CN106591458A - Method for evaluating activity of bio-feed - Google Patents
Method for evaluating activity of bio-feed Download PDFInfo
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- CN106591458A CN106591458A CN201611206096.2A CN201611206096A CN106591458A CN 106591458 A CN106591458 A CN 106591458A CN 201611206096 A CN201611206096 A CN 201611206096A CN 106591458 A CN106591458 A CN 106591458A
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- feedstuff
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The invention relates to a method for evaluating activity of a bio-feed. Concretely speaking, the invention relates to a method for evaluating activity of the bio-feed by taking intestinal flora as a target object, an aseptic animal is used for detecting the positive or negative fluctuation of the animal intestinal flora for through 16S rDNA sequencing, and the activity of the bio-feed is evaluated. The method can be used for evaluating activity of various bi-feeds, the effect of a detection substance by the intestinal flora can be detected through in-vivo and in-vitro experiment, and the superiority-inferiority of the sample can be reflected through flora fluctuation.
Description
Technical field
The present invention relates to the activity degree evaluation methodology of biological feedstuff, and in particular to the one kind with intestinal microbial population as target is biological
The evaluation methodology of feedstuff activity degree.
Background technology
1st, the anti-situation of feedstuff taboo is increasingly severe
Abuse of antibiotics destroys intestinal microbial population, the health of serious harm animals and humans, while antibiotic remainss also endanger
Food safety.Chemically cultivation will be development trend to biological cultivation, activated feed theory-compliant biological cultivation intension.Current
Food safety attention rate more and more higher, under the higher and higher background of antibiotic disabling cry, current chemical aquaculture model is more next
It is more hard to carry on.Supported with the biology that the active skull cap components such as microbial ecological agent, plant extract are main raw materials of feed additives
Grow pattern and would is that development trend.
2nd, activated feed appraisement system lacks
Biological feedstuff, the intension of nonreactive feedstuff, standard lack, and define with conventional feed unclear.Probiotic bacteria (unit), conjunction
Raw unit, plant extract isoreactivity biological feedstuff lack common evaluation criterion, assessment technique, how with containing antibiotic, high-copper,
The feedstuff of the chemokineses such as Gao Xin forms essence and distinguishes also unclear from standard, technology, application etc..Activated feed needs corresponding
Evaluation methodology is supporting the integral frameworks such as its theory, technology, application.
3rd, active factorses regulation and control intestinal microbial population will be development trend
Intestinal microbial population is the collective that a large amount of antibacterials of the existence in humans and animals intestinal are constituted, and it is known as second set of base of the mankind
Because of group, the barometer of human health status.The bread and cheese rich in active factorses of comprehensive research and development and application adjustment intestinal microbial population,
Clinical nutrition food, medicine and animal health product, have become the mankind and animal health industrial emphases and focus.Intestinal
System of colony will profound influence animal feed nutritive.Intestinal health is intestinal microbial population health, and pig is foster intestinal microbial population.
4. germfree animal and grand genomic sequencing technique
The flora transplanted in the flora mice of pig source can keep the characteristic of pig intestinal microbial population, be suitable to study the phase of pig intestinal microbial population
Interaction and metabolism and the barrier action to external source bacterium.This model is easy to control, it may be determined that mutual in terms of metabolism and ecology
Effect, is to study that intestinal microbial population drug resistance is produced, intestinal microbial population Tiny ecosystem provides one to barrier action of external source pathogen etc.
Plant suitable model.
Metagenomics technically include the grand gene order-checking two of the sequencing of 16S rDNA hypervariable regions and full-length genome
Individual level.The sequencing of 16S rDNA hypervariable regions can obtain the relevant informations such as microbial diversity, population structure, evolutionary relationship；And it is right
Microorganism species full-length genome is sequenced, further microbial gene function, each microorganism species can be cooperated relation,
The aspects such as the relation between flora and environment are furtherd investigate.
So far, not yet it is related to detect the intestinal of the germfree animal for transplanting pig source flora using technique of metagenome
Group's positively or negatively method of the fluctuation to evaluate the activity degree of activated feed, and the concept of feedstuff activity degree be also propose first and
Definition.
The content of the invention
A first aspect of the present invention is related to a kind of method that feedstuff activity degree is evaluated, and it passes through to detect feedstuff to animal intestinal
Evaluating feedstuff activity degree, wherein feedstuff is all feedstuffs or raw material to the positively or negatively regulating and controlling effect of flora, including biological is raised
Material, in some embodiments, detects feedstuff to microbial population of animal intestinal tract just using 16S rDNA or grand gene order-checkings method
To or negative regulation effect.If feedstuff has positive regulation to act on microbial population of animal intestinal tract, illustrate that this feedstuff is active, than
Such as biological feedstuff；If on intestinal microbial population without affecting or having inhibitory action, such as the feedstuff containing antibiotic is to intestinal for some feedstuffs
Flora is negative regulation, then the activity degree containing antibiotic feed is negative, i.e., without activity.
