centrifuge, and thalline is collected by centrifugation under conditions of 15000-20000 rpm.It is cold in order to reduce vacuum
Dry processing cost is freezed, the survival rate of lyophilized rear bacterium powder product is improved, 4 kinds of protection agent prescriptions are have selected（It is shown in Table 1）It is compared.
4 kinds of sterilized protective agents are compared 5 according to volume mass:1 is uniformly mixed with the thalline after centrifugation, and -70 DEG C of pre-freeze 4-5 h are put
In the cold-trap of -40 DEG C to -50 DEG C of vacuum freeze drier, below the Pa of vacuum 30 freezes 41 h or so.Take out bacterium
Powder, determines viable count, calculates survival rate.
Interpretation of result, protective agent 4 is suitable for Lactobacillus plantarum（Lactobacillus plantarum）WEI-70
The vacuum freeze drying processing of CGMCC No.11747 zymotic fluids, survival rate is up to 93%, and bacterium powder viable count is up to 5.0 × 1011-
7.0×1011CFU/g.Therefore bacteria protectant formula is defined as：Contain in every 1000 mL water：The g of skimmed milk power 100, paddy
The g of propylhomoserin sodium 10, the g of trehalose 10, the g of glycerine 30.
The protective agent of table 1. is numbered and is formulated
Forage plant lactobacillus WEI-70 CGMCC No.11747 can be used for adjusting animal and bird intestines microecological balance, can also
It is used to prevent as probiotics product or auxiliary treatment fowl bacterial diarrhoea.
Sequence table
<110>Beijing Vicaren Biological Technology Co., Ltd. of Beijing Vica Shengbang Biolog Technology Co., Ltd.
<120>One plant height activity forage plant lactobacillus and its cultural method and application
<160>1
<170>PatentIn version 3.5
<210>1
<211>1444
<212>DNA
<213>Lactobacillus plantarum
<400>1
TTAGCGGCTG GTTCCTAAAA GGTTACCCCA CCGACTTTGG GTGTTACAAA 50
CTCTCATGGT GTGACGGGCG GTGTGTACAA GGCCCGGGAA CGTATTCACC 100
GCGGCATGCT GATCCGCGAT TACTAGCGAT TCCGACTTCA TGTAGGCGAG 150
TTGCAGCCTA CAATCCGAAC TGAGAATGGC TTTAAGAGAT TAGCTTACTC 200
TCGCGAGTTC GCAACTCGTT GTACCATCCA TTGTAGCACG TGTGTAGCCC 250
AGGTCATAAG GGGCATGATG ATTTGACGTC ATCCCCACCT TCCTCCGGTT 300
TGTCACCGGC AGTCTCACCA GAGTGCCCAA CTTAATGCTG GCAACTGATA 350
ATAAGGGTTG CGCTCGTTGC GGGACTTAAC CCAACATCTC ACGACACGAG 400
CTGACGACAA CCATGCACCA CCTGTATCCA TGTCCCCGAA GGGAACGTCT 450
AATCTCTTAG ATTTGCATAG TATGTCAAGA CCTGGTAAGG TTCTTCGCGT 500
AGCTTCGAAT TAAACCACAT GCTCCACCGC TTGTGCGGGC CCCCGTCAAT 550
TCCTTTGAGT TTCAGCCTTG CGGCCGTACT CCCCAGGCGG AATGCTTAAT 600
GCGTTAGCTG CAGCACTGAA GGGCGGAAAC CCTCCAACAC TTAGCATTCA 650
TCGTTTACGG TATGGACTAC CAGGGTATCT AATCCTGTTT GCTACCCATA 700
CTTTCGAGCC TCAGCGTCAG TTACAGACCA GACAGCCGCC TTCGCCACTG 750
GTGTTCTTCC ATATATCTAC GCATTTCACC GCTACACATG GAGTTCCACT 800
GTCCTCTTCT GCACTCAAGT TTCCCAGTTT CCGATGCACT TCTTCGGTTG 850
AGCCGAAGGC TTTCACATCA GACTTAAAAA ACCGCCTGCG CTCGCTTTAC 900
GCCCAATAAA TCCGGACAAC GCTTGCCACC TACGTATTAC CGCGGCTGCT 950
GGCACGTAGT TAGCCGTGGC TTTCTGGTTA AATACCGTCA ATACCTGAAC 1000
AGTTACTCTC AGATATGTTC TTCTTTAACA ACAGAGTTTT ACGAGCCGAA 1050
ACCCTTCTTC ACTCACGCGG CGTTGCTCCA TCAGACTTTC GTCCATTGTG 1100
GAAGATTCCC TACTGCTGCC TCCCGTAGGA GTTTGGGCCG TGTCTCAGTC 1150
CCAATGTGGC CGATTACCCT CTCAGGTCGG CTACGTATCA TTGCCATGGT 1200
GAGCCGTTAC CCCACCATCT AGCTAATACG CCGCGGGACC ATCCAAAAGT 1250
GATAGCCGAA GCCATCTTTC AAGCTCGGAC CATGCGGTCC AAGTTGTTAT 1300
GCGGTATTAG CATCTGTTTC CAGGTGTTAT CCCCCGCTTC TGGGCAGGTT 1350
TCCCACGTGT TACTCACCAG TTCGCCACTC ACTCAAATGT AAATCATGAT 1400
GCAAGCACCA ATCAATACCA GAGTTCGTTC GACTGCATGT ATAG 1444
Claims (9)
1. one plant of forage plant lactobacillus（Lactobacillus plantarum）WEI-70 CGMCC No.11747.
