CN105779350A - Lactobacillus plantarum resistant to enteritis salmonella infection and application thereof - Google Patents
Lactobacillus plantarum resistant to enteritis salmonella infection and application thereof Download PDFInfo
- Publication number
- CN105779350A CN105779350A CN201610219161.9A CN201610219161A CN105779350A CN 105779350 A CN105779350 A CN 105779350A CN 201610219161 A CN201610219161 A CN 201610219161A CN 105779350 A CN105779350 A CN 105779350A
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- lactobacillus plantarum
- cgmcc
- lactobacillus
- milk
- plantarum cgmcc
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- A23C11/00—Milk substitutes, e.g. coffee whitener compositions
- A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
- A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
- A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
- A23C11/106—Addition of, or treatment with, microorganisms
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- A—HUMAN NECESSITIES
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
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Abstract
The invention provides lactobacillus plantarum resistant to enteritis salmonella infection and the application thereof and belongs to the technical field of microorganisms.The lactobacillus plantarum is preserved in the China General Microbiological Culture Collection Center (CGMCC) on Sep, 24th, 2015 with the preservation number of CGMCC No.11446.The lactobacillus plantarum CGMCC No.11446 has high acid and cholate resistance, and has a remarkable inhibition effect on growth of enteritis salmonella.Adhesion of enteritis salmonella to enterocyte HT-29 is strongly inhibited, and enteritis salmonella infection is prevented or relieved.The lactobacillus plantarum CGMCC No.11446 can be used for preparing pharmaceutical compositions for preventing or relieving enteritis salmonella, food containing activity lactobacillus and fermented food, and has broad application prospects.
Description
Technical field
The present invention relates to a kind of Lactobacillus plantarum that can intervene Salmonella enteritidis infection and application thereof, belong to microbial technique neck
Territory.
Background technology
Salmonella enteritidis (Salmonella enterica) is a kind of typical gram negative pathogenic bacteria, and this bacterium mainly causes the mankind
Enteritis and alimentary toxicosis and the gastroenteritis of poultry, human and livestock health in serious threat.Salmonella enteritidis is without specificity host
Pathogenic bacterium, this bacterium can directly cause poultry morbidity to cause serious economic loss, also can indirect pollution poultry as Salmonella
The carrier of bacterium, finally endangers the mankind.Mellroy SG etc. report, in the food poisoning that the developed countries such as America and Japan occur,
40%-80% is caused by fowl Salmonella, predominantly caused by Salmonella enteritidis.In March, 1989 World Health Organization (WHO)
(WHO) the urgent international conference of Salmonella enteritidis pollution problem in a poultry and fowl egg has been held specially.Global human is each
Plant in bacterial food poisoning disease, what Salmonella enteritidis caused rank the umber one in acute gastroenteritis (alimentary toxicosis), become
For an international sanitarian important research focus.Therefore, prevent and treat this pathogen all to have in people doctor, veterinary and public health
Highly important meaning.
The alimentary toxicosis pathogenesis that Salmonella enteritidis causes, it is considered that be by the endotoxin of substantial amounts of viable bacteria and release thereof altogether
With causing.First, Salmonella is amount reproduction in contaminated food products, is then ingested internal with the form of viable bacteria, and at intestinal
The most drastically breed, then stick to enterocyte and invade submucous tissue, causing inflammatory reaction.Subsequently, Salmonella is again
Enter blood circulation through lymphsystem, and then cause bacteremia.So it is husky to isolate enteritis in the blood of early infection patient
Door Salmonella.Owing to Salmonella enteritidis can discharge substantial amounts of endotoxin intracellular, it is taken as that what Salmonella enteritidis caused
Alimentary toxicosis is divided into and first infects two steps of being poisoned again.Viable bacteria thalline mainly acts on host intestine and causes inflammatory reaction, edema, hemorrhage
Etc. symptom, and endotoxin mainly acts on nervous system, blood circulation etc., makes vasomotor nerve benumb, and vascular tone drops
Low, thermoregulation function generation obstacle, cause fever, vomit and the symptom such as diarrhoea.Typically it is grown up infection, it is proposed that first stop more
Breath and supplementary moisture content, finally select the treatment means of antibiotic.If but infecting object is that infant, old people and resistance are more weak
Colony, and have the symptoms such as serious diarrhoea, fever, then antibiotic must be used to treat, otherwise may cause more serious
Septicemia etc., even have cause death danger.