In some embodiments, the step of feedstuff is detected to the positively or negatively regulating and controlling effect of microbial population of animal intestinal tract it
Before include with nonruminant originate flora set up animal model and with the feedstuff feeding animal model the step of, wherein
Flora is colonized in the intestinal of animal model, it is preferable that the step is carried out as follows：The feedstuff is fed into the animal model certain
Time, such as continuous 8 days, such as 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5
My god, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 70 days, 80 days, 90 days or more
For a long time, then using 16S rDNA or grand genomic sequencing techniques, in determining the feedstuff or factor pair animal model
Flora positively or negatively effect；Or the step of positively or negatively regulating and controlling effect of the detection feedstuff to microbial population of animal intestinal tract
Include the flora that nonruminant is originated being cultivated in intestinal flora isolated culturing device and being made with feedstuff or the factor before
With the step of, it is preferable that the intestinal flora isolated culturing device be in vitro chemostat, it is preferable that the step is carried out as follows：Profit
With intestinal flora isolated culturing device culture intestinal microbial population, by adding feedstuff or the factor and acting on certain hour, such as connect
Continuous effect 8 days, such as 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6
My god, 7 days, 8 days, 9 days, 10 days, 15 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 70 days, 80 days, 90 days or it is longer when
Between, then using 16S rDNA or grand genomic sequencing techniques, determine the feedstuff or factor pair isolated culture flora
Positively or negatively act on.
In some embodiments, the nonruminant is pig, and/or the animal model is mice, rat or suslik.
In some embodiments, the animal model is mice, it is preferable that animal model is that aseptic C57BL/6J is little
Mus, it is preferable that it is raised in Sterilized isolator.
In some embodiments, the step of method of the present invention includes detection feed microbe quantity, it can be adopted
The method of conventional plate count is carried out or the method that is sequenced using 16S rDNA is carried out.
In some embodiments, 16S rDNA sequence measurements refer to nine contained by detection bacterium 16S rDNA genes
" hypervariable region ", and/or grand gene order-checking refers to the genomic DNA for determining all microorganisms in whole environment.
In some embodiments, feedstuff include selected from probiotic bacteria, prebioticses, symphysis unit, plant extract, enzyme preparation and
One or more in acidulant.
In some embodiments, the abundance that beneficial symbiosis flora is shown to the positive acting of intestinal microbial population is raised, and/
Or the abundance of conditionality pathogenic bacterium is lowered, and/or external source pathogenic flora quantity is reduced or disappeared, otherwise is then negative role, excellent
Selection of land, beneficial fungal component mass selection from bacillus bifiduss such as bifidobacteria infantiies, bifidobacterium longum, lactobacilluss such as Lactobacillus plantarum,
Lactobacillus casei, bacillus acidophilus, Lactobacillus bulgaricus, Deshi Lactobacilluss, bacillus cereuss such as Bacillus licheniformis, hay bud
Spore bacillus, Bacillus coagulans, one or more of clostridium such as Clostridium butyricum, it is preferable that conditionality pathogenic bacterium are selected from enterococcus
Such as enterococcus faecalis, enterococcus faecalis, and/or Escherichia such as escherichia coli, it is preferable that external source pathogenic flora is selected from Salmonella such as
Salmonella choleraesuls, Salmonella enteritidis, salmonella typhi, and/or Escherichia such as pathogenic escherichia coli, clostridium is as produced
Gas capsular clostridium.
In some embodiments, the examination criteria of feedstuff can be set up according to methods described, wherein feedstuff activity degree is higher,
Intestinal microbial population is more in positive acting, and feedstuff activity degree is lower, and intestinal microbial population is more in negative role.Therefore, in some of the invention
Aspect, the present invention relates to the use of the examination criteria of the feedstuff of method of the present invention foundation.
In some embodiments, methods described is used to evaluate containing biological mildew removing agent, variety classes, the probiotic bacteria of function
Such as bacillus bifiduss, lactic acid bacteria, bacillus cereuss and yeast, prebioticses such as dextrinosan, oligofructose, oligomeric lactose, low
Chitosan, wooden oligosaccharide, enzyme such as protease, amylase, lipase, cellulase, hemicellulase, xylanase, phytic acid
Enzyme, pectase, plant extract such as tea polyphenols, resveratrol, Sanguinarine, Radix Rehmanniae, Radix Scutellariae, Herba Epimedii, Herba Plantaginiss, plant essence
Oil, antibiotic such as Calcium Oxytetracycline., kitasamycin, chlortetracycline, nosiheptide, enramycin, Avilamycin, bambermycin, olaquindox,
Heavy metal such as high-copper, Gao Xin, the feedstuff of pesticide residues.Those skilled in the art know, can be lived with method of the present invention evaluation
Property degree feedstuff and its contained composition be do not have it is conditional, as long as have optimization feedstuff activity degree demand, or worry be
To add or have been added to the activity degree into branch's impact feedstuff in feedstuff, it is possible to apply the method for the present invention.
