CN107574134A - A kind of caprophyl transplantation treatment Plays caprophyl lyophiled powder preparing technique - Google Patents
A kind of caprophyl transplantation treatment Plays caprophyl lyophiled powder preparing technique Download PDFInfo
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- CN107574134A CN107574134A CN201710985378.5A CN201710985378A CN107574134A CN 107574134 A CN107574134 A CN 107574134A CN 201710985378 A CN201710985378 A CN 201710985378A CN 107574134 A CN107574134 A CN 107574134A
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- caprophyl
- transplantation treatment
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Abstract
The invention discloses a kind of excrement transplantation treatment Plays caprophyl lyophiled powder preparing technique, comprise the following steps：(1) prepared by standard caprophyl liquid；(2) freeze drying protectant is prepared；(3) three-step approach freezes, vacuum drying；Wherein, freeze drying protectant is formulated as being separately added into 10 20% trehaloses in sterile saline, and 5 10% glycerine and 0.02 0.05% vitamin C mix, filtration sterilization.The special freeze drying protectant of the present invention can protect caprophyl not lose activity in freeze-drying process, improve caprophyl survival rate；Specifically lyophilized program can make caprophyl rapidly enter resting state, will not lose its activity again, effectively protect the stability of effective ingredient.Experiment shows that the freeze-dried powder prepared through technique provided by the invention effectively maintains the activity of caprophyl in excrement, substantially increases survival rate.
Description
Technical field
The present invention relates to field of medical technology, more particularly, to a kind of excrement transplantation treatment Plays caprophyl freeze-dried powder
Preparation technology.
Background technology
Caprophyl transplanting (fecal microbiota transplantation, FMT) at least starts from medical history of world
4th century of Christian era, but until just having obtained unprecedented fast development in recent years.2013, FMT was written into U.S. doctor first
Guide is learned, is infected for recurrent Clestridium difficile
The treatment of (clostridiumdifficile infection, CDI).Meanwhile its at home and abroad be used for it is inflammatory
The treatment of the outer disease of the enterals such as enteropathy, IBS, metabolic syndrome, has also caused extensive concern.It is clinical at present to carry out
Caprophyl transplantation treatment be all take donor fresh excreta prepare standard caprophyl liquid treated.This fresh caprophyl titer needs
Control is in 6h from feces collection to therapeutic process, and it is pressed for time, and because qualified donor is not easy to find, and Donor selection takes
With higher, these hinder the clinical practice of caprophyl transplanting.
Damage of the Freeze Drying Technique to biological tissue and eucaryotic cell structure and feature is smaller, it is rapidly entered dormancy shape
State, the stability of effective ingredient is effectively protected, therefore required caprophyl is prepared into caprophyl freeze-dried powder for the ease of transporting and storing,
But easily the activity of loss caprophyl, caprophyl survival rate are had a greatly reduced quality during freeze-dried powder is prepared.
The content of the invention
Present invention aim to address current caprophyl freeze-dried powder activity it is not high the defects of, invent a kind of caprophyl freeze-dried powder and prepare
Technique, it can effectively keep the activity of caprophyl.
To achieve these goals, the present invention provides a kind of excrement transplantation treatment Plays caprophyl lyophiled powder preparing technique,
Comprise the following steps：
(1) prepared by standard caprophyl liquid；
(2) freeze drying protectant is prepared；
(3) three-step approach freezes, vacuum drying；
Wherein, freeze drying protectant be formulated as 10-20% trehaloses are separately added into sterile saline, 5-10% is sweet
Oil and 0.02-0.05% vitamin Cs mix, filtration sterilization.
The concrete operations of above-mentioned steps (1) are：Qualified donor is screened, respectively aseptic collection excrement, after homogeneous processing, warp thread
Centrifuged after cloth filtering, discard supernatant and obtain caprophyl mud sediment thing.
The concrete operations of above-mentioned steps (3) are：Freeze drying protectant is added in caprophyl mud, is vortexed and mixes, take 1mL bacterium solutions
Caprophyl culture counting is carried out, remaining liq is placed in freeze dryer, sets lyophilized program：By room temperature fast cooling to 4 DEG C, 4 DEG C to-
40 DEG C of 1 DEG C of reductions per minute, -40 DEG C to -60 DEG C 5 DEG C of reductions per minute, when temperature drops to below -60 DEG C, will freeze
Sample be put into rapidly in freeze dryer bell jar, start vavuum pump pumping until sample drying.Lyophilized sample is placed in -80 DEG C of ice
Case, preserve three months.
