CN107812018A - A kind of inactivated probiotic product, preparation method and applications - Google Patents
A kind of inactivated probiotic product, preparation method and applications Download PDFInfo
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- CN107812018A CN107812018A CN201711116784.4A CN201711116784A CN107812018A CN 107812018 A CN107812018 A CN 107812018A CN 201711116784 A CN201711116784 A CN 201711116784A CN 107812018 A CN107812018 A CN 107812018A
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- A61K35/74—Bacteria
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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Abstract
The invention discloses a kind of inactivated probiotic, preparation method and applications, inactivated probiotic of the present invention, the active probiotic for clinical patient can be replaced, reach the effect of active probiotic product is equally even better, it still can efficiently stick intestinal epithelial cell after inactivation, suppress sticking for pathogenic bacteria, fermented supernatant fluid suppresses the growth and breeding of pathogenic bacteria in vitro, and then play health efficacy, furthermore, inactivated probiotic security is higher, phagus tolerans infects, quality stability is high, do not limited by bacterium amount, can and antibiotic synergism, do not produced, the advantages that influences of environmental factor such as transport, with wider application.The inactivated probiotic product of the present invention can preferably meet actual clinical practice.
Description
Technical field
The present invention relates to inactivated probiotic product, preparation method and applications.
Background technology
Probiotics is very extensive in clinical practice, can help to treat diarrhoea caused by a variety of causes and its caused disorderly
Disorderly, harmful bacteria growing can also be suppressed, infection caused by external pathogenic bacteria, regulation body immunity is reduced, suppress and delay certain
Process of a little cancers etc..The appearance of probiotics, Gospel is carried out to people's health care belt, the rehabilitation for many diseases has very well
Booster action, its clinical efficacy is obvious to all, unquestionable.Dominant mechanism is as follows：
1. biological killer's effect
The metabolite of probiotics, such as organic acid (such as acetic acid, propionic acid, lactic acid), bacteriocin, hydrogen peroxide isoreactivity material
Effect, decline intestinal pH, can suppress or kill pathogenic bacteria.
2. biological barrier effect
It is colonized between Intestinal epithelial cells, forms biomembrane spline structure, hinders the outer occupy-place and breeding for attacking bacterium.Closely
With reference to cooperatively occupying intestinal mucosa surface with other anaerobic bacterias, form biological barrier.It is and pathogenic by Reverse transcriptase
Microorganism contention sticks site in intestinal mucosa, so as to occupy-place, participates in the structure of intestines biological barrier, competes or repel the bacterium that passes by one's way
The field planting of bacterium is attacked outside.
3. immunization
Stimulating immune system, promote the secretion of immune factor, immune cell activated, promote developing, being ripe for immune system,
Be advantageous to kill pathogenic microorganisms, improve specific antibody IG A and produce.
Probiotics can be understood as " healthy bacterium ", it means that probiotics is generally benign to health without causing disease
Disease.But found in process of clinical application, probiotics may also cause body to produce adverse reaction, such as bacillary and fungoid
Septicopyemia, systemic infection, detrimental metabolic activity, excessive immune stimulation, gene transfer and gastrointestinal side effect etc. are inspired,
The possible serious adverse reaction of vigilant probiotics is especially needed in some special populations, such as premature and immunologic hypofunction
Person.