In some embodiments, 16S rDNA sequencings take following step to carry out：1) preparation of genomic DNA；2) it is high
Variable region primers are designed and PCR amplifications；3) Illumina sequencing libraries build (addition joint)；4) PCR primer Axy
PrepTM Mag PCR Normalizer do normalized；5) machine sequencing on：The library for building is loaded to cBot or cluster life
Into system, generate for cluster and Miseq sequencings；6) sequencing result is analyzed.In other embodiments, 16S rDNA sequencings can be with
Any 16S rDNA sequence measurements known in the art are taken to carry out.
In some embodiments, the present invention relates to the use of the feedstuff that the method for the present invention identifies acquisition, its have compared with
High activity degree, with increase positive biological feedstuff raw material or the factor, and/or with reduce negative sense feedstuff or because
Son.The positive biological feedstuff raw material or the factor and/or negative sense feedstuff or the factor are using method as above identification sieve
Choosing is obtained, and sets up DIFFERENT FEED raw database, activated feed equation is set up, for the calculating of feedstuff activity degree, so as to excellent
Changing the steps such as the formula of feedstuff, Jing pilot scales, animal experiment can obtain matured product.
In other words, the invention discloses a kind of biological feedstuff evaluation methodology with intestinal microbial population as target, is moved by aseptic
Thing, the method detection microbial population of animal intestinal tract of 16S rDNA sequencings positively or negatively fluctuate to evaluate the activity degree of biological feedstuff.Just
Include variety classes, the probiotic bacteria of function, prebioticses, enzyme, plant extract etc., negative sense feedstuff to biological feedstuff raw material or the factor
Raw material includes antibiotic, part chemokineses etc..
It is an object of the invention to using nonruminant such as pig source flora animal model such as mouse model, with 16S rDNA surveys
The impact of the method evaluation comparison difference factor pair intestinal microbial population of sequence, optimizes feed formula, such as evaluation comparison biology mildew removing agent
With impact of the chemical adsorptivity mildew removing agent to intestinal microbial population, to illustrate biological mildew removing agent advantage, optimization of C/C composites.
By taking mildew removing agent test as an example, testing result inside and outside synthesises sets up DIFFERENT FEED raw database, sets up activity and raises
Material equation, for the calculating of feedstuff activity degree.
For achieving the above object, the solution that provides in one embodiment of the present invention is：Using transplanting pig source bacterium
The mouse model of group, method evaluation comparison biology mildew removing agent and the chemical adsorptivity mildew removing agent being sequenced with 16S rDNA in vivo,
The external impact to intestinal microbial population, illustrates the advantage of biological mildew removing agent, optimization of C/C composites.Specifically include the mice of transplanting pig source flora
The foundation of model, the operation of in vitro Chemostat Model, 16S rDNA sequencings, biological mildew removing agent and chemical adsorptivity mildew removing agent are to intestinal
The comparison of the impact of road flora.
Beneficial effects of the present invention include：
(1) functional feed with intestinal microbial population as target, including the growth performance of lifting pig can be developed, piglet abdomen is treated
The functional feed for rush down, prevent prevention of sow constipation, improving growing and fattening Meat etc.,
(2) for researching and developing the activated feed of substitute antibiotics, poultry drug resistance is reduced, reduces antibiotic in feces of livestock and poultry residual
Stay, alleviate the ecological environmental pollution problems such as soil, water body,
The method of the present invention can be used for the assessment of various biological feedstuff activity degrees, and by experiment in vivo and vitro tester pair is detected
The effect of intestinal microbial population, with the superiority-inferiority of the fluctuation change reflection sample of flora.
Description of the drawings
Fig. 1 shows the overall technology route map of the present invention.
Specific embodiment
Definition
Biological feedstuff refers to that with feedstuff and feed additive as object fermented the novel fodder developed using microbial project
Resource and feed additive, mainly including fodder enzyme preparation, probiotic bacteria, bioactive oligopeptides and oligosaccharide etc..
Relative to biological feedstuff, the feedstuff containing chemokineses such as antibiotic, high-copper, Gao Xin, pesticide is not sent out intestinal microbial population
Wave positive regulation effect, i.e., inactive feedstuff.