2. a kind of fluid nutrient medium for forage plant lactobacillus WEI-70 CGMCC No.11747, it is characterised in that liquid
The main component of culture medium is as follows：
The g/L of maltodextrin 20, the g/L of corn starch 32, the g/L of sodium acetate trihydrate 3, the g/L of three hypophosphite monohydrate hydrogen dipotassium 2, lemon
Lemon acid ammonium 2 g/L, the g/L of bitter salt 0.6, the g/L of Manganous sulfate monohydrate 0.1, the g/L of Tween 80 1, pH value is 6.5.
3. a kind of forage plant lactobacillus WEI-70 CGMCC No.11747 cultural method, it is characterised in that inoculative proportion is
1%；Cultivation temperature is 34-37 DEG C；Stand or stuffiness culture, incubation time is 16-20 h.
4. a kind of forage plant lactobacillus WEI-70 CGMCC No.11747 are as the preparation method of probiotics product, its
It is characterised by, the zymotic fluid after strain is enriched with through first order seed shaking flask and 50 L fermentation tanks passes through high speed tubular type centrifugal collection bacterium
Mud, is well mixed with bacteria protectant, prior to -70 DEG C pre-freeze 4-5 h, is subsequently placed in vacuum freeze drier, freezes following
Freezed under trepang number：Vacuum is less than 30 Pa, and condenser temperature is -40 DEG C to -50 DEG C, freezes 41 h, is lived after drying
Property bacterium powder, will bacterium powder with crush crusher machine after mix with the maize cob meal of same granularity, acquisition probiotics product.
5. preparation method according to claim 4, it is characterised in that the rotating speed of the high speed tubular type centrifugal collection thalline
For 15000-20000 rpm.
6. preparation method according to claim 4, it is characterised in that the bacteria protectant formula is：Every 1000 mL water
In contain：The g of skimmed milk power 100, the g of sodium glutamate 10, the g of trehalose 10, the g of glycerine 30.
7. preparation method according to claim 4, it is characterised in that the mass volume ratio of the bacterium mud and bacteria protectant
For 1:5.
8. forage plant lactobacillus WEI-70 CGMCC No.11747 are as probiotics product in the regulation micro- life of animal and bird intestines
Application in state balance.
