CN106399135B - One plant height is laid eggs white yeast strain C20140911 and its breeding, cultural method and application - Google Patents
One plant height is laid eggs white yeast strain C20140911 and its breeding, cultural method and application Download PDFInfo
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Abstract
It lays eggs white yeast strain (Saccharomyces cerevisiae) C20140911 and its screening technique and application the invention discloses a plant height, and provides its breeding, cultural method and application.The invention has the benefit that its fermentation liquid protein content of yeast strain disclosed by the invention and type identify peptide fragment 241, protein group 121 much higher than bacterium germination is gone out altogether from the bacterial strain fermentation liquor；254.4% and 157.4% are improved than going out bacterium germination respectively；Not only protein content is high for the bacterial strain fermentation liquor, and the metabolic pathway participated in is also more, participates in more carbohydrate metabolism and energetic supersession；With uniquely with electron carrier activity and the active protein of transhipment, the functions such as bioelectric detecting, stimulate the reaction and positioning process may be exercised, protein function and the metabolic pathway of participation are far more than bacterium germination out in the bacterial strain fermentation liquor of the invention, bacterial strain of the invention can be used for preparing feed addictive and probiotics, improve breeding performonce fo animals, diarrhea of weaned piglets is reduced, effect is better than bacterium germination out.
Description
Technical field
The present invention relates to animal feed additive technical fields, and in particular to a plant height is laid eggs white yeast strain S yeast
(Saccharomyces cerevisiae) C20140911 and its breeding, cultural method and application.
Background technique
Saccharomycete is a kind of eukaryon unicellular microorganism, has typical eucaryotic cell structure, there is nucleus, endochylema film, cell
Wall, cytoplasm and other contents etc..Because yeast rich in good protein, amino acid, complete B family vitamin, with life knot
Several mineral materials and function dietary fiber existing for state form are closed, it is practical to be widely used in production.Yeast is that mankind's application is compared
It is early, and the microorganism being most widely used, people are frequently utilized that its fermentation manufactures various fermentation food and wine
Wine, as brewer's yeast is applied to Beer Brewage；The carrier of selenium-rich, Zinc-rich saccharomyces cerevisiae as organic trace element, makes microelement more
Easily absorb etc.；Yeast adds in feed mainly as single cell protein in animal husbandry, to reduce making for other protein raw materials
With.Meanwhile also there is the research by yeast culture (Yeast Culture, YC) as probiotics to report.Yeast Cultivation
Object is a kind of product of Yeast Cultivation, is referred to after the fermentation of strict control condition together with made from culture medium processing
Product, main component therein are the metabolites of culture medium, saccharomycete and saccharomycete.Protein in yeast metabolism product
Content is seldom, but it contains other " unknown somatomedins ", for adjusting the microecological balance of animal intestinal tract, improves feed
Utilization rate, increase premunition and anti-stress ability, promote growth, to improve the production performance of animal.Yeast culture exists
The application of animal husbandry, has many advantages, because it will not both interfere the effect of antibiotic, while can reduce making for antibiotic
With, call food safety, limit today of antibiotic usage, yeast product will have broader practice prospect.
Summary of the invention
The purpose of the present invention is to above-mentioned defect in the prior art, lay eggs white level to improve bacterial strain, to
Better probiotics and feed addictive are prepared, is that food safety and animal husbandry are raw to enhance its clinical use effect
Service is produced, a plant height is provided and lays eggs white yeast strain S yeast (Saccharomyces cerevisiae) C20140911
And its breeding, cultural method and application.
To achieve the goals above, technical solution provided by the invention an are as follows: plant height is laid eggs white yeast strain S yeast
(Saccharomyces cerevisiae) C20140911, in China Committee for Culture Collection of Microorganisms's commonly micro- life
The deposit number at object center is CGMCC No.9675, and the preservation time is on September 18th, 2014.
The rich protein yeast bacterial strain of above-mentioned high-biomass is in original saccharomyces cerevisiae Saccharomyces
By long-term domestication gained on the basis of cerevisia.
It lays eggs white yeast strain (Saccharomyces a second object of the present invention is to provide an above-mentioned plant height
Cerevisiae) the selection of C20140911, the selection be with 14g brewer's wort powdered medium, 1g peptone,
100ml distilled water is culture medium, and by culture medium high pressure sterilization, gnotobasis tune initial incubation liquid pH is 5.8, and access 1ml is initial
Set out bacteria culture fluid, and temperature is 27.3 DEG C, revolving speed 120r/min, shakes 48-72H, and constant-temperature table culture is passed on repeatedly；From low
The peptone of dosage 1% starts, and carries out growth tolerance domestication to yeast strain (Saccharomyces cerevisiae), by
Step increases peptone dosage to 8%, tests by multiple secondary screening and passage, filters out high yield protein yeast bacterial strain；
It lays eggs white yeast strain (Saccharomyces third object of the present invention is to provide an above-mentioned plant height
Cerevisiae) the cultural method of C20140911 is by malt extract medium 14.0g, peptone 8g, distilled water 100ml, height
Pressure sterilizing；Gnotobasis tune initial incubation liquid pH is 5.8, access kind daughter bacteria 1ml, is 27.3 DEG C in temperature, revolving speed 120r/
Min, constant-temperature table culture.
Fourth object of the present invention is laid eggs white yeast strain (Saccharomyces there is provided an above-mentioned plant height
Cerevisiae) application of the C20140911 in preparation animal high-protein feed additive.
High yield protein yeast bacterial strain disclosed by the invention, fermentation liquid protein content and type are higher than original bacterium germination out more
Strain.
High yield protein yeast bacterial strain fermentation liquor protein content disclosed by the invention is 0.329mg/ml, than the fermented liquid that sets out
0.304ml/mg improve 8.2%.
Peptide fragment 241, protein groups 121 are identified in high yield protein yeast bacterial strain fermentation liquor of the present invention altogether；It is original
Starting strain fermentation liquid identifies peptide fragment 68 altogether, protein groups 47, is higher by 254.4% and 157.4% respectively.
