CN110468076A - A method of the culture medium of enrichment urea substance, freeze-drying save protective agent of urea substance and preparation method thereof and save urea substance with freeze-drying - Google Patents
A method of the culture medium of enrichment urea substance, freeze-drying save protective agent of urea substance and preparation method thereof and save urea substance with freeze-drying Download PDFInfo
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Abstract
The application belongs to microorganism field, discloses a kind of culture medium of enrichment urea substance, the method that freeze-drying saves protective agent of urea substance and preparation method thereof and freeze-drying saves urea substance.The culture medium buffer capacity of enrichment urea substance of the present invention is stronger, is more advantageous to the enrichment of urea substance.Protective agent of the present invention itself is more stable, reduces contaminated risk during urea substance saves, while can expand preservation quantity, reduces human cost.It is saved protective agent of the present invention applied to urea substance, and urea substance stability is greatly improved.Freeze-drying of the present invention saves the method for urea substance by measurement bacteria suspension pH, can be monitored to the growth conditions of urea substance, can more intuitively quantify, be conducive to keep the stabilization between different batches.Freeze-drying preservation is further carried out to urea substance using the protective agent being lyophilized in advance, the preserving type being lyophilized twice will not be diluted urea substance bacteria suspension, can farthest be enriched with to it, effectively improve concentration after freeze-drying.
Description
Technical field
The invention belongs to microorganism fields, and in particular to a method of freeze-drying saves urea substance more particularly to a kind of richness
Collect the culture medium of urea substance, freeze-drying saves the method that protective agent of urea substance and preparation method thereof saves urea substance with freeze-drying.
Background technique
Urea substance is the independent new category separated from mycoplasma, including six kinds, wherein ureaplasma urealyticum
(M.urealyticum) have pathogenic.Ureaplasma urealyticum, also known as U. urealyticum are one of six kinds of Ureaplasmas, with people
Class urogenital infections have substantial connection, are common one of the bacterial parasites of human urogenital, can cause in certain circumstances
Disease.Ureaplasma urealyticum can utilize the urease of itself, the energy being metabolized after decomposing urea with offer.Ureaplasma urealyticum is in human body
Field planting can have secondary ascendant trend, that is, infect newborn by parent birth canal when giving a birth, reduce rapidly later；Since sexual life again
It is cumulative more.
Ureaplasma urealyticum is generally surface infection, does not invade blood mostly.Pathogenic mechanism is not still fully aware of, it is now recognized that
It may be related with aggressive enzyme and toxic product.Ureaplasma urealyticum can cause urinary system infection contamination and infertile under certain condition
Disease.Ureaplasma urealyticum plays the role of adhering to sperm, hinders the movement of sperm.Generate neuraminidase sample substance interference sperm and ovum
The combination of son, and have common antigen with Human sperm membrane, immunologic mjury can be caused to sperm and cause infertility.Disease caused by ureaplasma urealyticum
The most common are non gonococcal urethritis for disease, account for the 60% of non-bacterial urethritis.Ureaplasma urealyticum multiparasitization is in male urine
Road, prepuce of penis and vagina.If uplink infects, male prostatitis or epididymitis, women vaginitis, uterine neck can be caused
Inflammation, and fetus can be infected lead to miscarriage, premature labor and under-weight fetus, it can also cause the sense of neonatal respiratory tract and central nervous system
Dye.
In recent years, urogenital infections caused by ureaplasma urealyticum are paid more and more attention, and are to cause non-gonococcal urethritis
One of pathogen.Culture presevation is carried out to part urea substance, has ten to understanding the pathogen pathogenesis, diagnosis, treating
Divide important value and significance.But the common method for preserving effect of current urea substance is undesirable, and one of main cause is urea
Substance enrichment is extremely difficult.Urea substance is presently found minimum, the simplest cell with self-reproduction ability, gene
The smallest in group and prokaryotes, colony diameter only has 15~60 μm on solid medium, therefore can not be from solid culture
High vigor culture is directly acquired on base.When urea substance carries out Liquid Culture, meat soup clarification is not muddy, obtains strain concentration very
It is low, in addition urea substance itself can cause pH to increase and self-dissolving using urea etc., cause acquisition urea bovis culture concentration lower.