A lot of research report probiotic bacteria is had can effectively to control and reduce Salmonella field planting in poultry digestive tract.Oral mixing
Lactic acid bacteria can effectively reduce the invasion of Salmonella and reduce Salmonella enteritidis field planting in broiler caecum.Several factors energy shadow
Ring the effect of lactic acid bacteria suppression Salmonella, including process time, using dosage, bacterial activity and processing mode.Higgins etc.
Research finds, gavaging dosage is 106With 108Time, the positive detection rate of Salmonella can be significantly reduced, and do not reduce sramana
The field planting of Salmonella, shows that the treatment dosage of lactic acid bacteria can affect the field planting of Salmonella；Meanwhile, gavage viable bacteria and significantly reduce sand
The field planting of door Salmonella, and the lactic acid bacteria of supernatant culture and inactivation does not affect the field planting of Salmonella, illustrates that the activity of lactic acid bacteria is
Affect a key factor of lactic acid bacteria vivo efficacy.Except administered by oral gavage, add in drinking-water and spraying treatment also can effectively drop
The field planting of low Salmonella enteritidis.
Lactic acid bacteria is widely present among human intestine, natural fermentation milk product and vegetalitas fermented food such as Pickles, sauerkraut.
Long-Term Scientific Study result shows, probiotic bacteria is that human body is requisite and have the probiotics of important physiological function, and it is mainly given birth to
Reason function has: in preventing and treating lactose intolerance, recovery human body intestinal canal, colony balance safeguards that health, antitumor and prophylaxis of cancer are made
With, control the function such as toxins in human body level.Therefore lactic acid bacteria is explored the most permissible in the effect intervened during Salmonella enteritidis infects
Excavating the function of probiotic bacteria further, exploitation has the probiotic bacteria of higher health value, husky for utilizing meals strategy to intervene enteritis
Door Salmonella infects opens up the approach and solution made new advances.
Summary of the invention
It is an object of the invention to provide the Lactobacillus plantarum (Lactobacillus plantarum) that a kind of anti-Salmonella enteritidis infects,
It was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on 09 24th, 2015, protects
Hiding address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica (postcode: 100101), preservation
Numbered CGMCC No.11446, Classification And Nomenclature is Lactobacillus plantarum (Lactobacillus plantarum).
Described Lactobacillus plantarum CGMCC No.11446 has a following biological characteristics:
(1) thalline feature: in Gram-positive, cell is middle elongated rod shape, thalline about 0.5-1.2 μm width, and 2-5 μm is long,
Cheng Dan, paired or chaining, be formed without spore, and two ends are circular.
(2) colony characteristics: form obvious bacterium colony in MRS culture medium, diameter is between 0.2-2.6mm, circular, breast
White, translucent, surface wettability is smooth, neat in edge, not chromogenesis.
(3) growth characteristics: the minimum growth temperature of this bacterial strain is 18 DEG C, and maximum growth temperature is 40 DEG C, in temperature 30-37 DEG C
Lower growth is optimal, and tolerant the highest and minimum initial growth pH is 9.0 and 2.5, and the initial pH of the most suitable growth is 6.0；This
The period of delay of bright lactobacillus plantarum strain is relatively short, and about 4h initially enters exponential phase, and 12h just reaches stable phase.
Described Lactobacillus plantarum CGMCC No.11446 also has a following beneficial characteristics:
(4) growth of Salmonella enteritidis can well be suppressed；
(5) vitro inhibition Salmonella enteritidis sticks on enterocyte；
(6) having acid resistance, well-grown under pH3.0-9.0 environmental condition, under pH 2.5 environment, survival is good；
Described Lactobacillus plantarum CGMCC No.11446 can be used for preparation prevention, intervenes or treat the medicine that Salmonella enteritidis infects
Compositions.Described pharmaceutical composition is by Lactobacillus plantarum CGMCC No.11446 microbial inoculum and pharmaceutically acceptable carrier
Composition.Described pharmaceutically acceptable carrier be one or more be selected from the most normally used filler, binding agent,
Wetting agent, disintegrating agent, lubricant or correctives.Described pharmaceutical composition can be granule, capsule, tablet, pill
Or oral liquid formulation.
Described Lactobacillus plantarum CGMCC No.11446 can be used for the preparation food containing Lactobacillus or lactic acid bacteria fermented food.