Mass Spectrometric Identification of the present invention is 109 total to annotated albumen, in the high yield protein yeast bacterial strain fermentation liquor
63, peculiar albumen is identified, out the peculiar protein 11 of bacterium germination, close to 6 times of bacterium germination out；35, albumen is shared with bacterium germination out.
A plant height disclosed by the invention is laid eggs, and not only protein content is high for white yeast strain ferments liquid, protein function and participation
Metabolic pathway is far more than bacterium germination out.More carbohydrate metabolism and energetic supersession are participated in, with uniquely with electron carrier
Activity and the active protein of transhipment, may exercise the functions such as bioelectric detecting, stimulate the reaction and positioning process.
The peculiar albumen WEGO functional annotation clustering of the present invention shows 63 in the high yield protein yeast bacterial strain fermentation liquor
The peculiar albumen of kind, the albumen for participating in biological processes (biological progress) annotate for 105 totally, participate in molecular function
The albumen of (molecular function) annotates 77 kinds, the albumen note 41 of cytology component (Cellular component)
Kind.Peculiar albumen in 11 kinds of original bacterial strain fermentation liquor, the albumen for participating in biological processes (biological progress) are total
12 annotations, the albumen for participating in molecular function (molecular function) annotate 7 kinds, cytology component (Cellular
Component albumen note 5 kind).
The peculiar Protein G O functional annotation of the present invention shows in the peculiar albumen of the high yield protein yeast bacterial strain fermentation liquor,
The albumen of cell processes (cellular process) is primarily involved in biological processes (Biological Process) classification
33 kinds are annotated with, metabolic process (metabolic process) albumen is participated in and is annotated with 32 kinds, single creature process
(single-organism process) has 10 kinds of albumen annotations, (the cellular component of cell biological synthesis
Organization or biogenesis) there are 8 kinds, remaining participation has 5 kinds of positioning (localization), stress reaction
2 kinds of (response to stimulus), 6 kinds of biological regulation (biogical regulation) etc..Participate in molecular function
(Molecular Function's) mainly has catalytic activity (catalytic activity) albumen to annotate 30 kinds, in conjunction with egg
White 7 kinds of (binding) note 2, molecular structure albumen (structural molecule) annotates 6 kinds, remaining has transport protein
(transporter) 3 kinds, binding protein (the protein binding transcription with transcription factor activity
Factor activity) a kind, nucleic acid (the nucleic acid binding transcription with transcriptional activity
Factor activity) a kind of equal annotation；And antioxidant activity (ntioxidant activity) note a kind, electronics carry
1 kind of body activity (electron carrier activity).Participating in annotation albumen in Cellular Component classification has
18 kinds of cell (cell), 9 kinds of organelle (organelle), macromolecular compound (macromolecular complex) 9
Kind, 4 kinds of extracellular region (extracellular region), 4 kinds of cell membrane (membrane), film closing chamber (membrane-
Enclosed lumen) a kind.
In the peculiar albumen of starting strain fermentation liquid, generation is primarily involved in biological processes (Biological Process) classification
Journey of apologizing for having done sth. wrong (metabolic process) has 8 kinds of albumen annotations, single creature process (single-organism
Process) there are 5 kinds of albumen annotations, the albumen of cell processes (cellular process) is annotated with 4 kinds, remaining has participation to answer
The biosynthesis of sharp 2 kinds for reacting (response to stimulus), a kind for positioning (localization), cell
1 kind etc. of (cellular component organization or biogenesis).Molecular function (Molecular
Function) annotation albumen mainly has 6 kinds of participation catalytic activity (catalytic activity), binding protein in classification
(binding) 6 kinds, a kind of transport activity (transporter activity).Cell forms (Cellular Component)
Annotation albumen is made of participation cell (cell) in classification 2 kinds, a kind of organelle (organelle) composition, macromolecular
1 kind for closing object (macromolecular complex) composition, extracellular region (extracellular region) forms a kind, carefully
4 kinds of after birth (membrane) composition.
KEGG access annotation of the present invention shows the peculiar albumen of high yield protein yeast bacterial strain fermentation liquor in KEGG database
In be compared after, obtain the metabolic pathways of some of matching albumen, residual protein is limited because studying, and can not also look for
It to its matched information, matches albumen and participates in 24 metabolic pathways altogether: being respectively purine metabolism (Purine metabolism) 4
Kind, 4 kinds of glycolysis (Glycolysis/Gluconeogenesis), photosynthetic carbon fixation acts on (Carbon fixation in
Photosynthenic organisms) 2 kinds, 2 kinds of nitrogen metabolism (Nitrogen metabolism), alanine, asparagine
Acid and 2 kinds of glutamic acid metabolism (Alanine, asparatate and glutamate metabolism), protokaryon carbon solidification effect
2 kinds of (Carbon fixation pathways in prokaryotes), metabolism of pyruvate (Pyruvate metabolism)
1 kind, a kind of phosphoinositide metabolism (inositol phosphate metabolism), pantothenate and CoA biosynthesis
1 kind of (Pantothenate and CoA biosynthesis), glyoxylate and dicarboxylic acids approach (Glyoxylate and
Dicarboxylate metabolism) a kind, citrate recycles (a kind of Citrate cycle (TCA cycle), figured silk fabrics ammonia
1 kind of the biosynthesis (Valine, leucine and isoleucine biosynthesis) of acid, leucine and isoleucine,
1 kind of glycine, serine and threonine metabolism (Glycine, serine and threonine metabolism), amino sugar
It is metabolized a kind of (Amino sugar and nucleotide sugar metabolism) with nucleotide sugar, cysteine and egg
Propylhomoserin is metabolized a kind of (Cysteine and methionine metabolism), Cytochrome P450 drug metabolism (Drug
Metabolism-cytochrome P450) a kind, fructose and sweet dew glycometabolism (Fructose and mannose
Metabolism) a kind, a kind of arginine and Proline Metabolism (Arginine and proline metabolism), cell color
Plain P450 xenobiotics are metabolized a kind of (Metabolism of xenobiotics by cytochrome P450), naphthalene
It degrades a kind of (Naphthalene degradation), degradation (the Chloroalkane and of alkyl chloride and chloro-alkenes
Chloroalkene degradation) a kind, a kind of Fatty acid degradation (Fatty acid degradation), tyrosine metabolism
1 kind of (Tyrosine metabolism), a kind of Vitamin A Metabolism (Retinol metabolism).