Summary of the invention
In view of this, it is an object of the invention to the difficult, preservation effects for urea substance existing in the prior art enrichment
The problem of difference provides a kind of culture medium of enrichment urea substance, freeze-drying saves the protective agent of urea substance and freeze-drying saves urea substance
Method.
In order to achieve the object of the present invention, the present invention adopts the following technical scheme:
A kind of culture medium being enriched with urea substance, is made of following effective component:
The present invention also provides the preparation methods of above-mentioned culture medium, include the following steps:
(1) by Hepes/MOPS/MES choose any one kind of them buffer, tryptone, proteose peptone, sodium chloride, manganese sulfate,
Cholesterol, DNA add water to mix, and adjust pH to 6.0 ± 0.5,121 DEG C of autoclave sterilization 15min, and recovery room warm standby is used；
(2) by horse serum, cattle heart leachate, yeast extract, L-cysteine hydrochloride, urea liquid, glucose, blueness
Mycin G, amphotericin B, vitamin B1 and D-VB5 calcium are added above-mentioned basis and train after being filtered respectively with the filter in 0.22 μm of aperture
It supports and is stirred evenly in base.
The present invention also provides the protective agents that a kind of freeze-drying saves urea substance, by anhydrous trehalose, pancreas casein peptone, poly- second
Alkene pyrrolidone and water composition；
Each Ingredient Amount are as follows: anhydrous trehalose 100gL-1, pancreas casein peptone 50gL-1, PVP K30
20g·L-1, surplus be water.
The present invention also provides above-mentioned protectant preparation methods, and each ingredient mixing and water adding is prepared protective agent, 121 DEG C of height
Warm high pressure sterilization 15min, recovery room warm standby are used.
The present invention also provides a kind of method that freeze-drying saves urea substance, urea substance strain is seeded to above-mentioned enrichment urea substance
Culture medium in, 37 DEG C of culture 14-18h, when its pH value becomes 7.50~7.90, according to 1mL/ bottles of packing bacteria suspensions, every bottle
Bacteria suspension and isometric freeze-dried mixed preservation of above-mentioned protective agent.
Preferably, the lyophilisation condition that preservation is lyophilized described in the method for the freeze-drying preservation urea substance is -40 DEG C of pre-freezes
4h, -40 DEG C~-15 DEG C distillations 18h, -15 DEG C~28 DEG C dry 6h.
Further, in some embodiments, it is after being lyophilized that the freeze-drying, which saves protective agent described in the method for urea substance,
Protective agent.The protective agent mixed with packing bacteria suspension is freeze dried powder.Using the protective agent being lyophilized in advance to urea substance into
Row freeze-drying saves, and the method that the preserving type being lyophilized twice makes freeze-drying save urea substance will not carry out urea substance bacteria suspension dilute
It releases, urea substance is enriched with to a greater degree.
Preferably, protectant lyophilisation condition is -40 DEG C of pre-freeze 4h, -40 DEG C~-15 DEG C distillation 18h, -15 DEG C
~28 DEG C of dry 6h.
As shown from the above technical solution, the present invention provides a kind of culture medium for being enriched with urea substance, freeze-dryings to save urea substance
Protective agent and preparation method thereof the method that saves urea substance with freeze-drying.It is of the present invention enrichment urea substance culture medium relative to
Traditional culture medium, buffer capacity is stronger, can be reduced injury of the variation due to culture medium pH value to urea substance, is more advantageous to
The enrichment of urea substance.Protective agent of the present invention itself is more stable, can carry out autoclave sterilization processing, reduce urea substance
Contaminated risk during preservation, while preservation quantity can be expanded, it avoids frequent progress from saving, reduces human cost.
It is saved protective agent of the present invention applied to urea substance, and urea substance stability is greatly improved.Freeze-drying of the present invention saves urea
The method of substance can be monitored the growth conditions of urea substance, can more intuitively be quantified, be conducive to by measurement bacteria suspension pH
The stabilization between different batches is kept, difference between batch is reduced.Freeze-drying guarantor is further carried out to urea substance using the protective agent being lyophilized in advance
It deposits, the preserving type being lyophilized twice will not be diluted urea substance bacteria suspension, can farthest be enriched with to it, effectively
Improve concentration after being lyophilized.The protective agent appearance being lyophilized in advance is fluffy, favorable solubility when meeting water redissolution, and user of service is not necessarily to etc.