Caprophyl culture of the present invention, which counts, chooses Bifidobacterium conduct more sensitive to environmental condition and for probiotics
Testing index.
Above-mentioned caprophyl culture, which counts, to be comprised the following steps：Prepared by BBL culture plates, coated plate, and culture counts.
Concrete operations prepared by above-mentioned BBL culture plates are：BBL culture medium 70g, add 1ml Tween 80s, and heating is dissolved in
In 1000ml distilled water, 115 DEG C of sterilizing 20min；About 50 DEG C are cooled to, is poured onto in a diameter of 90mm sterile petri dish, often
Ware 10mL-15mL, it is placed in cooled and solidified under aseptic condition.
The concrete operations of above-mentioned coated plate are：The sample of acquirement is added to respectively in the sterile saline of 9mL volumes, multiple proportions
Dilution mixes, and takes 107With 108Two dilution factor coated plates.
The concrete operations that above-mentioned culture counts are：Culture plate is placed in anaerobism bag, is put in 37 DEG C of anaerobism trainings in incubator
After supporting 72h, bacterium colony counting is carried out.
The present invention carries out bacterium colony counting according to the methods of GB/T4789.34-2008.
Compared with prior art, beneficial effects of the present invention are：
(1) the special freeze drying protectant of the present invention can protect caprophyl not lose activity in freeze-drying process, improve caprophyl
Survival rate；
(2) lyophilized program of the invention can make caprophyl rapidly enter resting state, will not lose its activity again, effectively protect
The stability of effective ingredient is protected；
(3) the specific culture plate of the present invention, caprophyl freeze-dried powder is made to be reduced in coated plate counting process to caprophyl activity
Influence, make counting more accurate；
(4) caprophyl can be prepared into caprophyl freeze-dried powder by technique of the present invention, and at least be kept for three months, and survival rate can
Up to 92%.
Embodiment
The preferred embodiment of the present invention is more fully described below in conjunction with specific embodiment.
Embodiment 1
A kind of excrement transplantation treatment Plays caprophyl lyophiled powder preparing technique, comprises the following steps：
(1) prepared by standard caprophyl liquid：Screen qualified donor (age 25-35 year, no major disease, healthy male) 5 people, nothing
Bacterium gathers excrement 50g, after homogeneous processing, is centrifuged after filtered through gauze, discards supernatant and obtain caprophyl mud sediment thing.
(2) freeze drying protectant is prepared：100mL sterile salines are separately added into 15% trehalose, 7% glycerine and 0.05%
Vitamin C mixes, filtration sterilization.
(3) three-step approach freezes, vacuum drying：Freeze drying protectant is added in caprophyl mud, is vortexed and mixes, take 1mL respectively
Bacterium solution carries out Bifidobacterium culture and counted, and remaining liq is fitted into lyophilized pipe.Then lyophilized pipe is placed in freeze dryer, setting is frozen
Dry program：By room temperature fast cooling to 4 DEG C, 4 DEG C to -40 DEG C per minute to reduce by 1 DEG C, -40 DEG C to -60 DEG C 5 DEG C of reductions per minute.
When temperature drops to below -50 DEG C, the sample freezed is put into rapidly in freeze dryer bell jar, start vavuum pump pumping until
Sample drying.Lyophilized sample is placed in -80 DEG C of refrigerators, preserves three months.Every part of freeze-dried powder takes 1g to carry out bacterium colony training after three months
Support and count.
The operating procedure that above-mentioned caprophyl culture counts is：
1. prepared by BBL culture plates：BBL culture medium 70g are weighed, add 1ml Tween 80s, heating is dissolved in 1000ml distilled water
In, 115 DEG C of sterilizing 20min；When culture medium temperature after sterilizing is cooled to about 50 DEG C, sterile a diameter of 90mm training is poured into
Support in ware, per ware 10-15mL.(under aseptic condition) cooled and solidified is placed in Biohazard Safety Equipment.
2. coated plate：The sample of acquirement is added in 9ml sterile salines respectively, doubling dilution mixes, and takes 107With 108Two
Individual dilution factor coated plate, each each two of dilution factor are parallel.