Original peculiar Protein Information of bacterial strain fermentation liquor is compared in KEGG database, participates in 8 metabolism ways altogether
Diameter: wherein a kind of fructose sweet dew glycometabolism (Fructose and mannose metabolism), purine metabolism (Purine
Metabolism) a kind, a kind of glycolysis (Glycolysis/Gluconeogenesis), riboflavin is metabolized (Riboflavin
Metabolism) a kind, methane is metabolized a kind of (Methane metabolism), pentose phosphate pathway (Pentose phosphate
Pathway) a kind, a kind of aminobenzoic acid degradation (Aminobenzoate degradation), photosynthetic carbon fixation acts on (Carbon
Fixation in photosynthenic organisms) a kind).
The invention has the benefit that its fermentation liquid protein content of yeast strain disclosed by the invention and type are much higher than
Bacterium germination out.Protein content is 0.329mg/ml in the bacterial strain fermentation liquor, and the 0.304ml/mg than the fermented liquid that sets out is improved
8.2%.Identify peptide fragment 241, protein group 121 altogether from the bacterial strain fermentation liquor；The fermented liquid that sets out identifies peptide altogether
Section 68, improves 254.4% and 157.4% than going out bacterium germination respectively by protein group 47.The bacterium in the albumen identified
63, the peculiar albumen of fermentation liquid, the peculiar protein 11 of bacterium germination, shares 35, albumen with bacterium germination out out.The bacterial strain fermentation liquor is not only
Protein content is high, and the metabolic pathway participated in is also more, participates in more carbohydrate metabolism and energetic supersession；With unique
With electron carrier activity and the active protein of transhipment, bioelectric detecting, stimulate the reaction and positioning process etc. may be exercised
Function.Protein function and the metabolic pathway of participation are far more than bacterium germination out in the bacterial strain fermentation liquor of the invention.Bacterial strain of the invention can
Feed addictive and probiotics are used to prepare, breeding performonce fo animals are improved, reduce diarrhea of weaned piglets, effect is better than out
Bacterium germination.
Detailed description of the invention
Fig. 1 is the gel electrophoresis figure in molecular biology identification.
Fig. 2 is the chromatogram result of ESI Mass Spectrometric Identification.
Wherein, upper figure is original starting strain fermentation liquid chromatogram, and the following figure is bacterial strain fermentation liquor chromatogram of the present invention.
Fig. 3 is shared protein groups WEGO functional annotation clustering figure.
Fig. 4 is the Biological Progress classification chart in shared histone GO functional annotation.
Fig. 5 is the Molecular Function classification chart in shared histone GO functional annotation.
Fig. 6 is the Cell Component classification chart in shared histone GO functional annotation.
Fig. 7 is peculiar protein groups WEGO functional annotation clustering figure.
Fig. 8 is the Biological Progress classification chart in peculiar Protein G O functional annotation.
Fig. 9 is the Molecular Function classification chart in peculiar Protein G O functional annotation.
Figure 10 is the Cell Component classification chart in peculiar Protein G O functional annotation.
Specific embodiment
Embodiment 1:
Cultivate selection
1, one plant of richness protein yeast bacterial strain (Saccharomyces cerevisiae) C20140911, in the micro- life of China
The deposit number of object culture presevation administration committee common micro-organisms center is CGMCC No.9675, and the deposit date is 2014
September 18 days.
The rich protein yeast bacterial strain of above-mentioned high-biomass is in original saccharomyces cerevisiae Saccharomyces
By long-term domestication gained on the basis of cerevisia.
The selection of above-mentioned high protein bacterial strain is with 14.0g brewer's wort powdered medium, 1g peptone, the bis- steamings of 100ml
Water is that (high pressure sterilization, gnotobasis tune initial incubation liquid pH are that 5.8), access 1ml initially sets out bacteria culture fluid, permanent to culture medium
Warm shaking table culture, temperature are 27.3 DEG C, revolving speed 120r/min, shake 48-72H, pass on repeatedly.
The bacterial strain that high protein is produced for culture domestication, since the peptone of low dosage 1%, to yeast strain
Saccharomyces cerevisiae carries out growth tolerance domestication, is stepped up peptone dosage to 8%, by multiple secondary screening
It is tested with passage, filters out high-biomass richness protein yeast bacterial strain.
Condition of culture: malt extract medium (14g), peptone (8g), distilled water (100ml), high pressure sterilization；Gnotobasis
Adjusting initial incubation liquid pH is 5.8, constant-temperature table culture: temperature is 27.3 DEG C, revolving speed 120r/min.
Survival condition: constant temperature incubation 72h is detected, after 10 times of normal saline dilution, detects viable bacteria with blood cell counting plate
Number can reach 108/ml。
Strain is that brewing industry produces strain in the culture, is not animals and plants cause of disease；Brewer's wort, peptone in culture medium
It is all nontoxic, it does not pollute the environment.
Above-mentioned one plant of richness protein yeast bacterial strain (Saccharomyces cerevisiae) C20140911 is mainly used in making
In standby animal high-protein feed additive.(this part could be described in detail, how to be added, additive amount is how many, energy
Which kind of beneficial effect lamp brought)
Embodiment 2: the identification of strain
1. Morphological Identification:
Bacterium colony is milky in wort agar medium, and form is circle, and neat in edge is glossy, flat；It is micro-
When sem observation, how oval strain cell is or long avette, cell are budding, does not form mycelia, no ballistopore and the sub- spore of ascus
Son.