To can be used at any time, flexible operation and conveniently.Experiment shows steady using the method for the invention freeze-drying preservation urea substance preservation effect
Fixed, 42 months concentration is saved after being lyophilized not to be reduced, and is had good stability.
Specific embodiment
The invention discloses the protective agents and preparation method thereof that a kind of culture medium for being enriched with urea substance, freeze-drying save urea substance
The method for saving urea substance with freeze-drying.Those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.It is special
Not it should be pointed out that all similar substitutions and modifications are apparent to those skilled in the art, they all by
It is considered as and is included in the present invention.Method and product of the invention is described by preferred embodiment, and related personnel is obvious
The content of present invention can not departed from, method described herein is being modified in spirit and scope or appropriate changes and combinations, come
Implementation and application the technology of the present invention.
For a further understanding of the present invention, below in conjunction with the embodiment of the present invention, to the technical side in the embodiment of the present invention
Case is clearly and completely described, it is clear that and described embodiments are only a part of the embodiments of the present invention, rather than all
Embodiment.Based on the embodiments of the present invention, those of ordinary skill in the art institute without making creative work
The every other embodiment obtained, shall fall within the protection scope of the present invention.
Unless otherwise specified, reagent involved in the embodiment of the present invention is commercial product, can pass through business canal
Road purchase obtains.Wherein, the DNA is purchased from Sigma company.
Embodiment one compares different culture medium for the concentration effect of urea substance
By taking ureaplasma urealyticum ATCC33175 as an example, concentration effect of more several different culture mediums for urea substance.This hair
The bright culture medium being related to is respectively labeled as A1, A2, A3, A4 according to the different amounts of each component in formula labeled as A.The U.S.
The culture medium that CLSI M43-A (Page 24) is related to is labeled as B, " practical clinical microbiology is examined and map " (Page 120)
The culture medium being related to is labeled as C, and the culture medium that " microbiological culture media Quality Control and diagram " (Page 236) is related to is labeled as D.
Wherein, the formula of culture medium A is as shown in table 1.
The formula and preparation method of 1 culture medium A of table
Note: above-mentioned formula is the formulation dosage that total amount is 1L culture medium.Hepes/Mops/MES buffer is Hepes buffering
Liquid, Mops buffer, MES buffer can be optionally one of.
The preparation method of A culture medium:
(1) upper table 1 is pressed by Hepes/Mops/MES buffer, tryptone, proteose peptone, sodium chloride, manganese sulfate, gallbladder
Sterol, DNA add water to mix, and adjust pH to 6.0 ± 0.5,121 DEG C of autoclave sterilization 15min, and recovery room warm standby is used；
(2) upper table 1 is pressed by horse serum, cattle heart leachate, yeast extract, L-cysteine hydrochloride, urea liquid, Portugal
Step is added after being filtered respectively with the filter in 0.22 μm of aperture in grape sugar, benzyl penicillin, amphotericin B, vitamin B1 and D-VB5 calcium
(1) it is stirred evenly in the culture medium.
Prepare protective agent: anhydrous trehalose 100gL-1, pancreas casein peptone 50gL-1, PVP K30
20g·L-1, add water mixing constant volume, 121 DEG C of sterilizing 15min are placed in spare in gnotobasis.
50 μ L of ureaplasma urealyticum ATCC33175 strain is seeded to respectively in the above-mentioned several culture mediums of about 100mL, is placed in 37
DEG C culture 14h start, take 100 μ L to be placed in vial in gnotobasis every 30min, measure its pH with micro acidity meter
Value, until its pH value is 7.50~7.90, after it is mixed with ready protective agent according to the ratio of 1:1 after 1mL/ bottles of packing
Freeze-drying.