There are some researches show, the use of the bacteremic incidence of the patient of saccharomycete is about 1/5,600,000, and lactic acid bacteria is more up to
1/1000000.Generally, systemic infection incidence is up to 0.05%~0.4% caused by probiotics.Probiotics may cause
The generation of bacteremia, fungemia and septicopyemia, it triggers the degree of danger of infection, except having outside the Pass with individual factors, also
It is relevant with the species and dosage of probiotics.In the patient infected, be largely immunologic immaturity premature or it is immune by
The adult of damage, this is also the most important hazards of probiotics infections relating.Probiotics causes the hazards bag of general infection
Include, low immature, low or suppression, seriously disease, central vein catheter and intestinal epithelial barrier infringement is immunized.These hazards
Can clinically it run into, serious disease, various neoplastic hematologic disorders are commonly present different degrees of hypoimmunity, such case
Under, if improper using probiotics, the possibility of the risk of bacteremia caused by probiotics and meningococcal infection can increase greatly
Add.However, it often happens that in clinical practice, there are severe infections in patient, merge obvious symptom of digestive tract (such as abdomen
Rush down), at this time in addition to symptomatic treatment, it can usually use probiotics.In fact, it is many report show, serious disease when,
Patient due to disease stress, be often accompanied by the exposure of mucosal ulcer or submucosa, at this time the barrier function of enteron aisle is by serious
Challenge, if being likely to result in bacterial translocation and Secondary cases bacteremia using probiotics.It is acute heavy in addition to above-mentioned hazards
Disease Pancreatitis Patients can not only reduce infection rate using probiotics, may increase case fatality rate on the contrary, therefore probiotics does not apply to
With patients with severe acute pancreatitis.In addition, the intestinal perfusion deficiency caused by acute pancreatitis, mucosal inflammation, oxygen increase in demand
May be relevant with probiotics.
Therefore, the premature not yet to be reached maturity for immunity and the clinical patient of hypoimmunity, live probiotic
Bacterium is faced with the problem of security, and inactivated probiotic can provide a kind of selection for clinical practice.Comparatively speaking, shape is inactivated
The probiotics of state has higher security, the probiotics after inactivating will not growth and breeding in vivo, without worrying to infect, according to
The problems such as relying, will not also Pollution of Phage be caused to break out；Without worrying being mutated of viable bacteria body, improper position transfer and anti-
Property of medicine gene transfer equivalent risk, and quality stability is high, is influenceed without refrigeration, not by producing, the environmental factor such as transport, tool
There is wider application.
Therefore provide and be necessary for the inactivated probiotic of clinical patient, with solve existing viable bacteria based article to
The toxic side effect that this kind of special population such as premature, clinical patient is brought, such as bacillary and fungoid septicopyemia, whole body are sexy
Contaminate, inspire detrimental metabolic activity, excessive immune stimulation, gene transfer and gastrointestinal side effect etc., while work can also be overcome
Mushroom product by environmental condition (temperature, moisture, humidity, acid-base value etc.) influence quality stability it is poor, the shortcomings of storage need to be refrigerated.
The content of the invention
The invention aims to the viable bacteria for solving to find in process of clinical application body may be caused to produce bad
React, such as bacillary and fungoid septicopyemia, systemic infection, inspire detrimental metabolic activity, excessive immune stimulation, gene turn
Shifting and gastrointestinal side effect etc., the possible serious adverse reaction of vigilant probiotics is especially needed in some special populations, and
Provided by the present invention for the inactivated probiotic product of clinical patient, to inactivate based on lactobacillus cell and its fermentating metabolism product
Active ingredient is wanted, and confirms that inactivation lactobacillus acidophilus has complete eucaryotic cell structure by testing, can be attached to well
Intestinal epithelial cell, and can be to the pathogenic bacteria such as common causative such as Candida albicans, staphylococcus aureus, EHEC
The adhesion of bacterium produces significant antagonistic effect, and moreover, lactobacillus acidophilus fermented supernatant fluid can also suppress pathogenic bacteria
Such as staphylococcus aureus, the growth of EHEC.Therefore inactivated probiotic of the present invention can be used for clinical patient, prevent
With treatment pathogenic infection caused by relevant disease.
To achieve the above object, the present invention adopts the following technical scheme that：
For the inactivated probiotic product of clinical patient, include the component of following quality proportioning：Inactivated probiotic cell and
Tunning 25-50 parts, wherein inactivated probiotic cell 108~1011CFU/g, remaining is auxiliary material.
Inactivated probiotic product of the present invention, further, institute belong to national health and family planning committee member using probiotics
The edible bacterial strain that can be announced, including the strain available for food, the strain available for infant food or available for health care
It is one or more in the strain of food.
Inactivated probiotic product of the present invention, further, the auxiliary material includes but is not limited to excipient, protective agent, food
The one or more of additive or nutrition fortifier.