2. Physiology and biochemistry is identified:
Test method: it bacteria suspension method: takes and contains 2ml sterile water test tube in one, picked from the plate with transfer needle and purified list
A bacterium colony dilutes the uniform bacterial suspension that 0.5 Maxwell turbidity is made into sterile water, instills the micro biochemical identification for needing to test
Guan Zhong, every pipe 1 drip.(note: if content is semisolid or inclined-plane, then carrying out percutaneous puncture-inoculation)
Test result:
Fermentation test, glucose fermentation, galactolipin, sucrose, maltose and gossypose；Azymic: lactose, xylose, fibre
Tie up disaccharides and trehalose.
Carbon assimilation test, assimilation: glucose, maltose, sucrose, galactolipin and mannose；Do not assimilate: cellobiose, D-
Xylose, melibiose, trehalose and L-arabinose.
Nitrogen assimilation test, assimilation: (NH4)2SO4；Do not assimilate: KNO3。
Alcohols assimilation experiments, assimilation: ethyl alcohol；Do not assimilate: sorbierite.
It is above-mentioned the experimental results showed that, the bacterial strain be saccharomycete.
3. molecular biology identification:
3.1 test methods: bacterium solution culture --- DNA extraction --- PCR --- electrophoresis --- sequencing --- NCBI blast
It compares.
1. bacterium solution culture: yeast saves strain on picking inclined-plane, is added in cut-and-dried liquid malt extract medium 5ml,
23 DEG C of shaking table culture 48h, it is spare.
2. DNA is extracted: OMEGA BIO-TEK kit step extracts.
3. PCR: primer preparation: forward primer NL-1:5'-GCATAT CAA TAA GCG GAG GAAAAG-3', reversely
Primer NL-4:5'-GGT CCGTGTTTCAAGACGG-3', by the raw work preparation in Shanghai.
25 1.00 1.00 μ L of μ L, NL-4 of μ L:2 × Taq Master Mix12.50 μ L, NL-1 of reaction system, DNA profiling
L.00 μ L, ddH20 9.50 μ L, TOTAL25.00 μ L.
Reaction condition (35cycles): initial denaturation: 94 DEG C, 4min；Denaturation: 94 DEG C, 30s；Renaturation: 58 DEG C, 30s；T1:
72 DEG C, 45s；T2:72 DEG C, l0min；It saves: 15 DEG C, ∞.
4. deposition condition: 1.4% Ago-Gel carries out electrophoresis, point sample: 6 × Loading Buffer (point sample buffering
Liquid) 2.5 μ L, DNA5 μ L, deposition condition electrophoresis: is set after point sample as voltage 150V 25min.
5. the brighter product of electrophoretic band send to the raw work sequencing portion in Shanghai and is sequenced.
6. sequencing result is inputted into www.NCBI.nlm.nih.gov, using BLAST software, by the gene order measured with
The sequence of Genbank database carries out tetraploid rice.
3.2 test results:
1. electrophoretogram is as shown in Figure 1.(length about 600bp, 1,2 is yeast original strain, and 3,4 be the rich protein yeast of domestication
Bacterial strain)
2. BLAST comparing result: Saccharomyces cerevisiae S288c, NC_001144.5, chromosome
XII.
It is above-mentioned the results showed that bacterial strain provided by the invention be yeast strain.
Embodiment 3: strain protein assay
1. test method:
1 group of sample: the original yeast strain ferments liquid sample that sets out；2 groups: high yield protein yeast bacterial strain fermentation liquor.
1.1 sample treatments and SDS-PAGE:
Yeast fermentation broth 100ml is centrifuged 10min through 3000rpm, takes supernatant, then through 7830rpm, after 4 DEG C of centrifugation 30min
15ml sample is taken, 3KDa is dialyzed overnight, and dialyzate is 100mM ammonium bicarbonate soln.Sample 3KDa super filter tube after dialysis,
7830rpm, 4 DEG C of ultrafiltration to volume are less than 2ml.Bradford method measures concentration, takes 20 μ l to carry out SDS-PAGE electrophoresis, examines horse
The dyeing of this brilliant blue.
Enzymatic hydrolysis in 1.2 solution:
Every group takes 50 μ l samples, and DTT to final concentration of 10mM is added.IAA is added to final concentration of 50mM, is protected from light
30min.37 DEG C of LysC are added to react 3 hours.4 times of volume 25mM ammonium bicarbonate solns are added.4 37 DEG C of μ g Trypin are added
Reaction overnight, 0.1%TFA acidification terminate reaction.C18-SD Extraction Disk Cartridge desalting processing, vacuum
Freeze-drying.35 μ l 0.1%FA are added to redissolve precipitating.
1.3 capillary high performance liquid chromatographies:
It is separated using a nanoliter flow velocity HPLC liquid phase systems Easy nLC.Buffer: A liquid is 0.1% aqueous formic acid,
B liquid is 0.1% formic acid acetonitrile solution (acetonitrile 84%).Chromatographic column is balanced with 95% A liquid.Sample is by autosampler
It is loaded to loading column Thermo scientific EASY column (2cm*100 μm of 5 μm of-C18), then through analytical column
Thermo scientific EASY column (75 μm of * 100mm, 3 μm of-C18) separation, flow velocity 300nl/min.Related fluid
Phase gradient is as follows: -50 minutes 0 minute, B linear gradient was from 0% to 50%；- 54 minutes 50 minutes, B linear gradient from
50% to 100%；- 60 minutes 54 minutes, B liquid maintained 100%.