9. forage plant lactobacillus WEI-70 CGMCC No.11747 are raiseeed as probiotics product in prevention or auxiliary treatment
Application in fowl bacterial diarrhea.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610132132.9A CN107177522A (en) | 2016-03-09 | 2016-03-09 | One plant height activity forage plant lactobacillus and its cultural method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610132132.9A CN107177522A (en) | 2016-03-09 | 2016-03-09 | One plant height activity forage plant lactobacillus and its cultural method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107177522A true CN107177522A (en) | 2017-09-19 |
Family
ID=59830375
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610132132.9A Withdrawn CN107177522A (en) | 2016-03-09 | 2016-03-09 | One plant height activity forage plant lactobacillus and its cultural method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107177522A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107502575A (en) * | 2017-09-20 | 2017-12-22 | 中国农业科学院农产品加工研究所 | One plant of Lactobacillus plantarum with the high inhibitory activity of α glucuroides |
| CN109234182A (en) * | 2017-12-22 | 2019-01-18 | 浙江大学 | Lactobacillus plantarum ZJUF T34 and its application |
| CN111593010A (en) * | 2020-06-30 | 2020-08-28 | 黄河三角洲京博化工研究院有限公司 | Lactobacillus plantarum and method for producing fermented feed by using same |
| CN112094790A (en) * | 2020-11-17 | 2020-12-18 | 招远新兴化工有限公司 | Lactobacillus plantarum LP45 live bacterium preparation for regulating intestinal flora and application thereof |
| CN113403227A (en) * | 2021-06-08 | 2021-09-17 | 昆明理工大学 | Lactobacillus plantarum and preparation method and application thereof |
| CN114657106A (en) * | 2022-04-21 | 2022-06-24 | 青岛蔚蓝生物股份有限公司 | Lactobacillus plantarum and application thereof in preventing and treating acne |
| WO2022242446A1 (en) * | 2021-05-18 | 2022-11-24 | 上海市公共卫生临床中心 | Lactobacillus plantarum and use thereof in preparation of covid-19 vaccine immunopotentiator |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101878859A (en) * | 2010-07-01 | 2010-11-10 | 西北农林科技大学 | Microbial additive for silage fodder and preparation method thereof |
| CN102864096A (en) * | 2012-04-18 | 2013-01-09 | 北京和美科盛生物技术有限公司 | Lactobacillus plantarum and preparation method thereof for high-density culture and freeze-drying bacteria powder |
| CN103060228A (en) * | 2012-12-12 | 2013-04-24 | 广州立达尔生物科技股份有限公司 | Lactobacillus plantarum and micro-ecological preparation for silage |
| CN103392911A (en) * | 2013-08-06 | 2013-11-20 | 国家粮食局科学研究院 | Feed-use high-activity lactobacillus solid preparation and preparation method thereof |
| CN103981116A (en) * | 2013-12-24 | 2014-08-13 | 北京伟嘉人生物技术有限公司 | Culture method and application of enterococcus faecium |
| CN104152354A (en) * | 2013-05-14 | 2014-11-19 | 仙农生物科技(上海)有限公司 | Compound probiotics as well as production process and application thereof to leavening agent |
-
2016
- 2016-03-09 CN CN201610132132.9A patent/CN107177522A/en not_active Withdrawn
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101878859A (en) * | 2010-07-01 | 2010-11-10 | 西北农林科技大学 | Microbial additive for silage fodder and preparation method thereof |
| CN102864096A (en) * | 2012-04-18 | 2013-01-09 | 北京和美科盛生物技术有限公司 | Lactobacillus plantarum and preparation method thereof for high-density culture and freeze-drying bacteria powder |
| CN103060228A (en) * | 2012-12-12 | 2013-04-24 | 广州立达尔生物科技股份有限公司 | Lactobacillus plantarum and micro-ecological preparation for silage |
| CN104152354A (en) * | 2013-05-14 | 2014-11-19 | 仙农生物科技(上海)有限公司 | Compound probiotics as well as production process and application thereof to leavening agent |
| CN103392911A (en) * | 2013-08-06 | 2013-11-20 | 国家粮食局科学研究院 | Feed-use high-activity lactobacillus solid preparation and preparation method thereof |
| CN103981116A (en) * | 2013-12-24 | 2014-08-13 | 北京伟嘉人生物技术有限公司 | Culture method and application of enterococcus faecium |
Non-Patent Citations (2)
| Title |
|---|
| 徐致远等: "植物乳杆菌ST-Ⅲ对小鼠肠道菌群的调节", 《中国乳品工业》 * |
| 靳志敏等: "不同剂量植物乳杆菌对小鼠肠道菌群的影响", 《食品工业科技》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107502575A (en) * | 2017-09-20 | 2017-12-22 | 中国农业科学院农产品加工研究所 | One plant of Lactobacillus plantarum with the high inhibitory activity of α glucuroides |