The present invention also provides the preparation method of inactivated probiotic product, comprises the following steps：
1. choosing lactobacillus-fermented culture, ferment and reach 10 to cell number7More than CFU/mL；
After 2. fermented and cultured terminates, zymotic fluid is subjected to inactivation treatment；
3. the zymotic fluid after inactivation is centrifuged, separation obtains wet cell mud, by mass volume ratio 1:2~1:10 appropriate additions
Protective agent, inactivated probiotic thalline dry powder is obtained after being freeze-dried；
4. the zymotic fluid after inactivation is centrifuged, separation obtains fermented supernatant fluid, by volume mass than 10 after concentration:1~2:1
It is appropriate to add excipient, inactivation fermented supernatant fluid dry powder is obtained after being freeze-dried；
5. the dry powder that step 3 and step 4 are obtained is mixed, the inactivation containing thalline and metabolite of acquisition is prebiotic
Fungus powder product.
Or the zymotic fluid after the inactivation for being obtained step 2 is concentrated, the zymotic fluid after concentrating is by volume mass ratio
10:1~2:1 appropriate addition excipient, the inactivated probiotic powder containing thalline and metabolite obtained after being freeze-dried
Product.
Or volume mass is pressed after step 1 gained zymotic fluid is concentrated than 10:1~2:The 1 appropriate excipient that adds is dried
The inactivated probiotic powder product containing thalline and metabolite that inactivation obtains after drying.
Preferably, the protective agent and excipient are skimmed milk power, lactose, trehalose, disodium hydrogen phosphate, sucrose, dimension life
One or more in plain C, sodium glutamate, galactooligosaccharide, FOS, oligoisomaltose, starch.
The present invention relates to the method for one or more inactivated strains using probiotics, it is preferable that the ablation method can
To be heat inactivation, freezing inactivation or chemical ablation, and selected ablation method make it that inactivated strain is identical with vibrant bacterial strain
Or similar biological respinse attribute, possess even preferably effect identical with active probiotic.
Inactivated probiotic product of the present invention, wherein probiotics are unvital, but its cellular component is still protected
Hold and active probiotic is vibrant or the non-same or analogous biological respinse attribute of killed cells, possess it is identical with viable bacteria even more
Good effect, humans and animals can not only be efficiently attached to by inactivation treatment, the probiotic cell of inactivation after probiotics fermention
Intestinal epithelial cell, and pathogenic bacteria can be suppressed to sticking to intestinal epithelial cell, its fermentating metabolism product can also suppress
The growth and breeding of pathogenic bacteria.In addition, compared with viable bacteria, inactivated probiotic of the present invention at least has advantages below：Go out
Live probiotics will not growth and breeding in vivo, without worrying to infect, the problems such as dependence, Pollution of Phage will not be caused to break out,
Without worrying being mutated of viable bacteria body, the transfer of improper position and drug resistance gene transfer equivalent risk, so as to have higher peace
Quan Xing, and quality stability it is high (influenceed without refrigeration, not by producing, the environmental factor such as transport, to high temperature, drying and
The environment such as radiation have very strong resistance), harmful substance such as mycotoxin, heavy metal etc. can also may be effectively adsorbed, therefore
With wider application.
Brief description of the drawings
Cell integrity before and after Fig. 1 inactivations；
Fig. 2 inactivate to be contrasted with living lactobacillus acidophilus to intestinal epithelial cell Cao-2 Adhering capacity；
Fig. 3 inactivate antagonistic ability of the lactobacillus acidophilus to Candida albicans adhesive force；
Fig. 4 inactivate antagonistic ability of the lactobacillus acidophilus to staphylococcus aureus adhesive force；
Fig. 5 inactivate antagonistic ability of the lactobacillus acidophilus to EHEC adhesive force；
Bacteriostasis of Fig. 6 lactobacillus acidophilus fermented supernatant fluids to staphylococcus aureus；
Bacteriostasis of Fig. 7 lactobacillus acidophilus fermented supernatant fluids to EHEC.
Embodiment
It is for reference that invention described below embodiment, the details of one or more embodiments are provided below.Carry
It is to explain the present invention, rather than limitation of the present invention for each example.In fact, it will be apparent to those skilled in the art
, in the case of without departing from the scope or spirit of the invention, various modifications and changes can be carried out to the present invention.