1.4 ESI Mass Spectrometric Identifications:
Sample is carried out after capillary high performance liquid chromatography separates with Q-Exactive mass spectrograph (ThermoFinnigan)
Mass spectral analysis.Analyze duration: 60min, detection mode: cation, precursor scans range: 300-1800m/z, first mass spectrometric
Resolution ratio: 70,000at m/z 200, AGC target:3e6, level-one Maximum IT:10ms, Number of scan
Ranges:1, Dynamic exclusion:3.0s.The mass-charge ratio of the fragment of polypeptide and polypeptide is adopted in following manner
Collection: each full scan (full scan) acquires 10 fragment patterns storeds (MS2scan) afterwards, MS2Activation Type:HCD,
Isolation window:2m/z, second order ms resolution ratio: 17,500at m/z 200, Microscans:1, second level
Maximum IT:60 ms, Normalized collision energy:27eV, Underfill ratio:0.1%.
1.5 MASS SPECTRAL DATA ANALYSIS
Original document (MGF file) carries out database retrieval by Mascot software.Database is loaded in uniprot's under being
Database uniprot_saccharomyces_112806_20141020.fasta (accession sequence 112806, download time
For 2014-10-20).It is as follows to search library parameter setting: enzyme trypsin；Missed cleavage is set as 2；Static modifying
Set Carbamidomethy C；Dynamic embellishment sets Oxidation M.Peptides tolerance is set as 20ppm, ms/
Ms tolerance is set as 0.1Da, score > 20 Ion.
1.6 bioinformatic analysis
By the database UniProt knowledgebase of internal authority (Swiss-Prot/TrEMBL,
Www.expasy.org it) is carried out with Gene Ontology (GO) Database (http://www.geneontology.org/)
Retrieval, tentatively probes into the thin of the subcellular localization situation of the differentially expressed protein identified, the biological function of performance and participation
Born of the same parents' process.
2. test result
2.1 quantification of protein results:
Through SDS-PAGE electrophoresis, it was demonstrated that there are albumen abundant, sample protein matter quantitative result such as tables in two groups of fermentation liquids
Shown in 3-1.
Table 3-1
| Sample | 1 | 2 |
| Protein concentration (μ g/ μ l) | 0.304 | 0.329 |
2.2.2 ESI Mass Spectrometric Identification result:
Sample protein matter qualification result is counted as shown in table 3-2.
Table 3-2
| Sample | Protein group | Independent peptide fragment |
| 1 | 41 | 68 |
| 2 | 121 | 241 |
As can be seen that the protein chromatography absorption peak of two groups of samples is all relatively abundant from the two width chromatograms of Fig. 2, it was demonstrated that albumen
Content and type are more, and are analyzed by comparing it is found that 2 groups of absorbing proteins peak area is big compared with 1 group, and absorption peak is also more, just
Protein content in step 2 groups of samples of explanation is relatively abundant.
2.3 bioinformatic analysis --- GO analysis and KEGG metabolic pathway preliminary analysis:
It will be identified using localization sequence alignment program NCBI BLAST+ (ncbi-blast-2.2.28+-win32.ext)
To protein be compared with the protein sequence in NCBI nr database.According to similarity principle, resulting homologous egg
White functional information can be used for the functional annotation of target protein.We only retain before ranking 10 and Evalue≤1e-3
Aligned sequences carry out subsequent analysis.Resulting comparison similarity ranges are 50-100%, wherein most target protein sequence
Comparison similitude be 98% or more.
Mass Spectrometric Identification is 109 total to annotated albumen in initial data, wherein 1 group of peculiar protein 11,2 groups peculiar
63, albumen, two groups of shared 35, albumen.The protein sequence information batch extracting identified is from UniProtKB database
(http://www.uniprot.org) (version number: Release 2014_10), with the preservation of FASTA format.
In addition, by searching database UniProt knowledgebase (Swiss-Prot/TrEMBL,
Www.expasy.org) and Gene Ontology (GO) Database (http://www.geneontology.org/), right
The cell function process that this three histones matter is participated in has carried out preliminary analysis with the biocytology function being related to.
2.3.1 two groups of fermentation liquids share analysis of protein:
Using the Mapping function in Blast2GO (Version 2.8.0) to the comparison sequence of all albumen identified
The associated GO function entry of column extracts, and extracts altogether and wherein 27 protein sequence (77.14%) relevant 126
GO function entry, in this project, totally 25 protein sequences are annotated by 78 GO function entries, and average GO level is 5.41.
By complementary annotations, final annotation statistical result are as follows: totally 27 protein sequences are annotated by 91 GO function entries.
Shared histone WEGO functional annotation clustering:
In 35 protein of shared group, according to albumen WEGO functional annotation clustering, wherein participate in biological pathway
The albumen annotation of process (biological progress) accounts for 51%, the albumen of molecular function (molecular function)
Note 2 8%, cellular component analysis (cell component) annotation account for 20%.Using WEGO software to the albumen identified
The major function classification of matter completes the calculating of GO function degree of enrichment and graphicalization result is as shown in Figure 3.
Shared histone GO functional annotation
Wherein, as shown in figure 4, participating in metabolic process (metabolic in Biological Progress figure classification
Process) have 20 kinds of albumen annotation, the total BP quantity 39.2% of Zhan, cell processes (cellular process) have 15 hatching eggs
White annotation accounts for the 29.4% of BP sum, remaining to participate in biological regulation (biological regulation), signal path
(signaling), a variety of biological processes (single-organism process/multi-organism process), thin
Born of the same parents' original part component biosynthesis pathway (cellular component organization or biogenesis), stress be anti-
During answering (response to stimulus) etc.；In Molecular Function classification chart (Fig. 5), there is catalysis
Active (catalytic activity's) has 18 kinds of albumen annotations, accounts for the 64.3% of MF sum, residual protein participates in respectively
Binding protein (binding), molecular structure active (structural molecule activity), molecular transport activity
During (molecular transducer activity) etc.；As shown in fig. 6, in Cell Component classification, carefully
Born of the same parents' component (cell) totally 7 kinds of protein sequences, account for the 35% of CC total amount, remaining albumen participates in extracellular activity respectively
(extracellular region), organelle (organelle), cell membrane (membrane), macromolecular mixture
During (macromolecular complex) etc..