The described food containing Lactobacillus or lactic acid bacteria fermented food refer to directly add Lactobacillus plantarum CGMCC No.11446
Milk product, bean product or the fruit that microbial inoculum or the use bacteria fermentation containing Lactobacillus plantarum CGMCC No.11446 strain produce
Vegetables goods.Described milk product includes but not limited to fermentation milk, flavored fermented milk, sour milk beverage, sour cream or cheese；Described
Bean product include but not limited to bean milk, fermented bean curd, Semen Sojae Preparatum or bean sauce；Described fruit and vegetable product includes but not limited to Fructus Cucumidis sativi, recklessly
Radix Raphani, Radix Betae, Herba Apii graveolentis or Caulis et Folium Brassicae capitatae goods.
The present invention also provides for a kind of method preparing Lactobacillus plantarum CGMCC No.11446 microbial inoculum, is by containing described plant breast
The bacterium solution of bacillus CGMCC No.11446 prepared by normal freeze-drying technique or other technique preparation obtained by mycopowder system
Agent, wherein contains more than 106The active plant lactobacillus CGMCC No.11446 of CFU/g.
In one embodiment of the invention, it is provided that a kind of preferred described Lactobacillus plantarum CGMCC No.11446 microbial inoculum
Preparation method, comprises the following steps:
A. the preparation of culture medium: use in terms of culture medium gross mass 87.7% water by 10% skimmed milk, 0.5% glucose, 1.5%
Tryptone and 0.3% yeast extract dissolve, and then adjusting its pH is 6.8, obtains culture medium；
The most protectant preparation: use water and protective agent mixed raw material for obtaining containing 100g/L defatted milk powder, 30mL/L
Glycerol, 100g/L maltodextrin, 150g/L trehalose, the protective agent of 10g/L L-sodium；
C. Lactobacillus plantarum CGMCC No.11446 strain is inoculated into according to the inoculum concentration of 2-4% in terms of the quality of described culture medium
At temperature 110-120 DEG C in culture medium after sterilizing 8-12min, at temperature 37 DEG C, then cultivate 18h, at 4 DEG C with
6000r/min rotating speed is centrifuged 20min, abandons supernatant, adds pH 7.2 phosphate buffer and cleans 2-4 time, obtains thalline, then
Bacteria concentration is made to reach 10 with described protective agent is resuspended10More than CFU/mL；Then, this suspension is allowed to train in advance at temperature 37 DEG C
Support 60min, then carry out lyophilization and obtain described microbial inoculum.
The Lactobacillus plantarum CGMCC No.11446 of the present invention has acid and bile salt tolerance characteristic, has Salmonella enteritidis in vitro
Good rejection ability, it is possible to decrease the Salmonella enteritidis adhesion to enterocyte.Described Lactobacillus plantarum CGMCC No.
11446 can be used for preparation prevention or alleviate the pharmaceutical composition of Salmonella enteritidis infection, the food containing Lactobacillus and send out
Ferment food, has application prospect widely.
Accompanying drawing explanation
Fig. 1 is the physiological curve of Lactobacillus plantarum CGMCC No.11446.
Fig. 2 is the heterogeneity of the Lactobacillus plantarum CGMCC No.11446 fungistatic effect to Salmonella enteritidis.
Fig. 3 is Lactobacillus plantarum CGMCC No.11446 survival condition in Gl tract.
Fig. 4 is the effect that Lactobacillus plantarum CGMCC No.11446 suppression Salmonella enteritidis sticks on enterocyte.
Detailed description of the invention
Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and apparent.
But these examples are only exemplary, the scope of the present invention is not constituted any restriction.Those skilled in the art should understand that
It is the details of technical solution of the present invention and form can be modified or replace lower without departing from the spirit and scope of the present invention,
But these amendments and replacement each fall within protection scope of the present invention.Method, composition and the consumption related in following embodiment, as
Without specified otherwise, conventional method the most known to those skilled in the art.
The physiological curve experiment of embodiment 1 Lactobacillus plantarum CGMCC No.11446
MRS culture medium is culture medium known to those skilled in the art, and it contains peptone fish flour, yeast extract, Portugal
Grape sugar, sodium acetate, dibasic ammonium citrate, Tween 80, magnesium sulfate, manganese sulfate, pH 6.2-6.4.
The Lactobacillus plantarum CGMCC No.11446 of growth stable phase will be entered according in terms of MRS fluid medium quality 2%
Inoculum concentration be inoculated in MRS fluid medium, cultivate under the conditions of 37 DEG C, every 2h sample, measure OD600
Value, pH value and supernatant suppression Salmonella enteritidis activity, obtain Lactobacillus plantarum CGMCC No. as shown in Figure 1
The physiological curve of 11446.