Embodiment 1：The screening of different inactivation temperatures, inactivation time
Go deep into to probio research, it has been found that the probiotics of inactivated state possesses more in many aspects compared with viable bacteria
Big advantage.Inactivated probiotic is studied, the ablation method mainly used has heat inactivation, gamma-radiation inactivation, ultraviolet inactivation and acid
Inactivation.Heat inactivation is most important in ablation method and one kind for being most widely used, has security height, noresidue, easy
The advantages that operation.After environment temperature exceedes the maximum growth temperature of growth of probiotics, probiotic cell is may result in into distribution
Raw irreversible inactivation is dead.Different probiotics is different to the sensitiveness of temperature, and the heat resistance of probiotics is higher, inactivates institute
The temperature requirement needed is higher, and the time required for inactivating is longer.
Using the survival rate of probiotic cell after inactivation as index, inactivation technology is screened, while observes cell after inactivation
Integrality.
The cultural method of lactobacillus acidophilus：In an aseptic environment, inoculating lactobacillus acidophilus are in 300ml fluid nutrient mediums
In 500ml triangular flasks, it is placed in 37 DEG C of incubators, quiescent culture 24h (gently shakes up once) manually every 2h, after culture terminates
Carry out the measure of viable count.
Ablation method：Culture terminates, and is blown and beaten repeatedly uniformly with 10ml liquid-transfering guns, takes 10ml/ to manage (centrifuge tube), for inactivating
Experiment, is adjusted to experimental design temperature, in addition to control group, remaining centrifuge tube is placed in the water-bath of different temperatures by water-bath pot temperature
It is interior, concussion manually.According to experimental design inactivation time, centrifuge tube is taken out respectively after time arrival, is cooled down rapidly with purified water.Take
Sample carries out the measure of viable count.
Cell survival rate after the different inactivation parameter inactivations of table 1.
Cell survival rate after the different inactivation parameter inactivations of table 2.
Cell survival rate after the different inactivation parameter inactivations of table 3.
Cell survival rate after the different inactivation parameter inactivations of table 4.
Result such as table 1-4 understands that, when temperature reaches 75 DEG C, inactivation time reaches 10min, and lactobacillus acidophilus can be complete
Full inactivation, cell survival rate are all 0 ‰, and the cell integrity after being inactivated as shown in Figure 1 by micro- sem observation is good,
With indifference before inactivation, and cell integrity be lactic acid bacteria play prebiotic effect important prerequisite.Therefore follow-up test is optional
Select more than 75 DEG C, more than 10min inactivation technology.
Embodiment 2：Adhering capacity of the contrast inactivation with living lactobacillus acidophilus to intestinal epithelial cell Cao-2
The first step that pathogenic bacteria people invades host is to stick, and then expresses virulence factor, acts on infected host cell
Bring it about lesion, probiotics, which has, to be resisted foreign nationality bacterium and is colonized shown field planting resistance, i.e., probiotics by adhesin with
Mucosal epithelial cells acceptor sticks, and then occupy-place is colonized, to prevent pathogenic bacteria from sticking with mucosal receptors combination generation.Therefore it is prebiotic
Bacterium is the most important condition that probiotics plays its Mucosa for Protective Effect to sticking for mucosa cells, makes its field planting in enteron aisle, so as to
It is emulative on ecological niche to suppress sticking for other pathogenic bacteria, immune system is adjusted, repairs impaired intestinal mucosa.Traditional view
Think, only viable bacteria just has adhesive capacity, but is found according to the research of foreign countries, and inactivation lactic acid bacteria also has and viable bacteria identical
Adhering capacity.
By people's Colon and rectum gland cancer cell Caco-2 culture grow up to cell monolayer after, pancreatin digestion, by cell with every milliliter 1 ×
105The amount of individual cell is added in 6 hole histocyte culture plates, in CO under the conditions of 37 DEG C2It is incubated to growing up to list in incubator
Adhesion assay is carried out after confluent monolayer cells.By Caco-2 Tissue Culture Plates sterile PBS 2 times, the acidophilus breast that 1mL is prepared is added
Bacillus viable bacteria body suspension or inactivation lactobacillus acidophilus body suspension (108CFU/mL), each sample repeats 3 holes, in CO2In incubator
37 DEG C of culture 2h.Thalline suspension is removed afterwards, is washed 4 times with sterile PBS, to remove the lactobacillus acidophilus do not sticked, then often
30min is fixed in hole with absolute ethyl alcohol, after cleaning, carries out Gram's staining, is counted after drying.Microscope random observation 20 400
× the visual field, record the lactobacillus acidophilus number sticked on 100 cells.