KEGG access annotation:
These albumen are compared by inquiring in KEGG database, and the metabolism participated in has glycolytic pathway
Glycolysis/Gluconeogenesis (4), photosynthetic carbon fixation act on Carbon fixation in photosynthetic
Organisms (3), starch Sucrose Metabolism Starch and sucrose metabolism (2), methane are metabolized Methane
metabolism(1)。
2.3.2 two groups of peculiar analysis of protein of fermentation liquid:
Using the Mapping function in Blast2GO (Version 2.8.0) to the comparison sequence of all albumen identified
The associated GO function entry of column extracts, and extracts altogether and wherein 56 protein sequence (75.68%) relevant 296
GO function entry.In this project, totally 54 protein sequences are annotated by 182 GO function entries, and average GO level is 6.775.
By complementary annotations, final annotation statistical result are as follows: totally 59 protein sequences are annotated by 225 GO function entries.
Peculiar albumen WEGO functional annotation clustering:
Peculiar albumen WEGO functional annotation clustering is as shown in Figure 7: where totally 11 kinds of 1 group of peculiar albumen, participates in biology
The albumen of process (biological progress) annotates for 12 totally, accounts for 50.0%, participates in molecular function (molecular
Function albumen) annotates 7 kinds, accounts for 29.2%, the albumen note 5 kind of cytology component (cell component) accounts for
20.8%；2 groups of 63 kinds of peculiar albumen, the albumen for participating in biological processes (biological progress) annotate for 105, account for totally
46.1%, the albumen for participating in molecular function (molecular function) annotates 77 kinds, accounts for 33.8%, cytology component
1 kind of the albumen note 4 of (cell component), accounts for 17.9%.
Peculiar Protein G O functional annotation:
As seen in figs. 8-10, wherein in 1 group of peculiar albumen, be primarily involved in generation in Biological Process classification
Journey of apologizing for having done sth. wrong (metabolic process) has 8 kinds of albumen annotations, accounts for the 47.1% of BP total amount, single creature process
(single-organism process) has 5 kinds of albumen annotations, accounts for BP 29.4%, cell processes (cellular process)
Albumen be annotated with 4 kinds, account for BP 23.5%, remaining has stress reaction (response to stimulus) (2), positioning
(localization) (1), biosynthesis (the cellular component organization or of cell
Biogenesis) (1) etc.；Mainly there is catalytic activity (catalytic activity) in Molecular Function classification
(6), binding protein (binding) (6), the albumen such as transport activity (transporter activity) (1) annotation；
There is cell (cell) (2) albumen in Cellular Component classification, organelle (organelle) (1), macromolecular chemical combination
Object (macromolecular complex) (1), extracellular region (extracellular region) (1), cell membrane
(membrane)(4).
In 2 groups of peculiar albumen, mainly there is cell processes (cellular in Biological Process classification
Process albumen) is annotated with 33 kinds, accounts for the 34.7% of BP total amount, metabolic process (metabolic process) albumen note
32 kinds have been released, the 33.7% of BP total amount is accounted for, single creature process (single-organism process) there are 10 kinds of albumen notes
It releasing, (the cellular component organization or biogenesis) of cell biological synthesis has 8 kinds, remaining
Have positioning (localization) (5), stress reaction (response to stimulus) (2), biological regulation (biogical
Regulation) (6) etc.；Mainly there is catalytic activity (catalytic activity) egg in Molecular Function classification
White 30 kinds of annotation, accounts for the 45.5% of MF total amount, and 7 kinds of note 2 of binding protein (binding) accounts for the 40.9% of MF, remaining has point
Minor structure albumen (structural molecule) (6), transport protein (transporter) (3) have transcription factor activity
Binding protein (protein binding transcription factor activity) (1), with transcriptional activity
The annotation such as nucleic acid (nucleic acid binding transcription factor activity) (1)；And it is anti-oxidant
Active (antioxidant activity) (1), electron carrier activity (electron carrier activity) (1)；
Annotation albumen has 18 kinds of cell (cell) in Cellular Component classification, accounts for the 40.9% of CC total amount, organelle
(organelle) there are 9 kinds, account for the 20.5% of CC, macromolecular compound (macromolecular complex) (9), extracellular region
(extracellular region) (4), cell membrane (membrane) (4), film closing chamber (membrane-enclosed
lumen)(1)。
KEGG access annotation:
1 group of peculiar Protein Information is compared in KEGG database, obtains the metabolism of some of matching albumen
Approach, residual protein is limited because studying, and can not also find its matched information, this histone participates in 8 metabolic pathways: fruit altogether
Sugared sweet dew glycometabolism Fructose and mannose metabolism (1), purine metabolism Purine metabolism (1),
Glycolysis Glycolysis/Gluconeogenesis (1), riboflavin are metabolized Riboflavin metabolism (1), methane
It is metabolized Methane metabolism (1), pentose phosphate pathway Pentose phosphate pathway (1), aminobenzoic
Acid degradation Aminobenzoate degradation (1), photosynthetic carbon fixation act on Carbon fixation in
photosynthenic organisms(1)。
After being compared in 2 groups of peculiar albumen KEGG databases, matching albumen participates in 24 metabolic pathways: purine altogether
It is metabolized Purine metabolism (4), glycolysis Glycolysis/Gluconeogenesis (4), photosynthetic carbon fixation effect
Carbon fixation in photosynthenic organisms (2), nitrogen metabolism Nitrogen metabolism (2),
Alanine, asparatate and glutamic acid metabolism Alanine, asparatate and glutamate metabolism (2),
