CN107034141A - Gut flora fixer and preparation method thereof in human faecal mass - Google Patents
Gut flora fixer and preparation method thereof in human faecal mass Download PDFInfo
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- CN107034141A CN107034141A CN201710429297.7A CN201710429297A CN107034141A CN 107034141 A CN107034141 A CN 107034141A CN 201710429297 A CN201710429297 A CN 201710429297A CN 107034141 A CN107034141 A CN 107034141A
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Abstract
The present invention relates to gut flora fixer in a kind of human faecal mass and preparation method thereof, gut flora fixer in human faecal mass, per in 1000mL fixers, raw material components include：Tris HCl, EDTA and/or EDTA Na2, NaCl and ethanol, surplus is water.The present invention prepares fixer using materials such as Tris HCl, NaCl, EDTA as raw material components first, makes it have the function of fixed gut flora；Gut flora fixer in the human faecal mass that the present invention is provided, on the basis of Storing quality is ensured, can extend the holding time of the human excrement and urine of collection at normal temperatures, it is to avoid the human excrement and urine of collection is carried out into the inconvenience that cryopreservation is brought；Preparation method is simple, has a wide range of application.
Description
Technical field
The present invention relates to microbial technology field, and in particular to gut flora fixer and its preparation side in a kind of human faecal mass
Method.
Background technology
People's study on intestinal flora based on fecal sample, refers to by gathering human excrement and urine's sample, to the flora in excrement
Studied, its application mainly includes：(1) relation of research gut flora and human body diseases, because part disease is by enteron aisle
Bacterial metabolism thing influences, such as obesity, hypertension, diabetes etc.；Gut flora can be changed before part disease occurs, so that
It can be predicted to a certain extent；(2) change of research medicine rear intestinal flora, such as uses different antibiotic rear intestinal bacterium
The situation of change of group, and whether the effect of some antibiotic is than relatively low；Or the change using hormone medicine gut flora etc.；
The medicine operative condition for reaching enteron aisle can be studied；(3) relation that research gut flora absorbs with child nutrition, because enterobacteriaceae
Group can help food digestion, produce the material such as amino acid that human body needs, vitamin, short chain fatty acids etc., for improving food
Thing digests and to the trophism of people.
The common method of gut flora is studied by excrement to be had：Submethod is expanded and complete based on bacterium based on bacterial 16 S
Gene metagenome research methods, its process mainly includes：(1) fecal sample is gathered；(2) microorganism in excrement is extracted
DNA；(3) high-flux sequence is carried out to DNA；(4) data that analysis high-flux sequence is obtained obtain gut flora situation.Wherein
(1) step collection fecal sample, in order to ensure wherein microorganism (gut flora) various microorganism groups into it is constant, it will usually carry out
Stored frozen, the excrement that will be collected is put into -80 DEG C as far as possible low temperature refrigerators within the time as short as possible and preserved.Cause
For departing from intestinal environment, the residence time is more long in normal temperature, then gut flora, which is got over, to change, such as some bacterium number amounts
It can become many, some bacterium number amounts can tail off, in addition it is all dead.Due to the limitation such as experiment condition and experimental period, ordinary circumstance is not
The DNA that (2) step may be carried out immediately is extracted.So upon extracting, microorganism group in excrement into normal bowel ring
Different in border, research also just has no idea to obtain legitimate reading.Accordingly, it would be desirable to develop gut flora in new fixation human faecal mass
Method.
The content of the invention
For defect of the prior art, present invention aims at provide in a kind of human faecal mass gut flora fixer and its
Preparation method, on the basis of Storing quality is ensured, to extend the holding time of the human excrement and urine collected at normal temperatures, it is to avoid will
The human excrement and urine of collection carries out the inconvenience that cryopreservation is brought；Preparation method is simple, has a wide range of application.
To achieve the above object, the technical scheme that provides of the present invention is：
In a first aspect, the invention provides gut flora fixer in a kind of human faecal mass, in every 1000mL fixers, raw material
Component includes：400~600mmol Tris-HCl, 3~7mol NaCl, 50~150mmol EDTA and/or EDTA-Na2, it is remaining
Measure as water.