Stick experiment to be repeated 3 times, the average and standard deviation for sticking index are calculated with statistical method.
It is thin to people's Colon and rectum gland cancer by contrasting inactivation lactobacillus acidophilus and living lactobacillus acidophilus as shown in table 5 and Fig. 2
Born of the same parents Cao-2's sticks index, and result of the test shows, it is thin that the lactobacillus acidophilus of two states can attach to people's Colon and rectum gland cancer
Born of the same parents Cao-2, and the lactobacillus acidophilus after inactivation shows more preferable Adhering capacity, and good Adhering capacity is lactic acid
Bacterium carries out the important prerequisite that occupy-place field planting plays its Mucosa for Protective Effect, is sticked by adhesin and mucosal epithelial cells acceptor,
Then occupy-place is colonized, to prevent pathogenic bacteria from sticking with mucosal receptors combination generation.Therefore this example has by inactivating lactic acid bacteria
Good Adhering capacity shows that it can carry out occupy-place field planting in enteron aisle, and then prevents pathogenic bacteria from sticking intestinal epithelial cell
It is attached, immune system is adjusted, repairs impaired intestinal mucosa, is finally reached the effect of relevant disease caused by prevention and treatment pathogenic bacteria
Fruit.
Embodiment 3：Inactivation lactobacillus acidophilus attaches to intestinal epithelial cell Cao-2 antagonistic ability to pathogenic bacteria
Parkes Gareth et al. are in authoritative medical journal《Lancet》On summarize probiotics suppress diarrhoea occur machine
System, pathogenic bacteria are first had to attach to human body intestinal canal epithelial cell, and then raised growth is bred, in final intrusive body, so as to cause
Disease, once each approach that probiotics cut-out pathogenic bacteria are pathogenic, then can fundamentally suppress the generation of disease.Therefore suppress
It is that lactobacillus suppresses the pathogenetic key link of disease that pathogenic bacteria, which are sticked, and pathogenic entero becteria is sticked into site from intestinal epithelial cell and taken off
Fall, being capable of effectively prophylactic generation.
Antagonism is tested：
1. cell CaCo-2 preparation：Press《Cell recovery, freeze, pass on, cultivating, freezing standard practice instructions》Culture is thin
Born of the same parents, CaCo-2 cells are digested using pancreatin, 37 DEG C, 5min, are terminated using the IMDM culture mediums containing 10% hyclone (FBS)
Digestion.Then low speed centrifuge 1400r/min, 3min are centrifuged, and its concentration is adjusted by cell counting count board using IMDM nutrient solutions
For 1 × 105/mL, 2mL cell liquid is drawn respectively and is added in each hole of 6 well culture plates of built-in cover glass, 37 DEG C, 5%CO2
Cultivated in incubator, after 18-24h, CaCo-2 cell adherences on the cover slip, suction out solution, and 3 are cleaned using IMDM culture mediums
Secondary, preparation starts to test.
2. inactivate the cell CaCo-2 of lactobacillus acidophilus pair adhesion antagonistic effect：
Repel experiment：
Nonadherent cell CaCo-2 is cleaned using IMDM culture mediums.Inactivation lactobacillus acidophilus suspension is separately added into per hole
1mL, totally 2 hole；Simultaneously plus 1ml dilutions are in the cell culture plate well prepared, totally 1 hole, as only addition pathogenic bacteria
Control group, 37 DEG C, after cultivating 1h in 5%CO2 incubators, using sterile PBS liquid rinses 3 times, it is thermophilic to remove nonadherent inactivation
Lactobacillus lactis；Pathogenic bacteria suspension 1mL is added per hole, 37 DEG C, is terminated culture after cultivating 1h in 5%CO2 incubators, is used IMDM
Culture medium rinses 3 times, takes out cover glass, spontaneously dries, and then methanol fixes 30min, carries out Grain stain, each cover glass oil
50 intestinal cells of random counter under mirror, extrapolate the average adhesion antagonism index of each intestinal cell.