By accompanying drawing 1 it can be seen that Lactobacillus plantarum CGMCC No.11446, being placed in 37 DEG C of cultivations, 6h enters mid-log phase,
16h enters stable phase, and now OD600 value is maximum, and 22h enters decline phase.The pH value of fermented supernatant fluid is down to 4.50 from 6.24,
Keep constant after 18h.Start to produce antibacterial substance mid-term in logarithmic (log) phase, and reach the highest at stable phase later stage bacteriostatic activity, with
Rear holding is stable, and whole process is affected less by pH.Therefore, its result shows Lactobacillus plantarum CGMCC No.11446
Salmonella enteritidis is had good inhibitory activity.
The rejection ability analysis of Salmonella bacteria strain is tested by embodiment 2 Lactobacillus plantarum CGMCC No.11446
LB culture medium pour plate (every flat board 15mL, flat board is dressing plate 9mm), Salmonella enteritidis (Salmonella
Enterica) CCFM9161, Salmonella enteritidis (Salmonella enterica) CCFM9162, Salmonella enteritidis
(Salmonella enterica) CCFM9163 and Salmonella enteritidis (Salmonella enterica) CCFM9164 (above-mentioned experiment
Strain is all from Southern Yangtze University's food biotechnology center strain library) it is applied on flat board that (concentration is 1 × 108CFU/ flat board), vertical
I.e. put in 4 Oxford cups, and in the cup of Oxford, add 100 μ L lactobacillus ferment liquid, after spreading lh under the conditions of 4 DEG C,
Put into 37 DEG C of incubators and cultivate 24-28h, measure antibacterial ring size.It is repeated 3 times using MRS as negative control, experiment, its
Result of the test is shown in Table 1.
Table 1 suppresses other Salmonella enteritidis growth analyses
Note: "+" 10-11mm, " ++ " 11-15mm, " +++ " ＞ 15mm, "-" is without bacteriostatic activity.
The result of the test of table 1 shows, these a few strain Salmonella enteritidis are all had by Lactobacillus plantarum CGMCC No.11446 of the present invention
Significantly inhibit effect, and fungistatic effect is basically identical.
The component analysis of embodiment 3 Lactobacillus plantarum CGMCC No.11446 suppression Salmonella enteritidis
Carry out according to mode similarly to Example 2, utilize agar gel diffusion test to detect Lactobacillus plantarum CGMCC No. of the present invention
Viable bacteria that 11446 fermentation liquids, supernatant and PBS are resuspended and the suppression Salmonella enteritidis growing state of dead bacterium.Result of the test table
Bright (see Fig. 2), the fermentation liquid of Lactobacillus plantarum and cell-free supernatants have the ability significantly suppressing Salmonella bacteria growing,
And fermentation liquid shows higher bacteriostasis than cell-free supernatants.This result illustrates, antibacterial except having in supernatant
Outside material, viable bacteria also has certain fungistatic effect, and this may be relevant with competitiveness growth.
The impact of the fungistatic effect of supernatant is studied by pH.Use diluted sodium hydroxide solution or dilution heat of sulfuric acid by the present invention
Lactobacillus plantarum CGMCC No.11446 supernatant is adjusted to pH value 3.5,6.0 or 8.5, then carries out bacteriostatic test, and will
These bacteriostatic test results and use stock solution to carry out the result of the test that bacteriostatic test obtains to compare.
These result of the tests are shown in Table 2.
Table 2 pH impact on the bacteriostasis of supernatant
Note: "+" 10-11mm, " ++ " 11-15mm, " +++ " ＞ 15mm, "-" is without bacteriostatic activity.
From the results shown in Table 2, the pH of the supernatant stock solution of Lactobacillus plantarum CGMCC No.11446 of the present invention is 3.5
During with 6.0, supernatant stock solution has certain fungistatic effect.When the pH regulator of supernatant stock solution is to 3.5, fungistatic effect is best；
But, when the pH of supernatant stock solution is adjusted to 6.0, and fungistatic effect reduces therewith；When the pH of supernatant stock solution is adjusted to 8.0
Time, antibacterial loss is more than half.Illustrate that the antipathogenic composition in supernatant has certain dependency effect to sour environment.
Embodiment 4 Lactobacillus plantarum CGMCC No.11446 survival condition in Gl tract.
Manual simulation gastro-intestinal Fluid needs Fresh.Pepsin is dissolved in the PBS of pH 3.0 and makes its final concentration of 3g/L, warp
It is prepared as simulated gastric fluid after 0.22 μm membrane filtration.Trypsin is dissolved in the PBS of pH 8.0 and makes its final concentration of 1g/L,
It is prepared as simulated intestinal fluid after 0.22 μm membrane filtration.