In the further embodiment of the present invention, raw material components also include：1~3mol ethanol.
In the further embodiment of the present invention, per in 1000mL fixers, raw material components include：500mmol
Tris-HCl, 100mmol EDTA and/or EDTA-Na2, 5mol NaCl, 2mol ethanol, surplus is water.
In the further embodiment of the present invention, Tris-HCl pH value is 8.0.
In the further embodiment of the present invention, water is deionized water.
Second aspect, the invention provides the preparation method of gut flora fixer in above-mentioned human faecal mass, including following step
Suddenly：All raw material components are well mixed, liquid is fixed.
The third aspect, the invention provides the application method of gut flora fixer in above-mentioned human faecal mass, including following step
Suddenly：The excrement of collection is added in fixer, is kept in dark place.It should be noted that the excrement of collection is added into fixer
In, the addition of fixer, it is only necessary to submerge excrement.
In the further embodiment of the present invention, the time of preservation is 0~30 day.
In the further embodiment of the present invention, the temperature of preservation is not higher than 40 DEG C.
The third aspect, the present invention also protects gut flora fixer gut flora in human faecal mass is prepared in above-mentioned human faecal mass
Application in fixed product.
The technical scheme that the present invention is provided, with following beneficial effect：
(1) present invention uses Tris-HCl, NaCl, EDTA and/or EDTA-Na first2Fixer is prepared for main component,
Although Tris-HCl, NaCl, EDTA, EDTA-Na2It is common buffer composition, the extraction that its combination is frequently used for DNA is delayed
Fliud flushing, but do not had been reported that function of its combination with fixed gut flora both at home and abroad；
(2) gut flora fixer in the human faecal mass that the present invention is provided, Tris-HCl therein is buffer components, DNA
Meeting deprotonation, improves its dissolubility, the TE buffer solutions together formed with EDTA can be effectively protected in flora in the solution
DNA degradation；EDTA is metal-chelator, can chelate the minor metallic element needed for bacterium, such as magnesium so that this fermentoid can not
Play a role, so that the normal metabolic activity of bacterium is blocked, bacteria growing inhibiting, with certain bacteriostasis；NaCl master
It is bacteria growing inhibiting to act on；The main function of ethanol is bacteria growing inhibiting；
(3) gut flora fixer in the human faecal mass that provides of the present invention, can ensure Storing quality (enterobacteriaceae in excrement
Group's composition is stable) on the basis of, extend the holding time of the human excrement and urine collected at normal temperatures, it is to avoid by the human excrement and urine of collection
Carry out the inconvenience that cryopreservation is brought；
(4) gut flora fixer in the human faecal mass that the present invention is provided, directly can add excrement equipped with fixer
Preserve in pipe, then normal temperature is kept in dark place, it is not necessary to cryopreservation is carried out, using simple；
(5) gut flora fixes liquid and preparation method thereof simply in the human faecal mass that the present invention is provided, and production cost is low, can be controlled in
2~3 yuan/mL, with extremely wide application value.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Brief description of the drawings
The composition block diagram of gut floras of the Fig. 1 for the excrement in the embodiment of the present invention two after different disposal；
Fig. 2 be the embodiment of the present invention two in excrement after different disposal the principal component analysis of sequencing analysis result cluster
Figure；A-individual 1；B-individual 2；C-individual 3；
Microorganism Bifidobacteriums of the Fig. 3 for the excrement in the embodiment of the present invention three after different disposal is relatively rich
Spend variation diagram；
Fig. 4 is that Microbial biomass C lostridium relative abundance of the excrement after different disposal in the embodiment of the present invention three becomes
Change figure；
Microorganism Bifidobacteriums of the Fig. 5 for the excrement in the embodiment of the present invention three after different disposal is relatively rich
Spend variation diagram；
Fig. 6 is that Microbial biomass C ollinsella relative abundance of the excrement after different disposal in the embodiment of the present invention three becomes
Change figure；
Fig. 7 is that microorganism Eubacterium relative abundance of the excrement after different disposal in the embodiment of the present invention three becomes
Change figure；
Microorganism Faecalibacteriums of the Fig. 8 for the excrement in the embodiment of the present invention three after different disposal is relatively rich
Spend variation diagram；
Microorganism Fusobacterium relative abundances of the Fig. 9 for the excrement in the embodiment of the present invention three after different disposal
Variation diagram；
Microorganism Parabacteroidess of the Figure 10 for the excrement in the embodiment of the present invention three after different disposal is relatively rich
Spend variation diagram；
Microorganism Ruminococcus relative abundances of the Figure 11 for the excrement in the embodiment of the present invention three after different disposal
Variation diagram；
Figure 12 is that microorganism Bacteroides relative abundance of the excrement after different disposal in the embodiment of the present invention three becomes
Change figure；
Figure 13 is major function gene relative abundance of the excrement after different disposal in the embodiment of the present invention three with the time
The figure of change.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described.The following examples are only intended to illustrate the technical solution of the present invention more clearly, therefore is intended only as example, without
It can be limited the scope of the invention with this.