Displacement experiment：
Nonadherent cell CaCo-2 is cleaned using IMDM culture mediums.Pathogenic bacteria suspension 1mL is separately added into per hole, totally 2 hole；
The control group for only adding the bacteria suspension 1mL that causes a disease is set simultaneously, totally 1 hole；37 DEG C, after cultivating 1h in 5%CO2 incubators, use IMDM
Culture medium rinses 3 times, removes nonadherent pathogenic bacteria；It is separately added into inactivation lactobacillus acidophilus 1mL again per hole, control group is with 1ml
Dilution, which substitutes, inactivates lactobacillus acidophilus liquid, 37 DEG C, terminates culture after cultivating 1h in 5%CO2 incubators, uses IMDM culture mediums
Rinsing 3 times, taking-up cover glass, is spontaneously dried, then methanol fixation 30min, Grain stain, is counted at random under each cover glass oil mirror
50 intestinal cells of number, extrapolate the average adhesion antagonism index of each intestinal cell.
Competition experiments：
Nonadherent CaCo-2 is cleaned using IMDM culture mediums.Inactivation lactobacillus acidophilus suspension 1mL is separately added into per hole, altogether
2 holes；Pathogenic bacteria suspension 1mL is added simultaneously to mix, and is set and only added 1ml dilutions and the control group of 1ml pathogenic bacteria, totally 1
Hole；37 DEG C, culture is terminated after 1h is cultivated in 5%CO2 incubators, is washed 3 times using IMDM culture mediums, takes out cover glass, is done naturally
Dry, then methanol fixes 30min, Grain stain, 50 intestinal cells of random counter under each cover glass oil mirror, extrapolates each
The average adhesion antagonism index of intestinal cell, and calculate cell adherence inhibiting rate.
As shown in Figure 3, it is known that compared with control group, after addition inactivation lactobacillus acidophilus, Candida albicans is to cell
CaCo-2 Adhering capacity significantly declines, and shows that the Adhering capacity of Candida albicans, suppression can be suppressed by inactivating lactobacillus acidophilus
It is that lactobacillus suppresses the pathogenetic key link of disease that pathogenic bacteria processed, which are sticked, and then prevents disease caused by candida albicans infection.
Candida albicans (Candida albicans) is the essential condition pathomycete for causing immunocompromised host infection.
In spite of the treatment of antifungal drug, but the case fatality rate of Fungemia is still high, and the particularly bacteremic case fatality rate of candida albicans is high
Up to 40% -80%.Have been reported that, systemic Candida albicans infection case fatality rate is 68.9%.And the primary bar that Candida albicans is pathogenic
Part is exactly to stick.The pathogen can both invade skin and mucous membrane, and and can involves internal organ.Most common two kinds of syndrome is mucous membrane skin
Skin candidiasis (such as oropharynx candidiasis or thrush, esophagitis and vaginitis) and invasion or deep organ's candidiasis
(such as candidemia, chronic dissemination or liver and spleen candidiasis, endocarditis etc.).Therefore inactivation involved in the present invention is thermophilic
Lactobacillus lactis can prevent or treat relevant disease caused by oily candida albicans infection, compared to active bacteria formulation, antibiotic, inactivation
Lactobacillus acidophilus is applied to clinical patient by with more security, less side effect, reduces the generation of infection.
As shown in Figure 4, it is known that compared with control group, after addition inactivation lactobacillus acidophilus, staphylococcus aureus is to thin
Born of the same parents CaCo-2 Adhering capacity significantly declines, and shows that the adhesion energy of staphylococcus aureus can be suppressed by inactivating lactobacillus acidophilus
Power, and then prevent disease caused by infection of staphylococcus aureus.
Staphylococcus aureus is a kind of important pathogen of the mankind, is the representative of gram-positive bacteria, can cause many
Severe infections.The epidemiology of staphylococcus aureus typically has following features：Season distribution, it is more common in spring and summer；Poisoning food
Kind class is more, such as milk, meat, egg, fish and its product.In addition, poisoning caused by leftovers, fried egg, glutinous rice cakes and bean jelly etc.