Herein activated feed is basically identical with the implication of biological feedstuff, bioactive feed, is used interchangeably, and encloses
For fermentable.Activated feed emphasizes have positive regulation to act on microbial population of animal intestinal tract.Activity degree refers to activated feed
One quantizating index of positive regulation is played to microbial population of animal intestinal tract.Specifically, lived based on the feedstuff under intestinal microbial population target
Property degree evaluation methodology, carry out activity degree evaluation experimental to DIFFERENT FEED or raw material, screen suitable object of reference, formed data base and
Activated feed equation, for the calculating of feedstuff activity degree.
Microbial population of animal intestinal tract refers to the miscellaneous microorganism lived away from home in animal intestinal, combines according to a certain percentage,
Condition each other between each bacterium, depend on each other for existence, a kind of ecological balance is formed on quality and quantity.The antibacterial of these huge numbers substantially can be with
It is divided into three big class：Probioticss, harmful bacteria and neutral bacterium.
The flora in nonruminant source refers to the microorganism species in healthy nonruminant intestinal.Nonruminant be relative to
For complex stomach animal, the animal of body only one of which gastric gland is referred to, for example, pig, chicken, people etc..Pig source flora refers to health pig
Microorganism in intestinal.
Phenotype of the phenotype and/or symptom of the microbial population of animal intestinal tract that the present invention is utilized on animal generally refers to young animal
Whether suffer from diarrhoea, female animal whether constipation, growth performance of growing and fattening animal etc., such as the phenotype on pig refers generally to piglet is
No diarrhoea, sow whether constipation, growth performance of growing-finishing pig etc..Grand gene order-checking is referred in the whole environment to sample
The genomic DNA of all microorganisms carries out high-flux sequence, analyzes the multiformity and abundance of micropopulation, seek microorganism with
The new, gene with specific function is excavated and studied to environment, the relation between microorganism and host.
Animal model is referred to for studying the non-human species for understanding that certain particular organisms phenomenon is used, so that in the mould
The discovery obtained in type animal can provide the understanding of associated biomolecule activity in other organisms.In the present invention, animal model is used
In the flora in transplanting nonruminant such as pig source.Animal model used by the present invention can be any animal mould known in the art
Type, such as mice, rat, suslik etc., the animal model is preferably aseptic, and raises in gnotobasiss.
16S rDNA are 16S ribosomal DNA, are the ingredients of protokaryon ribosome 30S small subunits, average molecular
Quality is about 0.6MDa, and length is about 1540nt, and 16S rDNA are one section of sequences the most frequently used in systematic bacteriology research, comprising
Variable region and conserved region.In the present invention, the variable region in 16S rDNA genes, especially hypervariable region, such as 9 " high
Degree variable region ", is used for PCR amplification sequencings.9 " hypervariable regions " of 16S rDNA are well known in the art.
16S rDNA sequencings are that the specific hypervariable region of directed toward bacteria small subunit ribosome 16SrDNA genes enters performing PCR expansion
Increase sequencing, and the sequencing data for obtaining is compared with existing 16S rDNA data bases, so as to reflect group
Evolutionary relationship and community diversity between species composition, species.
Prebioticses (bacterium) refer to the based food composition that growth of probiotics can be promoted to breed, used as the substrate of proliferation of probiotics
Can not be digested and assimilated by animal body, be fermented in large intestine.Prebioticses mainly include various oligosaccharide kind materials, and such as Isomalt is low
Polysaccharide, oligofructose, oligomeric lactose, oligo-chitosan, wooden oligosaccharide etc..
Probiotic bacteria refers to that Jing animals and human body are ingested sufficient amount of and confirm micro- life favourable to host health, living
Thing.Mainly include several big class such as lactic acid bacteria, bacillus cereuss and yeast.Such as bacillus bifiduss, Lactobacillus plantarum, lactobacillus casei
And bacillus acidophilus etc..
Symphysis unit refers to the biological preparation that probiotic bacteria is used in combination with prebioticses, and the prebioticses in symphysis unit must promote interior
The probiotic bacteria for containing, i.e., both combine needs to play synergism.
The plant extract that the present invention can be used includes tea polyphenols, resveratrol, Sanguinarine, Radix Rehmanniae, Radix Scutellariae, excessive sheep
The leaves of pulse plants, Herba Plantaginiss, plants essential oil (Herba Origani oil, Herba Lysimachiae foenum-graeci quintessence oil, rosemary ethereal oil, Peppermint essential oil, star anise oil etc.).
The enzyme preparation that the present invention can be used includes protease, amylase, lipase, cellulase, hemicellulase, wood
Dextranase, phytase, pectase etc..
The acidulant that the present invention can be used includes citric acid, Pfansteihl, fumaric acid, formic acid, acetic acid, propanoic acid, butanoic acid, mountain
Pears acid, malic acid, tartaric acid, benzoic acid etc..