3. culture counts：Culture plate is placed in anaerobic zone, is put in incubator and carries out bacterium colony after 37 DEG C of Anaerobic culturel 48h
Count.
The caprophyl count results of 1 five groups of parallel laboratory tests of embodiment are shown in Table 1：
The Bifidobacterium of table 1 sum (lg10n)
Bifidobacteria viable bacteria sum average is 6.54 ± 0.31log in sample before lyophilized10Cfu, freeze after preserving three months
Bifidobacteria viable bacteria sum average is 6.02 ± 0.51log10Cfu, survival rate 92%.
As a result prove, the freeze-dried powder prepared through technique provided by the invention effectively maintains the activity of caprophyl in excrement, greatly
Improve survival rate greatly.
Claims (8)
1. a kind of excrement transplantation treatment Plays caprophyl lyophiled powder preparing technique, it is characterised in that comprise the following steps：
(1) prepared by standard caprophyl liquid；
(2) freeze drying protectant is prepared；
(3) three-step approach freezes, vacuum drying；
Wherein, freeze drying protectant is formulated as being separately added into 10-20% trehaloses in sterile saline, 5-10% glycerine and
0.02-0.05% vitamin Cs mix, filtration sterilization.
2. excrement transplantation treatment Plays caprophyl lyophiled powder preparing technique according to claim 1, it is characterised in that described
The concrete operations of step (1) are：Aseptic collection donor excrement, after homogeneous processing, is centrifuged after filtered through gauze, is discarded supernatant and is obtained
To caprophyl mud sediment thing.
3. excrement transplantation treatment Plays caprophyl lyophiled powder preparing technique according to claim 1, it is characterised in that described
The concrete operations of step (3) are：Freeze drying protectant is added in caprophyl mud, is vortexed and mixes, takes 1mL bacterium solutions to carry out caprophyl culture
Count, remaining liq is placed in freeze dryer, sets lyophilized program：By room temperature fast cooling to 4 DEG C, 4 DEG C to -40 DEG C drops per minute
Low 1 DEG C, -40 DEG C to -60 DEG C 5 DEG C of reductions per minute, when temperature drops to below -60 DEG C, the sample freezed is put rapidly
Enter in freeze dryer bell jar, start vavuum pump pumping until sample drying, lyophilized sample is placed in -80 DEG C of refrigerators, preserves three months.
4. excrement transplantation treatment Plays caprophyl lyophiled powder preparing technique according to claim 3, it is characterised in that described
Caprophyl culture, which counts, chooses Bifidobacterium that is more sensitive to environmental condition and being probiotics as Testing index.
5. excrement transplantation treatment Plays caprophyl lyophiled powder preparing technique according to claim 4, it is characterised in that described
Caprophyl culture, which counts, to be comprised the following steps：Prepared by BBL culture plates, coated plate, and culture counts.
6. excrement transplantation treatment Plays caprophyl lyophiled powder preparing technique according to claim 4, it is characterised in that described
BBL culture plates prepare concrete operations be：BBL culture medium 70g, add 1ml Tween 80s, and heating is dissolved in 1000ml distilled water
In, 115 DEG C of sterilizing 20min；50 DEG C are cooled to, is poured onto in a diameter of 90mm sterile petri dish, per ware 10mL-15mL, is put
The cooled and solidified under aseptic condition.
7. excrement transplantation treatment Plays caprophyl lyophiled powder preparing technique according to claim 4, it is characterised in that described
The concrete operations of coated plate are：The sample of acquirement is added to respectively in the sterile saline of 9mL volumes, doubling dilution mixes, and takes
107With 108Two dilution factor coated plates.