By Lactobacillus plantarum CGMCC No.11446 with 0.85% normal saline resuspended after, will in the simulated gastric fluid (pH 3.0)
Its bacterial concentration is adjusted to 1 × 109CFU/mL.Viable count is counted respectively after being placed on 37 DEG C of cultivation 2h after mixing.At mould
After intending gastric juice cultivates 3h, take 1mL culture fluid and add to 9mL simulated intestinal fluid (pH 8.0), in 37 DEG C of trainings after mixing
Support.Viable count is detected respectively at 6h.48h, the survival rate below equation of lactic acid bacteria is cultivated with MRS solid medium 37 DEG C
Calculate:
Survival rate (%)=[log CFU N1/log CFU N0] × 100%
N1=through simulating the viable count of lactobacillus after gastro-intestinal Fluid processes, N0The viable count of=untreated front lactic acid bacteria.
These result of the tests are as shown in Figure 3.
As can be seen from Figure 3: Lactobacillus plantarum CGMCC No.11446 is after the simulated gastric fluid process of 2 hours, and it is deposited
Living and can reach more than 90%, after bacterium solution continues on through the simulated intestinal fluid process of 6 hours, its survival rate still may be up to more than 80%, says
Bright Lactobacillus plantarum CGMCC No.11446 has higher survival rate in intestinal, has and deeply carries out zooperal diving
Power.
Embodiment 5 Lactobacillus plantarum CGMCC No.11446 suppression Salmonella enteritidis sticks to the analysis on enterocyte
Experiment
1) prevention experiment: washed 3 times by the HT-29 cell PBS liquid cultivated to monolayer, adds 1mL Lactobacillus plantarum suspension
(concentration is 3 × 108CFU/mL), temperature 37 DEG C and 5%CO2Cultivate 1h under conditions of incubator, then wash with PBS liquid
Wash 2 times, unadsorbed Lactobacillus plantarum is removed, adds 1mL Salmonella enteritidis (3 × 108CFU/mL), continue
Temperature 37 DEG C and 5%CO21h is cultivated under conditions of incubator；With PBS, cell monolayer is rinsed 3 times, fix 30 with methanol
After min, then carry out Gram’s staining.100 cells of 20 visual field numbers looked at random by each sample, with viscous on average each cell
Attached Salmonella enteritidis number evaluates its adhesion situation.
2) intervention experiment: by 1mL Lactobacillus plantarum suspension (3 × 108And 1mL Salmonella enteritidis (3 × 10 CFU/mL)8
CFU/mL) join together in HT-29 cell, temperature 37 DEG C and 5%CO22h is cultivated under conditions of incubator；Use PBS
Cell monolayer is rinsed 3 times, after fixing 30min with methanol, then carries out Gram’s staining.Each sample looks for 20 to regard at random
Wild several 100 cells, evaluate its adhesion situation with the Salmonella enteritidis number adhered on average each cell.
3) Experiment on therapy: after first 1mL Salmonella enteritidis being cultivated 1h together with HT-29 cell, washs with PBS liquid
2 times, remove unadsorbed Salmonella enteritidis, add 1mL Lactobacillus plantarum (3 × 108CFU/mL) continue temperature 37 DEG C
With 5%CO21h is cultivated under conditions of incubator.With PBS, cell monolayer is rinsed 3 times, after fixing 30min with methanol,
Carry out Gram’s staining again.100 cells of 20 visual field numbers looked at random by each sample, with the enteritis adhered on average each cell
Salmonella number evaluates its adhesion situation.
Adhesion rate (%)=[N1/N0] × 100%
N1The adhesion sum of Salmonella enteritidis, N after=treated group of process0The adhesion sum of=positive control Salmonella enteritidis
(meansigma methods as 100%).
These result of the tests are as shown in Fig. 4 and Biao 3.
Table 3 Lactobacillus plantarum CGMCC No.11446 on Salmonella enteritidis on each cell adhere to number impact
From table 3 and Fig. 4 it can be seen that intervention group result shows the fermentation of Lactobacillus plantarum CGMCC No.11446 of the present invention
Clearly, viable bacteria and dead bacterium be respectively provided with suppression Salmonella enteritidis and adhere to the ability of HT-29 cell, but the inhibition of viable bacteria is best.
Prophylactic tria result shows, Lactobacillus plantarum CGMCC No.11446 fermentation supernatant of the present invention and dead bacterium show close suppression
The adhesion HT-29 cell of Salmonella enteritidis, but the inhibition of viable bacteria is poor.Therapeutic test result shows, plant of the present invention
The adhesiving effect of the fermentation supernatant suppression Salmonella enteritidis of lactobacillus CGMCC No.11446 is best.