Probioticss in intestinal are mainly various bacillus bifiduss, lactobacilluss etc., are the indispensable key elements of health,
Various vitamin can be synthesized, the digestion of food is participated in, promote intestinal peristalsis promoting, suppress the growth of pathogenic flora, decomposition are harmful to, have
Noxious material etc..
Conditionality pathogenic bacterium in the intestinal i.e. antibacterial with dual function, such as escherichia coli, enterococcus, in positive reason
It is beneficial to health under condition, once proliferation out of control, or it is transferred to other positions of body from intestinal, it is possible to cause many problems.
External source pathogenic bacterium in intestinal include Salmonella, staphylococcus aureuses, pathogenic escherichia coli etc..They belong to
External source flora kind, once eat by mistake entering intestinal, then can cause the symptoms such as diarrhoea, alimentary toxicosis.
The potency of feed formula refers to that a certain material causes effect unit of biological respinse, can be detected with physico-chemical method,
Can be determined with biological detecting method；Or the mark of biological product activity (quantity) height, generally using biology method measuring.It is main
To include energy digestibility, Ileal amino acid digestibility, protein digestibility, fat digestibility, digestibility of fiber, ash, calcium, phosphorus
Etc. index.
Mildew removing agent is referred to can be adsorbed mycotoxin (such as vomitoxin, 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone, aflatoxin) or drop
A kind of feed additive of solution.
In vitro Chemostat Model refers to a kind of can simulate intestinal, continuous intestinal flora isolated culturing device for flowing.It
Temperature-controlling system, automatic transport fresh culture and output liquid waste system and environment needed for intestinal microbial population growth including fermentation tank is protected
Barrier system.
Intestinal microbial population culture with culture medium suitably selected according to demand.Such as lactic acid bacteria MRS culture medium, greatly
Enterobacteria LB culture medium.
Operational taxonomic unit (OTU) is sorting of operation unit (OTU), for species taxonomy and species relative abundance analyze it is basic
Unit.Generally, if between sequence, such as the similarity of different 16S r RNA sequences just can be less than 98%
It is defined as an OTU, and each OTU corresponds to a different 16SrDNA sequence, that is, each OTU corresponds to one not
Same antibacterial (microorganism) is planted.
Pasitive Regulation Effect of Genseng refers to the probiotic bacteria of variety classes, function；Prebioticses；Enzyme；Plant extract etc. is to intestinal
The positive regulation of group, the abundance that can make the beneficial symbiosis flora such as bacillus bifiduss, lactobacilluss is raised, the bar such as enterococcus, enterobacteria
The abundance of part pathogenic bacterium is lowered, and the external source such as Salmonella and pathogenic escherichia coli pathogenic flora disappears.
Negative regulation effect refers to the impact to intestinal microbial population such as antibiotic, part chemokineses, causes bacillus bifiduss, breast
The abundance of the beneficial symbiosis flora such as acidfast bacilli is lowered, and the abundance of the conditionality pathogenic bacterium such as enterococcus, enterobacteria is raised, Salmonella
Raise with the external source pathogenic flora abundance such as pathogenic escherichia coli.
The present invention will be further illustrated by following non-limiting examples below, it is as well known to those skilled in the art, not
In the case of spirit of the invention, many modifications can be made to the present invention, such modification also falls into the scope of the present invention.
Following experimental techniques if no special instructions, are conventional method, the experiment material for being used if no special instructions,
Easily can obtain from commercial company.
Embodiment
The foundation of the mouse model of the transplanting pig of embodiment of the method 1 source flora
Aseptic C57BL/6J mices (being provided by Third Military Medical University's Experimental Animal Center) are raised in Third Military Medical University's base
In Chu Bu experimental zoologies teaching and research room Sterilized isolator, feeding environment：20～24 DEG C, humidity 40%～70%, strict 12h illumination
12h is dark.Feedstuff from Beijing Austria of section pull together feed corporation,Ltd sale co60 irradiation big and small mouse upgrowths breed material, bedding and padding Jing
Co-60 γ 50kGy x ray irradiation xs are sterilized, and mouse cage, drinking-water and drinking bottle etc. adopt autoclave sterilization (121 DEG C, 60min).
The aseptic C57BL/6J mices of 30 4 week old are fed into 0.1mL swine excrement suspensions (from Healthy Youth pig, without gastroenteropathy, drink
Food and living habit are normal, and do not feed within 2 months before this experiment starts the early morning the 1st of the pig of any antibiotic and probiotic bacteria
Secondary fresh excreta), treat that flora is colonized 3 weeks, that is, obtain the mice of transplanting pig source flora.