Concentration mensuration after freeze-drying: the culture medium recommended using CLSI is carried out dense according to the method for 10 times of continuous gradient dilutions
Degree measurement (color changing units measurement), specific measuring method are carried out according to " manufacture and application of microbiological culture media ", i.e. every bacterium
Strain freeze-dried powder 1mL sterile water redissolves, and takes 0.2mL to add in 1.8mL culture solution after being completely dissolved, therefrom takes again after mixing
0.2mL is added in 1.8mL culture solution, is successively diluted to 10-1、10-2……10-10, paraffin oil is added dropwise, is placed in 37 DEG C of incubators
Culture, observes and records culture medium color change daily, until no longer until discoloration, the last one gradient of discoloration as terminal,
That is a color changing units.Same sample does 3 repetitions, is averaged.Such as the last gradient of discoloration is 10-8, concentration is
108CCU/mL.As a result compare such as table 2:
2 different culture medium of table compares the concentration effect of urea substance
| Culture medium | A1 | A2 | A3 | A4 | B | C | D |
| Concentration (CCU/mL) after freeze-drying | 107 | 107 | 107 | 107 | 106 | 105 | 105 |
By 2 result of table as it can be seen that different culture medium A1, A2, A3, A4 of the different amounts of each component in culture medium A are enriched with
Effect is better than culture medium B, C, D.
Embodiment two compares protective agent difference adding method for the concentration effect of urea substance
By taking ureaplasma urealyticum ATCC33175 as an example, compare protective agent difference adding method to concentration after the freeze-drying of urea substance
It influences.
Culture medium A 1 is prepared according to the formula and method of embodiment 1.
Prepare protective agent: anhydrous trehalose 100gL-1, pancreas casein peptone 50gL-1, PVP K30
20g·L-1, 121 DEG C of sterilizing 15min, a part be placed in it is spare in gnotobasis, a part aseptically according to 1mL/ bottles
It is lyophilized after packing spare.
50 μ L of ureaplasma urealyticum ATCC33175 strain is seeded in the above-mentioned culture medium A of about 100mL, 37 DEG C of cultures are placed in
14h starts, and takes 100 μ l to be placed in vial in gnotobasis every 30min, measures its pH value with micro acidity meter, until
Its pH value is 7.50~7.90, a part of bacteria suspension and liquid protectant mixed according to the ratio of 1:1 after 2mL/ bottle dispense after freeze
Dry, a part of bacteria suspension is sub-packed in the protective agent after above-mentioned freeze-drying according to 1mL/ bottles, and concussion is frozen after making it completely dissolved
Dry (being lyophilized twice).
Concentration mensuration after freeze-drying.Method is as in the first embodiment, its result such as the following table 3:
3 protective agent difference adding method of table compares the concentration effect of urea substance
| Protective agent adding method | Bacteria suspension: protective agent=1:1 | It is lyophilized twice |
| Concentration (CCU/mL) after freeze-drying | 107 | 108 |
By 3 result of table as it can be seen that protective agent is added the enrichment for being more advantageous to urea substance by the way of being lyophilized twice.
According to the method described above, protective agent difference adding method is compared to dense after the freeze-drying of urea substance using A2, A3, A4 culture medium
The influence of degree, as a result similar to A1 culture medium, protective agent is added by the way of being lyophilized twice is more advantageous to urea substance
Enrichment.
Embodiment three, different urea substance preservation effect compare
4 plants of different urea substances are saved using store method of the present invention.4 plants of urea substances are ureaplasma original respectively
Body ATCC33175, ATCC27618, ureaplasma parvum ATCC27813, ATCC700970.4 plants of urea substances save three batches.
Culture medium A 1 is prepared according to the formula and method of embodiment 1.
Prepare protective agent: anhydrous trehalose 100g.L-1, pancreas casein peptone 50g.L-1, PVP K30
20g.L-1, 121 DEG C of sterilizing 15min, aseptically according to 1mL/ bottles packing after be lyophilized it is spare.