All these results clearly demonstrate, and Lactobacillus plantarum CGMCC No.11446 of the present invention is equal in different processing modes
Salmonella enteritidis is adhered to enterocyte and has inhibitory action, but inhibition exists certain diversity, comprehensively analyzes intervention
Group and treatment group effect are preferable.
Embodiment 6 utilizes the Lactobacillus plantarum CGMCC No.11446 of the present invention preparation Lac Bovis seu Bubali containing this bacterium
By raw milk defatted milk at 95 DEG C of thermal sterilization 20min, it is subsequently cooled to 4 DEG C, adds Lactobacillus plantarum of the present invention
CGMCC No.11446 microbial inoculum so that it is concentration reaches 106More than CFU/mL, stored refrigerated at 4 DEG C, then obtain containing this
The Lac Bovis seu Bubali of invention Lactobacillus plantarum CGMCC No.11446 viable bacteria.
Embodiment 7 utilizes the Lactobacillus plantarum CGMCC No.11446 of the present invention preparation bean milk containing this bacterium
Use soft water soaking soybean, at temperature 80 DEG C, soak 2h, then remove soybean cover.Then, drain immersion water, then add
Boiling water defibrination, and under the temperature conditions higher than 80 DEG C, it is incubated 12min.The slurry obtained filters with 150 eye mesh screens, then enters
Row centrifugation, the centrifugal liquid obtained is thick bean milk, then heats it up temperature 140-150 DEG C, then by rapid for thick for heat bean milk
Importing vacuum cooled room and carry out evacuation, the smell substance in described thick bean milk is discharged rapidly along with water vapour.Through vacuum outgas
After, its temperature is down to about 37 DEG C, then accesses the Lactobacillus plantarum CGMCC No.11446 microbial inoculum of the present invention so that it is concentration reaches
To 106More than CFU/mL, stored refrigerated at temperature 4 DEG C, then obtain containing Lactobacillus plantarum CGMCC No.11446 of the present invention
The bean milk of viable bacteria.
Embodiment 8 utilizes the Lactobacillus plantarum CGMCC No.11446 of the present invention preparation beverage made of fruits or vegetables containing this bacterium
Select fresh vegetables to squeeze the juice after cleaning, then carry out flash pasteurization, after at temperature 140 DEG C, high-temperature hot sterilizes 2 seconds,
Cool to temperature about 37 DEG C immediately, then access Lactobacillus plantarum CGMCC No.11446 microbial inoculum prepared by the present invention so that it is concentration
Reach 106More than CFU/mL, stored refrigerated at temperature 4 DEG C, then obtain containing Lactobacillus plantarum CGMCC of the present invention
The beverage made of fruits or vegetables of No.11446 viable bacteria.
Embodiment 9 utilizes Lactobacillus plantarum CGMCC No.11446 of the present invention to prepare capsule goods
The Lactobacillus plantarum CGMCC No.11446 of the present invention is cultivated 24h in MRS culture medium, in temperature 4 DEG C and 4000
Centrifugal 20min under conditions of r/min, thalline pH7.2 phosphate buffer rinses twice, uses the resuspended thalline of skimmed milk to make
Whole cell concentration reaches 1 × 1010-3×1010CFU/mL.Bacteria suspension is joined in the sodium alginate soln of 3%, is sufficiently stirred for,
Cell is homogeneously dispersed in sodium alginate soln, is then expressed to this mixed liquor in the calcium chloride solution of 2% form glue
Grain, static solidification 30min, micelle is collected by filtration, micelle collection obtained carries out lyophilization 48h, obtains planting containing the present invention
The powder of thing lactobacillus CGMCC No.11446, is encased in this powder in the capsule for medicine sold in the market,
To described capsule goods.
Embodiment 10 utilizes Lactobacillus plantarum CGMCC No.11446 of the present invention to prepare for the microbial inoculum producing fermented food
Lactobacillus plantarum CGMCC No.11446 is inoculated in 115 DEG C of sterilizings 10 according to the inoculum concentration of culture matrix gauge 3%
In the culture medium of min, this culture medium is by 10% enzyme hydrolysis skimmed milk, 0.5% glucose, 1.5% pancreas in terms of this culture medium gross mass
Peptone, 0.3% yeast extract and excess water composition, pH6.8.Then, allow inoculation Lactobacillus plantarum CGMCC No.11446
Culture medium at temperature 37 DEG C, cultivate 18h, at 4 DEG C, be centrifuged 20min with 6000r/min rotating speed, abandon supernatant, add pH
7.2 phosphate buffers clean 2-4 time, obtain thalline, then reach concentration 10 with protective agent is resuspended10CFU/mL.Described guarantor
Protect agent and contain 100g/L defatted milk powder, 30mL/L glycerol, 100g/L maltodextrin, 150g/L trehalose and 10g/L L-
Sodium glutamate.