Protokaryon carbon solidification effect Carbon fixation pathways in prokaryotes (2), metabolism of pyruvate Pyruvate
Metabolism (1), phosphoinositide metabolism inositol phosphate metabolism (1), pantothenate and CoA biology close
At Pantothenate and CoA biosynthesis (1), glyoxylate and dicarboxylic acids approach Glyoxylate and
Dicarboxylate metabolism (1), citrate recycle Citrate cycle (TCA cycle) (1), and valine is bright
Biosynthesis Valine, leucine the and isoleucine biosynthesis (1) of propylhomoserin and isoleucine, sweet ammonia
Acid, serine and threonine metabolism Glycine, serine and threonine metabolism (1), amino sugar and nucleosides
Sour glycometabolism Amino sugar and nucleotide sugar metabolism (1), cysteine and Methionine metabolism
Cysteine and methionine metabolism (1), Cytochrome P450 drug metabolism Drug metabolism-
Cytochrome P450 (1), fructose and sweet dew glycometabolism Fructose and mannose metabolism (1), arginine
With Proline Metabolism Arginine and proline metabolism (1), the metabolism of Cytochrome P450 xenobiotics
Metabolism of xenobiotics by cytochrome P450 (1), naphthalene degradation Naphthalene degradation
(1), the degradation Chloroalkane and chloroalkene degradation (1) of alkyl chloride and chloro-alkenes, fatty acid
It degrades Fatty acid degradation (1), tyrosine is metabolized Tyrosine metabolism (1), Vitamin A Metabolism
Retinol metabolism(1)。
3 conclusions:
Peptide fragment 68, protein groups 47 are identified in 1 group altogether；2 groups identify peptide fragment 241, protein groups 121 altogether.It is former
It is 109 total that annotated albumen is identified in beginning data, wherein 1 group of peculiar protein 11,2 groups 63, two groups, peculiar albumen
Shared 35, albumen.Illustrate there is protein abundant in two groups of fermentation liquids, but the high yield protein yeast bacterial strain in the invention
Protein content and type are above out bacterium germination in fermentation liquid.
2 groups of fermentation liquids compared with 1 group in not only protein content it is relatively abundant, and the metabolic pathway participated in is also more.Meanwhile fermentation liquid
In generally existing higher carbohydrate metabolism and energetic supersession, also participate in affect a small amount of biological pathways etc., 2 groups have
Uniquely there is electron carrier activity and the active protein of transhipment, may exercise bioelectric detecting, stimulate the reaction and position into
The functions such as journey.Protein function and the metabolic pathway of participation are far more than bacterium germination out in the bacterial strain fermentation liquor of the invention.
Embodiment 4: response phase method optimum culture condition
1. the selection of response surface analysis factor level
Table 4-1 is shown as response surface analysis factor and level.
Table 4-1
The design of 3.2 response surface experiments and result
Box-Behnken experimental design and result are as shown in table 4-2.Table 4-2 is tried using Design-Expert software
It tests data and carries out multiple regression analysis, obtain the quadratic regression model of saccharomycete clump count Yu each variable factors are as follows: Y=2.87
+0.13A-0.085B+0.015C+0.025AB+5.000E-003AC+0.000BC+2.000 E-003A2-0.29B2+
2.000E-003C2。
Table 4-2
| Test serial number | A | B | C | Preparation clump count/(108CFU/mL) |
| 1 | 0 | 0 | 0 | 2.89 |
| 2 | 0 | 1 | 1 | 2.61 |
| 3 | 0 | -1 | 1 | 2.75 |
| 4 | 0 | -1 | -1 | 2.68 |
| 5 | -1 | 0 | 1 | 2.76 |
| 6 | 0 | 0 | 0 | 2.88 |
| 7 | 0 | 0 | 0 | 2.86 |
| 8 | 0 | 1 | -1 | 2.56 |
| 9 | -1 | -1 | 0 | 2.57 |
| 10 | 0 | 0 | 0 | 2.78 |
| 11 | 1 | 0 | 1 | 3.08 |
| 12 | 1 | -1 | 0 | 2.86 |
| 13 | -1 | 0 | -1 | 2.78 |
| 14 | 1 | 0 | -1 | 2.98 |
| 15 | -1 | 1 | 0 | 2.45 |
| 16 | 1 | 1 | 0 | 2.76 |
| 17 | 0 | 0 | 0 | 2.89 |
Variance analysis is carried out to model with software, as a result as shown in table 4-3.
Table 4-3
The confirmatory experiment of 3.3 model equations:
The extreme point of regression model, i.e. optimal culture condition are found out by software Design-Expert.V8.0.6: initial
PH is 5.8, and cultivation temperature is 27.26 DEG C, liquid amount 100.00mL, and saccharomycete clump count theoretical prediction maximum value is
2.89*108CFU/mL.For the reliability for examining Responds Surface Methodology, optimal culture condition confirmatory experiment is selected, it is contemplated that practical
It is easy to operate, adjust optimal culture condition are as follows: initial pH is 5.8, and cultivation temperature is 27.30 DEG C, liquid amount 100.00mL.
With this condition, 3 parallel tests are carried out, saccharomycete clump count is 3.00*108CFU/mL.It is close with theoretical expectation values, it says
The bright model be it is believable, Responds Surface Methodology is feasible for the optimization of yeast bacteria preparation condition of culture.
Optimal culture condition are as follows: initial pH is 5.8, and cultivation temperature is 27.30 DEG C, liquid amount 100.00mL
Influence of the embodiment 5:C20140911 bacterial strain fermentation liquor to intestine of young pigs microbiota
1 materials and methods
1.1 material
1.1.1 reagent and instrument
C20140911 fermentation liquid；Original yeast fermentation broth；Mai Kangkai (Hangzhou microorganism reagent Co., Ltd)；SS agar
(Hangzhou microorganism reagent Co., Ltd)；MRS(OXOID,ENGLAND)；Blood plate (Lanzhou Rongchang County biological products company).
1.1.2 experimental animal
Test carries out on Yuzhong pig farm, chooses 9 nest of 20d piglet of health, 6, every nest, totally 54 (Durocs × about
Gram × the white tri-crossbreeding of length), 3 groups are randomly divided into, every group of 3 nests, every nest is 1 repetition.Animal routinely carries out pipe during test
Reason is freely eaten, and free water, diarrhea takes drugless medication during test.