8. excrement transplantation treatment Plays caprophyl lyophiled powder preparing technique according to claim 4, it is characterised in that described
Cultivating the concrete operations counted is：Culture plate is placed in anaerobism bag, is put in incubator after 35 DEG C of -37 DEG C of Anaerobic culturel 72h,
Carry out bacterium colony counting.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949572A (en) * | 2018-08-21 | 2018-12-07 | 卓源健康科技有限公司 | A kind of caprophyl freezen protective liquid and its store method |
| CN108949571A (en) * | 2018-06-12 | 2018-12-07 | 浙江工商大学 | A kind of method of human excrement and urine bacterium standard sample preparation, preservation and recycling |
| CN109125357A (en) * | 2018-10-24 | 2019-01-04 | 南京益恒寿生命科技有限公司 | A kind of probiotics that treating intestinal irritable syndrome-caprophyl liquid complex liquid |
| CN109294952A (en) * | 2018-10-23 | 2019-02-01 | 上海市第十人民医院 | A kind of preparation method of intestines bacteria composition |
| CN109628311A (en) * | 2018-12-28 | 2019-04-16 | 美霖佳生物医学科技(苏州)有限公司 | Freeze drying protectant, freeze-dried powder, capsule and preparation method thereof for protecting enterobacteriaceae |
| CN109679877A (en) * | 2019-01-17 | 2019-04-26 | 广州承葛生物科技有限公司 | A kind of preparation method of high activity caprophyl capsule |
| CN110065688A (en) * | 2019-04-22 | 2019-07-30 | 刘坚 | A kind of production packing method of pig manure source bio-feritlizer |
| CN110540941A (en) * | 2019-07-16 | 2019-12-06 | 上海交通大学 | method for extracting and preserving bacterial liquid from excrement |
| CN112195103A (en) * | 2020-10-13 | 2021-01-08 | 深圳未知君生物科技有限公司 | Freeze-drying protective agent, coprinus comatus freeze-dried product and preparation method thereof |
| CN112472810A (en) * | 2020-12-09 | 2021-03-12 | 佛山市朗芯生物科技有限公司 | Fecal bacteria freeze-drying protective agent and application thereof in fecal bacteria transplantation |
| CN112656766A (en) * | 2021-01-19 | 2021-04-16 | 赣州善健生物科技有限公司 | Fecal fungus freeze-drying and capsule preparation process for fecal fungus transplantation treatment |
| CN113209039A (en) * | 2021-04-22 | 2021-08-06 | 重庆晶云生物科技有限公司 | Enterobacter capsule and preparation method thereof |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949571A (en) * | 2018-06-12 | 2018-12-07 | 浙江工商大学 | A kind of method of human excrement and urine bacterium standard sample preparation, preservation and recycling |
| CN108949572A (en) * | 2018-08-21 | 2018-12-07 | 卓源健康科技有限公司 | A kind of caprophyl freezen protective liquid and its store method |
| CN109294952A (en) * | 2018-10-23 | 2019-02-01 | 上海市第十人民医院 | A kind of preparation method of intestines bacteria composition |
| CN109125357A (en) * | 2018-10-24 | 2019-01-04 | 南京益恒寿生命科技有限公司 | A kind of probiotics that treating intestinal irritable syndrome-caprophyl liquid complex liquid |
| CN109628311A (en) * | 2018-12-28 | 2019-04-16 | 美霖佳生物医学科技(苏州)有限公司 | Freeze drying protectant, freeze-dried powder, capsule and preparation method thereof for protecting enterobacteriaceae |
| CN109679877A (en) * | 2019-01-17 | 2019-04-26 | 广州承葛生物科技有限公司 | A kind of preparation method of high activity caprophyl capsule |
| CN110065688A (en) * | 2019-04-22 | 2019-07-30 | 刘坚 | A kind of production packing method of pig manure source bio-feritlizer |
| CN110540941A (en) * | 2019-07-16 | 2019-12-06 | 上海交通大学 | method for extracting and preserving bacterial liquid from excrement |
| CN112195103A (en) * | 2020-10-13 | 2021-01-08 | 深圳未知君生物科技有限公司 | Freeze-drying protective agent, coprinus comatus freeze-dried product and preparation method thereof |
| CN112195103B (en) * | 2020-10-13 | 2021-04-23 | 深圳未知君生物科技有限公司 | Freeze-drying protective agent, coprinus comatus freeze-dried product and preparation method thereof |
| CN112472810A (en) * | 2020-12-09 | 2021-03-12 | 佛山市朗芯生物科技有限公司 | Fecal bacteria freeze-drying protective agent and application thereof in fecal bacteria transplantation |
| CN112656766A (en) * | 2021-01-19 | 2021-04-16 | 赣州善健生物科技有限公司 | Fecal fungus freeze-drying and capsule preparation process for fecal fungus transplantation treatment |
| CN113209039A (en) * | 2021-04-22 | 2021-08-06 | 重庆晶云生物科技有限公司 | Enterobacter capsule and preparation method thereof |
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Application publication date: 20180112 |