Then, allow this suspension preculture 60min at temperature 37 DEG C, then carry out lyophilization, obtain described plant breast bar
Bacterium CGMCC No.11446 microbial inoculum.Utilize in microbial inoculum prepared by said method containing more than 106The active plant breast bar of CFU/g
Bacterium CGMCC No.11446.This microbial inoculum can be directly used for preparing the food such as milk product of the present invention, bean product or fruit and vegetable product,
Directly add or as fermenting agent, it is possible to prepare pharmaceutical composition with respective carrier.
Embodiment 11 utilizes Lactobacillus plantarum CGMCC No.11446 of the present invention to prepare cultured milk
After fresh milk sugaring is dissolved, under the conditions of temperature 65 DEG C with 20MPa, carry out homogenizing, be then incubated at temperature 95 DEG C and kill
Bacterium 5min, then reduce the temperature to 35 DEG C, adds Lactobacillus plantarum CGMCC No.11446 microbial inoculum of the present invention, business dry powder is sent out
Ferment agent Lactobacillus bulgaricus and the mixed vaccine of business dry powder leaven streptococcus thermophilus composition, their mass ratio is l:1:1,
The inoculum concentration of described mixed vaccine is the 0.03% of fresh milk weight, and mixing, heat-preservation fermentation at temperature 35 DEG C, after curdled milk, in temperature
Cold preservation 24h at 4 DEG C, obtains described cultured milk.
Embodiment 12 utilizes Lactobacillus plantarum CGMCC No.11446 of the present invention to prepare tablet
Weigh the Lactobacillus plantarum CGMCC No.11446 mycopowder preparation 25.7 of the present invention using freeze-drying method to prepare respectively
Weight portion, starch 55.0 weight portion, cellulose derivative 4.5 weight portion, carboxymethyl starch sodium 12.0 weight portion, Pulvis Talci 0.8
Weight portion, sucrose 1.0 weight portion and water 1.0 weight portion, mixing, use conventional method to make wet granular, then use tablet machine
Carry out tabletting, then use small drug drying machine to be dried, then be packaged to be the tablet of the present invention.
Embodiment 13 utilizes Lactobacillus plantarum CGMCC No.11446 of the present invention to prepare pill
Weigh the Lactobacillus plantarum CGMCC No.11446 mycopowder preparation 32.2 of the present invention using freeze-drying method to prepare respectively
Weight portion, microcrystalline Cellulose 48.0 weight portion, polyvinylpyrrolidone 4.5 weight portion, calcium carbonate 10.0 weight portion, stearic acid
Magnesium 2.8 weight portion, lecithin 1.3 weight portion and ethanol 1.2 weight portion.Mix homogeneously, the refined honey adding convention amount makes this
The pill of invention.
The acid resistance of embodiment 14 Lactobacillus plantarum CGMCC No.11446
The Lactobacillus plantarum CGMCC No.11446 of freezen protective is inoculated in MRS culture medium, trains at temperature 37 DEG C
Support 24h, then after MRS culture fluid Secondary Culture 2～3 times, take the cultivation of 1mL Lactobacillus plantarum CGMCC No.11446
Liquid, is inoculated in the MRS fluid medium of 19mL difference pH value (3.0-9.0) respectively, cultivates 24h at temperature 37 DEG C,
Test result indicate that, Lactobacillus plantarum CGMCC No.11446 its survival rate in the environment of pH3.0-9.0 still can reach 90%
Above.As can be seen here, Lactobacillus plantarum CGMCC No.11446 has the toleration of good acid or alkali environment, is conducive to application
In the sweat of different acid or alkali environments.
Use training method as recited above to cultivate and obtain Lactobacillus plantarum CGMCC No.11446 culture, its thalline
Clean twice with 1.0mL pH7.2PBS (phosphate buffer), more resuspended with 1.0mL pH7.2 phosphate buffer, it is resuspended
Lactobacillus thalline mixes with 9.0mL pH 2.5 simulated gastric fluid, then cultivates at temperature 37 DEG C, respectively start (0h) and
Sample during 3h, carry out plate count with the cast cultivation of MRS agar culture medium, measure viable count and calculate its survival rate.