Experimental method in following embodiments, is conventional method unless otherwise specified.Examination used in following embodiments
Material is tested, is to be commercially available from routine biochemistry reagent shop unless otherwise specified.Quantitative test in following examples,
Three repetition experiments are respectively provided with, data are the average value or mean+SD of three repetition experiments.
Embodiment one：Gut flora fixer and preparation in human faecal mass
The present embodiment provides gut flora fixer in a kind of human faecal mass, in fixer described in per 1000mL, raw material components
Including：500mmol Tris-HCl (pH value is 8.0), 100mmol EDTA, 5mol NaCl, 2mol ethanol, surplus is deionization
Water.
Preparation method includes：1000mmol/L Tris-HCl solution (pH value is 8.0) is first prepared, measures 500mL's
Tris-HCl solution, adds 100mmol EDTA, 5mol NaCl and 2mol ethanol, then adds deionized water to 1L, mixing is equal
It is even, it is fixed liquid.
Embodiment two：Bacterial 16 S amplicon research gut flora composition
1st, the excrement for once draining same individual is divided into 30 parts of collections, then by the excrement of collection be respectively put into containing
In the human faecal mass that the embodiment of the present invention one of equivalent is prepared in 30 collection tubes of gut flora fixer, fixed liquid energy leaching
No excrement (similarly hereinafter).
30 samples are placed into 0~9 day (representing within the 0th day the collection same day) at normal temperatures, 3 pipes are frozen daily as repetition,
Refrigerator freezing is placed into, after 9 days all excrement frozen are carried out with DNA extractions, sequencing and analysis, and (concrete operations are that this area is normal
Operating method).
The composition block diagram for analyzing rear intestinal flora is as shown in Figure 1.Different colours represent not of the same race in gut flora in way
The microorganism of class.It is the sample (daily in triplicate) repeated daily after number of days, decimal point that abscissa, which is,.Ordinate is microorganism
Relative abundance.As shown in Figure 1, from the 0th day to the 9th day gut flora microorganism group into change it is small, this change main source
Caused by collection point.As a result illustrate that gut flora fixer effect is fine in the human faecal mass that the present invention is provided.
2nd, gut flora fixer progress excrement is fixed in the human faecal mass provided using the present invention, while using external product
It is fixed, most long room temperature carries out bacterial 16 S amplicon research gut flora composition after 36 days.Due to need excrement compared with
It is many, therefore Different Individual progress feces collection is chosen, concrete scheme and result are as follows：
1 (A) of individual：Gut flora fixer in the human faecal mass prepared using the embodiment of the present invention one containing equivalent
Collection tube 36, gather placed under 36 parts of excrement that same individual is once drained, normal temperature respectively, from the 0th day (collection the same day)
Start to freeze 3 pipes in each 3 days, all freeze and sequencing analysis are carried out after finishing；
2 (B) of individual：Using the collection tube 15 of the external product containing equivalent, same individual is gathered respectively and is once drained
15 parts of excrement, place under normal temperature, 3 pipes frozen week about, is frozen altogether 5 weeks, is all frozen and sequencing analysis are carried out after finishing；
3 (C) of individual：Using the collection tube 15 of the external product containing equivalent, same individual is gathered respectively and is once drained
15 parts of excrement, place under normal temperature, 3 pipes frozen week about, is frozen altogether 5 weeks, is all frozen and sequencing analysis are carried out after finishing.