Also have been reported that.Infection of the upper respiratory tract patient's nasal cavity bacterial bearing rate 83%, staphylococcus aureus is most common in mankind's suppurative infection
Pathogen, local suppurative infection can be caused, can also cause pneumonia, pseudomembranous enteritis, pericarditis etc., or even septicemia, septicopyemia
The general infections such as disease.
As shown in Figure 5, it is known that compared with control group, after addition inactivation lactobacillus acidophilus, EHEC is to cell
CaCo-2 Adhering capacity significantly declines, and shows that the Adhering capacity of EHEC can be suppressed by inactivating lactobacillus acidophilus, be entered
And prevent disease caused by infection due to Escherichia coli.
EHEC can cause infectious diseases：1. extra intestinal infection：Urethral infection, bacteremia, cholecystitis, lung
2. scorching, neonatal meningitis infects in enteron aisle：Acute diarrhea, chronic diarrhea, hemorrhagic enteritis.
Embodiment 4：Rejection ability of the lactobacillus acidophilus metabolite to pathogenic bacteria growth and breeding
Probiotics is sticked by suppressing pathogenic bacteria with pathogenic bacteria direct competitive binding site；Antibacterial material is produced to suppress to cause a disease
Bacteria growing；Field planting of the pathogenic bacteria in enteron aisle can be suppressed by changing the means such as body immune system, and probiotics produces some points
Son can be to intestinal epithelial cell renewal and function have an impact.
Human body takes in a large amount of pathogenic bacteria by modes such as diet daily, and these pathogenic bacteria may be colonized in enteron aisle, but not
Have and cause pathogenic, main cause is that a large amount of lactobacillus floras present in enteron aisle can produce various antibacterial substances and suppress pathogenic bacteria
Amount reproduction, so as to avoid it from causing disease.
Metabolite suppresses pathogenic bacteria growth test in vitro:
1. the preparation of lactobacillus acidophilus fermented supernatant fluid
Lactobacillus acidophilus 37 DEG C of Anaerobic culturel 24h in MRS culture mediums, activation three generations is standby, by the MRS of lactobacillus acidophilus
Nutrient solution is fitted into centrifuge tube, and 15min is centrifuged with 4 000r/min rotating speed.Then it is degerming with filtering with microporous membrane, obtain
Clear liquid.
2. the preparation for the bacteria suspension that causes a disease
It is inoculated with pathogenic bacteria 10ml fluid nutrient mediums, puts 37 DEG C of incubator culture 24h；Scrape in above-mentioned 2-5 plate
Fresh lawn fully mixes into 10ml physiological saline and bacteria suspension is made, and determines its OD600Value, is counted by spectrophotometer
Number, 1 × 10 is adjusted to by its final concentration5CFU/mL, used in 2 hours.
3. agar diffusion method determines the bacteriostasis of lactobacillus acidophilus culture supernatant
The above-mentioned μ l of pathogenic bacteria suspension 400 prepared are taken to be added in pathogenic bacteria solid culture ware respectively, with triangular form glass
Glass rod coating is uniform, and under aseptic condition after ventilation drying surface liquid, the Oxford cup of sterilizing is placed on into culture with aseptic nipper
On base, gently pressurize, it is contacted tight with culture medium, each flat board places 3 Oxford cups, respectively into 2 Oxford cups
250 μ l lactobacillus acidophilus culture supernatants are added dropwise, the μ l of lactobacillus acidophilus liquid culture medium 250 are added dropwise into 1 Oxford cup and do sky
White control, after 37 DEG C are cultivated 24h, antibacterial circle diameter is measured, and taken pictures, each every kind of bacteria suspension of sample makees 3 flat boards.
From fig. 6 it can be seen that compared with control group, lactobacillus acidophilus fermented supernatant fluid is to pathogenic bacteria Staphylococcus aureus
Bacterium can produce obvious inhibitory action.Be probably due to lactobacillus acidophilus ferment produce antibacterial substance for example lactic acid, hydrogen peroxide,
Antibacterial peptide etc., so as to which the growth to pathogenic bacteria staphylococcus aureus produces significant inhibition, and then prevent or treat because
Relevant disease caused by infection of staphylococcus aureus.