The in vitro Chemostat Model of embodiment of the method 2 runs
Sending out in vitro Chemostat Model (the explanation composition model according to Application Number (patent) is 200710028346.2)
Intestinal microbial population culture medium 500ml of sterilizing is injected in fermentation tank, is sealed with wax, make whole system be in aseptic, closed environment.The company of unlatching
The peristaltic pump of fresh culture is connect with 35ml.h-1Flow velocity runs, and conveying fresh culture is continuously replenished nutrient substance, and waste liquid is with same
Uniform flow is removed, it is ensured that culture fluid is constant.Accurately weigh swine excrement sample and (Healthy Youth pig is collected in, without gastroenteropathy, drink
Food and living habit are normal, and do not feed within 2 months before this experiment starts the early morning the 1st of the pig of any antibiotic and probiotic bacteria
Secondary fresh excreta) 5g, with 0.85% normal saline, 5 times of dilutions of sterilizing, fully filter after homogenate, take 3 parts of samples of equivalent and mix
It is even, in being inoculated in the fermentation tank for balancing 24h.The culture medium prescription (g/L) of wherein intestinal microbial population culture is：Peptone
3.0g, milk cheese albumen 3.0g, lipoprotein 0.6g, pectin 0.6g, xylan 0.6g, starch 5.0g, L-Cysteine 0.4g,
Cholesterol 0.25g, chenodeoxy cholic acid 0.25g, cholic acid 0.25g, arabogalactan 0.6g, KH2PO42.0g, N
aHCO30.1g, CaCl2.2H2O 0.45g, MgSO4.7H2O 0.5g, protohemin 0.01g.
The 16S rDNA of embodiment of the method 3 are sequenced
Extract sample STb gene, Bian 16S rDNA gene V3-V4Variable region is expanded as target, universal primer sequence
Row：341F-806R forward primers (5'-3')：ACTCCTACGGGRSGCAGCAG(341 F)(SEQ ID NO：1), reverse primer
(5'-3')：GGACTACVVGGGTATCTAATC(806 R)(SEQ ID NO：2) (with reference to Sun, W.et al.Mechanism
and Effect of Temperature on Variations in Antibiotic Resistance Genes during
Anaerobic Digestion of Dairy Manure.Sci.Rep.6,30237；doi:10.1038/srep30237
(2016) .), to expand and carry out machine sequencing after the PCR primer purification for completing.Whole sequencing flow process is by the sharp next biological section in Shanghai
Skill company limited completes, including DNA extraction and design of primers etc., carry out upper machine sequencing using Illumina HiSeq PE250.
By carrying out OUT (operational taxonomic unit (OTU)) cluster analyses to sequencing result, reflect that the species composition of group, thing macroevolution are closed
System and community diversity.
The active factorses evaluation of embodiment 1
1.1 laboratory animals and process：
Pig source flora mouse model (established as described above) feeds respectively biological mildew removing agent (addition 2g/kg), chemistry and takes off mould
Agent (addition 2g/kg), normal feedstuff.
Wherein described biological mildew removing agent is referred to using all kinds of mycotoxins in biological mode efficient degradation feedstuff, degraded
Product is nontoxic.(concrete composition is bacillus subtilises >=1 × 109CFU/g, moisture≤10.0%, lactic acid bacteria >=2.5 ×
108CFU/g, vitamin A >=100000IU/kg, Vitamin E >=800IU/kg).
Described chemical mildew removing agent refers to montmorillonite adsorbent, and main component is hydrated aluminum silicate calcium (HSCAS).
The formula of normal feedstuff is given in Table 1.
The SPF mice normal feedstuff of table 1
1.2 main agents and instrument:
DNA extraction kit (Qiagen, article No.：51504), (common anaerobic culture medium (GAM), is purchased from Qingdao to culture medium
GaoKeYuan Hai Bo Bioisystech Co., Ltd), in vitro chemostat is (according to the explanation that Application Number (patent) is 200710028346.2
Composition model), PCR amplification instrument (Invitrogen, model：12346-086), ultra cold storage freezer (- 80 DEG C), superclean bench,
Microscope etc.
1.3. feedstuff or product micro organism quantity are detected
Routinely microorganism the method for plate culture count or 16S rDNA are sequenced, to content of microorganisms in feedstuff or product
It is measured.
Biological mildew removing agent composition：
Bacillus subtilises >=1 × 109CFU/g, moisture≤10.0%, lactic acid bacteria >=2.5 × 108CFU/g vitamin A >=
100000IU/kg, Vitamin E >=800IU/kg (Ao Fei Bioisystech Co., Ltd of Shenzhen hundred takes off mould treasured).