Survival rate is viable count logarithm value when 3h and the ratio of viable count logarithm value during at 0h in this culture fluid, represents with %.
Test result indicate that, Lactobacillus plantarum survival rate in the simulated gastric fluid environment of pH2.5 is more than 90%.As can be seen here, plant
Thing lactobacillus CGMCC No.11446 has acid resistance, and under the environment (gastric environment) of pH2.5, survival is good.
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any person skilled in the art,
Without departing from the spirit and scope of the present invention, all can do various changes and modification, therefore protection scope of the present invention should be with
What claims were defined is as the criterion.
Claims (10)
1. the Lactobacillus plantarum (Lactobacillus plantarum) that an anti-Salmonella enteritidis infects, its deposit number is CGMCC
No.11446。
2. the pharmaceutical composition containing Lactobacillus plantarum described in claim 1, it is characterised in that described pharmaceutical composition be by
Lactobacillus plantarum CGMCC No.11446 microbial inoculum and pharmaceutically acceptable carrier composition.
3. pharmaceutical composition as claimed in claim 2, it is characterised in that described pharmaceutically acceptable carrier is a kind of or many
Plant the most normally used filler, binding agent, wetting agent, disintegrating agent, lubricant or correctives.
4. pharmaceutical composition as claimed in claim 2, it is characterised in that described pharmaceutical composition is granule, capsule, sheet
Agent, pill or oral liquid formulation.
5. pharmaceutical composition as claimed in claim 2, it is characterised in that described Lactobacillus plantarum CGMCC No.11446 microbial inoculum
It is by normal freeze-drying technique or other technique system by the bacterium solution containing described Lactobacillus plantarum CGMCC No.11446
Standby obtained mycopowder preparation, containing more than 10 in described microbial inoculum6The active plant lactobacillus CGMCC No. of CFU/g
11446。
6. the purposes of Lactobacillus plantarum described in claim 1, it is characterised in that described Lactobacillus plantarum CGMCC No.11446 uses
In the pharmaceutical composition that preparation prevention or alleviation Salmonella enteritidis are infected.
7. the purposes of Lactobacillus plantarum described in claim 1, it is characterised in that described Lactobacillus plantarum CGMCC No.11446 uses
Food or the lactic acid bacteria fermented food of Lactobacillus is contained in preparation.
8. the purposes of Lactobacillus plantarum as claimed in claim 7, it is characterised in that the described food containing Lactobacillus or breast
Acid bacteria fermentation food is directly to add Lactobacillus plantarum CGMCC No.11446 microbial inoculum or use containing Lactobacillus plantarum
Milk product, bean product or the fruit and vegetable product that the bacteria fermentation of CGMCC No.11446 strain produces.
9. the purposes of Lactobacillus plantarum as claimed in claim 8, it is characterised in that described milk product includes but not limited to fermentation
Breast, flavored fermented milk, sour milk beverage, sour cream or cheese；Described bean product include but not limited to bean milk, fermented bean curd,
Semen Sojae Preparatum or bean sauce；Described fruit and vegetable product includes but not limited to Fructus Cucumidis sativi, Radix Dauci Sativae, Radix Betae, Herba Apii graveolentis or Caulis et Folium Brassicae capitatae goods.
10. the method utilizing Lactobacillus plantarum described in claim 1 to prepare microbial inoculum, comprises the following steps:
A. the preparation of culture medium: use in terms of culture medium gross mass 87.7% water by 10% skimmed milk, 0.5% glucose, 1.5%
Tryptone and 0.3% yeast extract dissolve, and then adjusting its pH is 6.8, obtains culture medium；
The most protectant preparation: use water and protective agent mixed raw material for obtaining containing 100g/L defatted milk powder, 30mL/L
Glycerol, 100g/L maltodextrin, 150g/L trehalose, the protective agent of 10g/L L-sodium；
C. Lactobacillus plantarum CGMCC No.11446 strain is inoculated into according to the inoculum concentration of 2-4% in terms of the quality of described culture medium
At temperature 110-120 DEG C in culture medium after sterilizing 8-12min, at temperature 37 DEG C, then cultivate 18h, at 4 DEG C
Under be centrifuged 20min with 6000r/min rotating speed, abandon supernatant, add pH 7.2 phosphate buffer and clean 2-4 time, obtain
Thalline, then make bacteria concentration reach 10 with described protective agent is resuspended10More than CFU/mL；Then, allow this suspension in temperature
Preculture 60min at spending 37 DEG C, then carry out lyophilization and obtain described microbial inoculum.
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