| CN107502575B (en) * | 2017-09-20 | 2020-12-22 | 中国农业科学院农产品加工研究所 | Lactobacillus plantarum with high alpha-glucosidase inhibition activity |
| CN109234182A (en) * | 2017-12-22 | 2019-01-18 | 浙江大学 | Lactobacillus plantarum ZJUF T34 and its application |
| CN109234182B (en) * | 2017-12-22 | 2020-08-04 | 浙江大学 | Lactobacillus plantarum ZJUFT34 and application thereof |
| CN111593010A (en) * | 2020-06-30 | 2020-08-28 | 黄河三角洲京博化工研究院有限公司 | Lactobacillus plantarum and method for producing fermented feed by using same |
| CN111593010B (en) * | 2020-06-30 | 2022-07-26 | 黄河三角洲京博化工研究院有限公司 | Lactobacillus plantarum and method for producing fermented feed by using same |
| CN112094790A (en) * | 2020-11-17 | 2020-12-18 | 招远新兴化工有限公司 | Lactobacillus plantarum LP45 live bacterium preparation for regulating intestinal flora and application thereof |
| CN112094790B (en) * | 2020-11-17 | 2021-03-02 | 河北一然生物科技有限公司 | Lactobacillus plantarum LP45 live bacterium preparation for regulating intestinal flora and application thereof |
| WO2022242446A1 (en) * | 2021-05-18 | 2022-11-24 | 上海市公共卫生临床中心 | Lactobacillus plantarum and use thereof in preparation of covid-19 vaccine immunopotentiator |
| CN113403227A (en) * | 2021-06-08 | 2021-09-17 | 昆明理工大学 | Lactobacillus plantarum and preparation method and application thereof |
| CN114657106A (en) * | 2022-04-21 | 2022-06-24 | 青岛蔚蓝生物股份有限公司 | Lactobacillus plantarum and application thereof in preventing and treating acne |
| CN114657106B (en) * | 2022-04-21 | 2023-11-21 | 青岛蔚蓝生物股份有限公司 | Lactobacillus plantarum and application thereof in preventing and treating acne |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103981117B (en) | One plant height resistance enterococcus faecium and its cultural method and application | |
| CN106399196B (en) | One lactobacillus plantarum HEW-A490 and its application | |
| CN103981118B (en) | A kind of bacillus subtilis feed addictive and its preparation method and application | |
| CN107177522A (en) | One plant height activity forage plant lactobacillus and its cultural method and application | |
| CN106282072B (en) | Compound lactobacillus microecological preparation and preparation method and application thereof | |
| CN101338283B (en) | Lactobacillus casei and applications thereof in solid-state fermentation | |
| CN101273757B (en) | Lactobacillus reuteri lyophilized preparation and method for preparing the same | |
| CN107164269A (en) | A kind of lactobacillus paracasei, preparation and its application in pig feed | |
| CN103421715B (en) | Lactobacillus rhamnosus and application thereof | |
| CN105543126A (en) | Lactobacillus plantarum JM113 and application thereof | |
| CN104651268A (en) | Lactobacillus plantarum and application thereof | |
| CN106754551A (en) | A kind of bacterium amount lactobacillus preparation high and preparation method and application | |
| CN101974463B (en) | Lactobacillus reuteri and composite viable bacteria preparation thereof | |
| CN106906154A (en) | One lactobacillus plantarum strain and its feed addictive and feed | |
| CN107034163A (en) | A kind of novel lactic piece coccus and its application | |
| CN105368755A (en) | Acid-yielding Enterococcus faecium, bacteriostatic microecological preparation and application thereof | |
| CN109679882A (en) | One Enterococcus faecalis DT1-1 and its application | |
| CN102391962B (en) | Enterococcus faecium preparation agent and manufacturing technology thereof | |
| CN102061272B (en) | Lactobacillus salivarius SIL1 and application thereof | |
| CN104611256B (en) | A kind of microorganism lyophilized formulations and preparation method thereof | |
| CN112980735B (en) | Clostridium butyricum, microbial inoculum, application of clostridium butyricum and microbial inoculum and preparation method of microbial inoculum | |
| CN104673726A (en) | Porcine lactobacillus acidophilus freeze-drying preparation and application thereof | |
| CN107312732A (en) | A kind of probiotic feed additive | |
| CN108611299A (en) | One plant of lactobacillus plantarum for producing bacteriostatic peptide and its application | |
| CN106119152A (en) | The bacillus acidophilus of a kind of high-yield lactic acid rhzomorph and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20170919 |
|
| WW01 | Invention patent application withdrawn after publication |