1.4. activity in vivo factor evaluation
With above-mentioned pig source flora mouse model as laboratory animal, by above-mentioned 16S rDNA detection methods, above-mentioned life is detected
Positively or negatively regulating and controlling effect of the thing mildew removing agent with above-mentioned chemical mildew removing agent to intestinal microbial population.
1.4.1 laboratory animal and process
30 mices (the aseptic week old of C57BL/6J mices 7, male and female half and half) are divided into into 3 groups, 10 per group.Control group fed
Normal feedstuff, biological mildew removing agent group feeding normal feedstuff+biology mildew removing agent (addition 2g/kg), chemical mildew removing agent group feeds base
Plinth feedstuff+chemistry mildew removing agent (addition 2g/kg).Experimental period is 8 weeks.1.4.2 sample collection
Feed and take within the 8th week after mildew removing agent its fresh excreta sample (3 parts) and take pictures, loaded in the aseptic EP pipes of 2mL, cold storage
It is stand-by in -80 DEG C.
1.4.3DNA extract
Stool in mice 1mg is taken, in 2mL centrifuge tubes, 1mL lysates is added；Referring in particular to the feces genomic DNA of Tiangeng
The explanation of extracts kit is carried out.
1.4.4 16S rDNA are sequenced
As stated above, 3 repetitions of per group of random selection, totally 9 sample sequencings.
1.5 external activity factor evaluations
In vitro chemostat with Pigs Inoculated intestinal microbial population is sequenced as model by 16S rDNA, the biological mildew removing agent of detection with
Positively or negatively regulating and controlling effect of the chemical mildew removing agent to intestinal microbial population.
1.5.1 in vitro chemostat packet and process
Using 3 sets of in vitro Chemostat Models, it is divided into blank control group, chemical mildew removing agent group, biological mildew removing agent group, in vitro mould
After type Pigs Inoculated intestinal microbial population 14d, chemical mildew removing agent group addition chemistry mildew removing agent (addition 2g/L), biological mildew removing agent group addition
Biological mildew removing agent (addition 2g/L), continuous action 8 days.
1.5.2 sample collection
After feces inoculation fermentation tank, plus mildew removing agent continuous action gather culture fluid after 8 days, loaded on the aseptic EP pipes of 2mL in,
Cold storage is stand-by in -80 DEG C, for bacterial number detection and Bacterial community analysis.
1.5.3DNA extract and 16S rDNA sequencings
Culture fluid 0.5mL is taken, in 2mL centrifuge tubes, 1mL lysates is added；Referring in particular to the feces genomic DNA of Tiangeng
The explanation of extracts kit is carried out.
1.5.4 16S rDNA are sequenced
As stated above, 3 repetitions of per group of random selection, totally 9 sample sequencings.
1.6 technology paths, referring to accompanying drawing 1.
1.7 experiment in vivo and vitro results
It is sequenced by 16S rDNA, reflects evolutionary relationship and community diversity between species composition, the species of group.Determine
Positive activation or inhibitory action of two mildew removing agent to intestinal microbial population：
(1) compared with other two groups, the beneficial fungal component such as bacteroid, bacillus bifiduss, lactobacilluss in biological mildew removing agent group
The abundance of group is raised, and makes fungal component account for overwhelming dominance；
(2) compared with other two groups, in biological mildew removing agent group under the abundance of conditionality pathogenic bacterium such as enterococcus, Escherichia
Adjust.
(3) external source such as Salmonella and pathogenic escherichia coli pathogenic flora is not detected by three groups, if any pathogenic
Bacterium, adds and pathogenic bacterium abundance can be reduced or eliminated after activated feed.
The results are shown in Table 2.
Table 2 tests the relative abundance of related microorganisms monoid
Sequence table
<110>Hai Jinzhuo Bioisystech Co., Ltd before Shenzhen
<120>The evaluation methodology of biological feedstuff activity degree
<130> LZ1605770CN01
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>341F-806R forward primers
<400> 1
actcctacgg grsgcagcag 20
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>Reverse primer
<400> 2
ggactacvvg ggtatctaat c 21
Claims (10)
1. a kind of method that feedstuff activity degree is evaluated, it passes through to detect that positively or negatively regulation and control of the feedstuff to microbial population of animal intestinal tract are made
For evaluating feedstuff activity degree, wherein feedstuff is preferably biological feedstuff, it is preferable that using 16S rDNA or grand gene order-checking sides
Method detects positively or negatively regulating and controlling effect of the feedstuff to microbial population of animal intestinal tract.