It can be seen from figure 7 that compared with control group, lactobacillus acidophilus fermented supernatant fluid is to pathogenic bacteria EHEC energy
Enough produce inhibitory action.It is probably to ferment to produce antibacterial substance such as lactic acid, hydrogen peroxide, antibacterial peptide etc. due to lactobacillus acidophilus,
So as to which the growth to pathogenic bacteria EHEC produces inhibition, and then prevent or treat the phase because of caused by EHEC
Related disorders.
Pathogenic infection causes the mechanism of relevant disease to be that pathogenic bacteria will attach to human body intestinal canal epithelial cell first, then
Raised growth is bred, and toxin is produced, so as to cause disease.And the present invention inactivated probiotic product be exactly based on verification experimental verification its
The pathogenic each approach of pathogenic bacteria can be cut off such as to be colonized, grow, fundamentally prevent and treat relevant disease.
Claims (8)
1. a kind of inactivated probiotic product, include the component of following quality proportioning：Inactivated probiotic cell and tunning 25-50
Part, wherein 108~1011CFU/g of inactivated probiotic cell, remaining is auxiliary material.
The probiotics includes strain, the strain available for infant food or the bacterium available for health food available for food
It is one or more in kind；The auxiliary material is the one or more of excipient, protective agent, food additives or nutrition fortifier.
2. a kind of preparation method of inactivated probiotic product, comprises the following steps：
(1) chooses lactobacillus-fermented culture, ferments and reaches more than 107CFU/mL to cell number；
(2) after fermented and cultureds terminate, zymotic fluid is subjected to inactivation treatment；
(3) centrifuges the zymotic fluid after inactivation, and separation obtains wet cell mud, by mass volume ratio 1:2~1:10 appropriate add are protected
Agent is protected, inactivated probiotic thalline dry powder is obtained after being freeze-dried；
(4) centrifuges the zymotic fluid after inactivation, and separation obtains fermented supernatant fluid, by volume mass than 10 after concentration:1~2:1 is suitable
Amount adds excipient, and inactivation fermented supernatant fluid dry powder is obtained after being freeze-dried；
(5) is mixed the dry powder that step 3 and step 4 obtain, the inactivated probiotic containing thalline and metabolite of acquisition
Powder product.
3. a kind of preparation method of inactivated probiotic product, comprises the following steps：
(1) chooses lactobacillus-fermented culture, ferments and reaches more than 107CFU/mL to cell number；
(2) after fermented and cultureds terminate, zymotic fluid is subjected to inactivation treatment；
(3) zymotic fluid after the inactivation that is obtained step 2 is concentrated, and the zymotic fluid after concentrating is by volume mass than 10:
1~2:1 appropriate addition excipient, the inactivated probiotic powder system containing thalline and metabolite obtained after being freeze-dried
Product.
4. a kind of preparation method of inactivated probiotic product, comprises the following steps：
Lactobacillus-fermented culture is chosen, ferments and reaches more than 107CFU/mL to cell number；Volume is pressed after gained zymotic fluid is concentrated
Mass ratio 10:1~2:The 1 appropriate inactivation containing thalline and metabolite for adding excipient and obtained after drying inactivation is dried
Probiotic powder product.
5. inactivated probiotic product according to claim 1, it is characterised in that the protective agent and excipient are defatted milk
Powder, lactose, trehalose, disodium hydrogen phosphate, sucrose, vitamin C, sodium glutamate, galactooligosaccharide, FOS, oligomeric different wheat
One or more in bud sugar or starch.
A kind of 6. preparation method of inactivated probiotic product according to Claims 2 or 3 or 4, it is characterised in that the tax
Shape agent is skimmed milk power, lactose, trehalose, disodium hydrogen phosphate, sucrose, vitamin C, sodium glutamate, galactooligosaccharide, oligomeric fruit
One or more in sugar, oligoisomaltose or starch.
7. the preparation method of a kind of inactivated probiotic product according to Claims 2 or 3 or 4, it is characterised in that described to go out
Activating method is heat inactivation, freezing inactivation or chemical ablation.
8. a kind of application of inactivated probiotic product, it is substituted for the active probiotic of clinic.
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