As a result it is as follows：Species composition using principal component method (by analyzing sequencing result) to sample is divided
Analysis, gut flora constitutes more similar sample, and gathering must be closer, and gut flora constitutes more dissimilar sample, and sample room is thinner
Far.As seen from Figure 2,3 (C) of 1 (A) of individual 30 samples more individual 2 (B) and individual 15 samples are so poly- that more to concentrate,
Illustrate that 1 (A) of individual 30 samples are closely similar.As a result also illustrate, gut flora fixer in the human faecal mass that the present invention is provided
It is more excellent in fixed gut flora state aspect effect compared with external product.
Embodiment three：Bacterium full genome metagenome is studied
Gut flora fixer progress excrement is fixed in the human faecal mass provided using the present invention, and most long room temperature is after 36 days
Carry out in bacterium full-length genome metagenome researchs (concrete operations are operating method commonly used in the art), observation gut flora
Microorganism and the situation of change of functional gene, concrete scheme and result are as follows：
The excrement that same individual is once drained is divided into 39 parts of collections, then by the excrement of collection be respectively put into containing etc.
In the human faecal mass that the embodiment of the present invention one of amount is prepared in 39 collection tubes of gut flora fixer, fixed liquid energy submergence
Placed under excrement (similarly hereinafter), normal temperature.3 pipes were taken to be frozen every 3 days, 36 days altogether (the collection same day is also required to freeze 3 pipes), entirely
Portion freezes finish after carry out bacterium full genome metagenome research.
As a result it is as follows：1st, Fig. 3-Figure 12 be shown 10 kinds of microorganisms of content highest (including：Bifidobacterium、
Clostridium、Bifidobacterium、Collinsella、Eubacterium、Faecalibacterium、
Fusobacterium, Parabacteroides, Ruminococcus, Bacteroides), its relative abundance is respectively at 36 days
In situation of change.Abscissa is different samples, is arranged that (i.e. A01, A02, A03 are represented the 3rd day and frozen sample according to number of days
Product, B01, B02, B03 are represented the 6th day and are frozen sample, and the rest may be inferred), ordinate is the relative abundance of the bacterium.The smoothness table of line
Show the change of correspondence bacterial abundance.It can be seen that line is relatively smooth, illustrate that the microbiology turbidity of this in gut flora is little.
Explanation is needed, due to mixing that can not be very, therefore the deviation on collection point occurs, fluctuation thus also occurs, but exist
In the range of statistics.As a result illustrate that gut flora fixer effect is fine in the human faecal mass that the present invention is provided.
2nd, Figure 13 is the figure that functional gene abundance is changed over time：Abscissa is different samples, is arranged according to number of days,
Ordinate is the relative abundance of the genoid, and line is more smooth, changes smaller.As seen from Figure 13, amino acid metabolism (Amino
Acid Metabolism) related gene varied less in the case of 36 days in room temperature.As a result the human excrement that the present invention is provided is illustrated
Just middle gut flora fixer effect is fine.