2. the method for claim 1, it is in detection feedstuff to the positively or negatively regulating and controlling effect of microbial population of animal intestinal tract
Include that the flora originated with nonruminant is set up animal model and feeds the step of the animal model with the feedstuff before step
Suddenly, wherein flora is colonized in the intestinal of animal model, it is preferable that the step is carried out as follows：The feedstuff is fed into the animal
Model certain hour, such as continuous 8 days, then using 16S rDNA or grand genomic sequencing techniques, determine the feedstuff or
The positively or negatively effect of the flora in factor pair animal model；Or in detection feedstuff to the positive or negative of microbial population of animal intestinal tract
Include to before the step of regulating and controlling effect by nonruminant originate flora cultivate in intestinal flora isolated culturing device and with
The step of feedstuff or the factor are acted on, it is preferable that the intestinal flora isolated culturing device is in vitro chemostat, it is preferable that
The step is carried out as follows：Using intestinal flora isolated culturing device culture intestinal microbial population, by adding feedstuff or the factor simultaneously
Effect certain hour, such as continuous action 8 days, then using 16S rDNA or grand genomic sequencing techniques, determine the feedstuff former
The positively or negatively effect of material or factor pair isolated culture flora.
3. method as claimed in claim 2, wherein nonruminant are pig, and/or the animal model is mice, rat or ground
Mus, it is preferable that animal model is mice, it is preferable that animal model is aseptic C57BL/6J mices, it is preferable that it is raised in nothing
In bacterium isolator.
4. the method as described in any one of Claim 1-3, the step of it also includes micro organism quantity in detection feedstuff, preferably
Ground, using colony counting method or 16S rDNA sequence measurements the step is carried out.
5., such as the method for any one of claim 1 to 4, wherein 16S rDNA sequence measurements refer to detection bacterium 16S rDNA genes
Nine contained " hypervariable regions ", and/or grand gene order-checking refers to the genome for determining all microorganisms in whole environment
DNA。
6. the method as described in any one of claim 1 to 5, wherein feedstuff include being carried selected from probiotic bacteria (unit), symphysis unit, plant
Take one or more in thing, enzyme preparation and acidulant.
7. the method as described in any one of claim 1 to 6, wherein showing beneficial fungal component to the positive acting of intestinal microbial population
The abundance of group is raised, and/or the abundance of conditionality pathogenic bacterium is lowered, and/or external source pathogenic flora quantity is reduced or disappeared, otherwise
It is then negative role, it is preferable that beneficial fungal component mass selection is from bacillus bifiduss such as bifidobacteria infantiies, bifidobacterium longum, lactic acid bar
Bacterium such as Lactobacillus plantarum, lactobacillus casei, bacillus acidophilus, Lactobacillus bulgaricus, Deshi Lactobacilluss, bacillus cereuss such as lichens
Bacillus cereuss, bacillus subtilises, Bacillus coagulans, one or more of clostridium such as Clostridium butyricum, it is preferable that conditionality is caused
Pathogenic bacteria is selected from enterococcus such as enterococcus faecalis, enterococcus faecalis, and/or Escherichia such as escherichia coli, it is preferable that external source pathogenic flora
It is as big in caused a disease selected from Salmonella such as Salmonella choleraesuls, Salmonella enteritidis, salmonella typhi, and/or Escherichia
Enterobacteria, clostridium such as bacillus perfringens.
8. the method as described in any one of claim 1 to 7, wherein the examination criteria of feedstuff can be set up according to methods described, its
Middle feedstuff activity degree is higher, and intestinal microbial population is more in positive acting, and feedstuff activity degree is lower, and intestinal microbial population is more in negative role.
9. the method as described in any one of claim 1 to 8, it can be used to evaluate containing biological mildew removing agent, variety classes, function
Probiotic bacteria such as bacillus bifiduss, lactic acid bacteria, bacillus cereuss and yeast, prebioticses such as dextrinosan, oligofructose, oligomeric
Lactose, oligo-chitosan, wooden oligosaccharide, enzyme such as protease, amylase, lipase, cellulase, hemicellulase, xylan
Enzyme, phytase, pectase, plant extract for example tea polyphenols, resveratrol, Sanguinarine, Radix Rehmanniae, Radix Scutellariae, Herba Epimedii, Herba Plantaginiss,
Plants essential oil, antibiotic for example Calcium Oxytetracycline., kitasamycin, chlortetracycline, nosiheptide, enramycin, Avilamycin, bambermycin,
Olaquindox, heavy metal such as high-copper, Gao Xin, the feedstuff of pesticide residues.
10. the method as described in any one of claim 1 to 9, wherein 16S rDNA sequencings take following step to carry out：1) gene
The preparation of group DNA；2) hypervariable region design of primers and PCR are expanded；3) Illumina sequencing libraries build (addition joint)；4)
PCR primer does normalized with Axy PrepTM Mag PCR Normalizer；5) machine sequencing on：On the library for building
Sample to cBot or cluster generate system, generate for cluster and Miseq sequencings；6) sequencing result is analyzed.
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