4 plants of 50 μ L-100 μ L of urea substance strain are seeded in the above-mentioned culture medium A of about 100mL respectively, are placed in 37 DEG C of cultures
14h starts, and takes 100 μ l to be placed in vial in gnotobasis every 30min, measures its pH value with micro acidity meter, until
Its pH value is 7.50~7.90, is sub-packed in the protective agent after above-mentioned freeze-drying according to 1mL/ bottles, and concussion makes it completely dissolved laggard
Row freeze-drying.Concentration mensuration after freeze-drying, method as in the first embodiment, concentration mensuration the results are shown in Table 4.
The preservation effect of the different urea substances of table 4 compares
| First | Second batch | Third batch | |
| ATCC33175(CCU/mL) | 108 | 108 | 108 |
| ATCC27618(CCU/mL) | 107 | 107 | 107 |
| ATCC27813(CCU/mL) | 109 | 109 | 109 |
| ATCC700970(CCU/mL) | 108 | 108 | 108 |
By 4 result of table as it can be seen that 4 plants of different urea substances are good using store method of the present invention progress preservation effect,
And save three batches of effect stabilities.
According to the method described above, it using the preservation effect of the more different urea substance of A2, A3, A4 culture medium, is as a result trained with A1
Seemingly, 4 plants of different urea substances are good using store method of the present invention progress preservation effect for feeding base phase.
Example IV, Detection of Stability
4 plants of reference cultures (ureaplasma urealyticum ATCC33175, ATCC27618, ureaplasma parvum ATCC27813,
ATCC700970 freeze-drying rear stability verifying) is carried out using store method of the present invention.
More than three plants of reference culture freeze-dryings of above-described embodiment are saved and are placed in≤- 20 DEG C of environment, and respectively 0 month, 3
Concentration mensuration is carried out to it within a month, 6 months, 9 months, 12 months ..., 48 months, 51 months, concentration, which does not reduce, is considered as stabilization.
Its result such as the following table 5.
5 Detection of Stability result of table
By 5 result of table as it can be seen that 4 plants of reference cultures save 42 months concentration after being lyophilized according to the present invention program does not drop
It is low, it has good stability.
Claims (8)
1. a kind of culture medium for being enriched with urea substance, is made of following effective component:
2. the preparation method of culture medium described in claim 1, includes the following steps:
(1) choose any one kind of them buffer, tryptone, proteose peptone, sodium chloride, manganese sulfate, gallbladder of Hepes/MOPS/MES is solid
Alcohol, DNA add water to mix, and adjust pH to 6.0 ± 0.5,121 DEG C of autoclave sterilization 15min, and recovery room warm standby is used；
(2) by horse serum, cattle heart leachate, yeast extract, L-cysteine hydrochloride, urea liquid, glucose, penicillin
G, above-mentioned basal medium is added after being filtered respectively with the filter in 0.22 μm of aperture in amphotericin B, vitamin B1 and D-VB5 calcium
In stir evenly.
3. a kind of freeze-drying saves the protective agent of urea substance, by anhydrous trehalose, pancreas casein peptone, polyvinylpyrrolidone and water group
At；
Each Ingredient Amount are as follows: anhydrous trehalose 100gL-1, pancreas casein peptone 50gL-1, PVP K30 20g
L-1, surplus be water.
4. each ingredient mixing and water adding is prepared protective agent, 121 DEG C of high temperature and pressure by protectant preparation method described in claim 3
Sterilize 15min, and recovery room warm standby is used.
5. a kind of method that freeze-drying saves urea substance, urea substance strain are seeded in culture medium described in claim 1,37 DEG C of cultures
14-18h, when its pH value becomes 7.50~7.90, according to 1mL/ bottles of packing bacteria suspensions, every bottle of bacteria suspension and isometric right
It is required that the freeze-dried mixed preservation of protective agent described in 3.
6. according to the method described in claim 5, the lyophilisation condition that the freeze-drying saves is -40 DEG C of pre-freeze 4h, -40 DEG C~-15
DEG C distillation 18h, -15 DEG C~28 DEG C dry 6h.
7. according to the method described in claim 5, the protective agent is the protective agent after freeze-drying.
8. according to the method described in claim 7, protectant lyophilisation condition is -40 DEG C of pre-freeze 4h, -40 DEG C~-15 DEG C
Distil 18h, -15 DEG C~28 DEG C dry 6h.
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