The technical scheme that the present invention is provided, with following beneficial effect：(1) present invention first using Tris-HCl,
NaCl, EDTA and/or EDTA-Na2 are that main component prepares fixer, although Tris-HCl, NaCl, EDTA, EDTA-Na2 are equal
For common buffer composition, it combines the Extraction buffer for being frequently used for DNA, but does not have been reported that it was combined both at home and abroad
Function with fixed gut flora；(2) gut flora fixer in the human faecal mass that provides of the present invention, Tris-HCl therein is
Buffer components, DNA meeting deprotonations in the solution, improve its dissolubility, the TE buffer solutions together formed with EDTA can have
DNA degradation in the protection flora of effect；EDTA is metal-chelator, can chelate the minor metallic element needed for bacterium, such as magnesium,
Prevent this fermentoid is from playing a role, so as to block the normal metabolic activity of bacterium, bacteria growing inhibiting, with certain antibacterial
Effect；NaCl main function is bacteria growing inhibiting；The main function of ethanol is bacteria growing inhibiting；(3) present invention is provided
Human faecal mass in gut flora fixer, can on the basis of Storing quality (in excrement gut flora composition stable) is ensured,
Extend the holding time of the human excrement and urine collected at normal temperatures, it is to avoid the human excrement and urine of collection is carried out what cryopreservation was brought
Inconvenience；(4) gut flora fixer in the human faecal mass that the present invention is provided, directly can add the preservation equipped with fixer by excrement
Guan Zhong, then normal temperature be kept in dark place, it is not necessary to cryopreservation is carried out, using simple；(5) in the human faecal mass that the present invention is provided
Gut flora fixes liquid and preparation method thereof simply, and production cost is low, can be controlled in 2~3 yuan/mL, with extremely wide application value.
It should be noted that unless otherwise indicated, technical term or scientific terminology used in this application should be this hair
The ordinary meaning that bright one of ordinary skill in the art are understood.Unless specifically stated otherwise, otherwise illustrate in these embodiments
Part and relative step, numerical expression and the numerical value of step are not limit the scope of the invention.It is illustrated and described herein
In all examples, unless otherwise prescribed, any occurrence should be construed as merely exemplary, not as limitation, because
This, other examples of exemplary embodiment can have different values.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to foregoing embodiments for pipe, it will be understood by those within the art that：Its according to
The technical scheme described in foregoing embodiments can so be modified, or which part or all technical characteristic are entered
Row equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme, it all should cover among protection scope of the present invention.
Claims (10)
1. gut flora fixer in a kind of human faecal mass, it is characterised in that in fixer described in per 1000mL, raw material components bag
Include：
400~600mmol Tris-HCl, 3~7mol NaCl, 50~150mmol EDTA and/or EDTA-Na2, surplus is water.
2. gut flora fixer in human faecal mass according to claim 1, it is characterised in that the raw material components are also wrapped
Include：
1~3mol ethanol.
3. gut flora fixer in human faecal mass according to claim 2, it is characterised in that fixer described in per 1000mL
In, raw material components include：
500mmol Tris-HCl, 100mmol EDTA and/or EDTA-Na2, 5mol NaCl, 2mol ethanol, surplus is water.
4. gut flora fixer in the human faecal mass according to claim any one of 1-3, it is characterised in that：
The pH value of the Tris-HCl is 8.0.
5. gut flora fixer in human faecal mass according to claim 4, it is characterised in that：
The water is deionized water.
6. the preparation method of gut flora fixer in the human faecal mass described in claim any one of 1-5, it is characterised in that including
Following steps：
All raw material components are well mixed, the fixer is obtained.
7. the application method of gut flora fixer in the human faecal mass described in claim any one of 1-5, it is characterised in that including
Following steps：
The excrement of collection is added in the fixer, is kept in dark place.
8. the application method of gut flora fixer in human faecal mass according to claim 7, it is characterised in that：
The time of the preservation is 0~30 day.
9. the application method of gut flora fixer in human faecal mass according to claim 7, it is characterised in that：
The temperature of the preservation is not higher than 40 DEG C.
10. gut flora fixer gut flora in human faecal mass is prepared is consolidated in the human faecal mass described in claim any one of 1-5
Application in fixed output quota product.
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| CN201710429297.7A CN107034141A (en) | 2017-06-08 | 2017-06-08 | Gut flora fixer and preparation method thereof in human faecal mass |
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Cited By (3)
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| CN108192827A (en) * | 2017-12-29 | 2018-06-22 | 深圳谱元科技有限公司 | A kind of intestinal flora sample room-temperature extender and its preparation method and application |
| CN111826371A (en) * | 2019-04-15 | 2020-10-27 | 上海锐翌生物科技有限公司 | Excrement nucleic acid preservation solution and preparation method and application thereof |
| CN112522362A (en) * | 2020-12-25 | 2021-03-19 | 南京申友生物技术有限公司 | Preservation solution for preserving bacterial DNA in fecal sample at normal temperature |
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