1.2. experimental design
Test repeats single factor experiment for 3 processing 3, and 3 processing are respectively the 1st group (C20140911 zymotic fluid group), the 2nd group
(original yeast strain ferments liquid group) and the 3rd group (blank control group), experimental animal is observed continuously 3 days since 20d, is no different
Normal then start oral fermentation liquid, continuous 5 days, 28d wean was selected in each processing repeats at random respectively at 23d, 28d, 35d, 42d
1 cut open killing, and ligatures to ileum, caecum, colon both ends, takes intestinal contents in certain site sterile.Content -20
DEG C anaerobism saves, and detects for microbiota.
1.3 test method
1.3.1 conventional method separation counts
Intestinal contents about 0.2g is fetched respectively, is added in about 1.8ml physiological saline, is shaken up；Caecum and colonic contents are about
0.5g is added in 4.5ml physiological saline, shakes up then 10 times of gradient dilutions of physiological saline, 0.1ml is taken to be added to different plates
Every part of sample of differential counting is done twice in parallel, is chosen the culture dish of clump count (CFU) between 30~300 and is counted, calculates average
Value, calculates the bacterial population of every gram of content, and indicate with its logarithm.Including lactobacillus (MRS), Bifidobacterium (training
Support base self-control), Escherichia coli (Mai Kangkai), salmonella (SS) and aerobic bacteria sum and anaerobic bacteria total (blood plate)
Measurement.
2 results
Influence of 2.1 fermentation liquids to ileum microbiota
2.1.1 to the influence of Escherichia coli quantity
Influence of each processing group of table 5-1 to piglet ileal contents Escherichia coli
Note: 1) data unit is the wet content of lgCFU/g in table；2) data are mean value ± standard error in table；3) shoulder infuses word
Matrix shows the comparison of identical intestinal segment, identical age in days；Similarly hereinafter.
It tests 1 group and 2 groups of ileal contents coliform counts of test decreases with age in days increase, control group increases with age in days
Add raising.It tests 1 group, 2 groups of test and tests 1 group and test 2 with significant difference (p < 0.05) (p < 0.01) when 42 age in days of control group
Group difference is not significant.
2.1.2 to the influence of salmonella quantity
During the test, 1 group is tested to increase with 2 groups of ileal contents Salmonella bacterium numbers of test with age in days and reduce, it is right
Increase according to salmonella quantity after group wean, and is consistently higher than other two groups.Compared with the control group, 28d, 35d are poor for two test groups
Different not significant, 42d difference is extremely significant (p < 0.01), but indifference conspicuousness between the two.
Influence of each processing group of table 5-2 to piglet ileal contents salmonella
2.1.3 to the influence of aerobic bacteria quantity
Influence of each processing group of table 5-3 to the total aerobic bacteria of piglet ileal contents
During the test, it tests 1 group and 2 groups of ileum aerobic bacteria quantity of test is integrally on a declining curve, and control group is then
Increase with age in days and rises.1 group is tested with 2 groups of test without significant difference；Test 1 group compared with the control group, 28d, 35d difference
It is not significant, 42d significant difference (p < 0.05)；Test 2 groups compared with the control group, 28d, 35d difference is not significant, 42d significant difference
(p<0.05)。
From the above it can be seen that each processing group is not significant on the difference of ileum harmful bacteria influence in 28d, 35d,
Tested when 42d 1 group, test 2 groups of harmful bacteria quantity be substantially less than control group (p < 0.05), and test 1 group and test 2 groups between
Difference is not significant.Thus illustrate that bacterial strain fermentation liquor of the present invention is similar to the effect of original yeast fermentation broth, can be effectively reduced
Evil bacterium number amount
2.1.4 to the influence of bifidobacteria
Influence of each processing group of table 5-4 to piglet ileal contents Bifidobacterium
By table 5-4 it is found that ileal contents Bifidobacterium number, 28 ages in days test 1 group be significantly higher than control group (p <
0.05), difference is not significant between other age in days each groups；But as the increase each group of age in days is in rising trend
2.1.5 to the influence of Bacillus acidi lactici quantity
Influence of each processing group of table 5-5 to piglet ileal contents Bacillus acidi lactici
By table 5-5 it is found that ileal contents Bacillus acidi lactici number is with the increase of age in days, 2 groups of 1 group of test and test are presented
The trend of liter.1 group is tested during entire test and 2 groups of test is significantly higher than control group (p < 0.05)；Test 1 group and 2 groups of test
Difference is not significant.
2.1.6. to the influence of anaerobic bacteria quantity
Influence of each processing group of table 5-6 to the total anaerobic bacteria of piglet ileal contents
By table 5-6 it is found that during the whole test process, the total anaerobism bacterium number of ileal contents is tested with the increase of age in days
1 group and 2 groups of in rising trend, control group held stationaries of test.It tests 1 group and 2 groups of total anaerobism bacterium numbers of test is consistently higher than control
Group, but difference is not significant between each group.
Influence of 2.2 fermentation liquids to cecum microorganisms fauna
2.2.1 to the influence of Escherichia coli quantity
Influence of each processing group of table 5-7 to piglet cecal content Escherichia coli
In caecum, as age in days increases, 2 groups of Escherichia coli quantity of 1 group of test and test are declined, and control group exists
Decline when 28d, then begin to rise, control group is above 2 groups of 1 group of test and test during entire test.Test 1 group and examination
It is not significant to test difference between 2 groups, with control group 35d significant difference (p < 0.05), 42d difference is extremely significant (p < 0.01)；Test 2
Group and control group 35d, 42d difference are extremely significant (p < 0.01).
2.2.2 to the influence of salmonella quantity
Influence of each processing group of table 5-8 to salmonella in cecal content
In test, 1 group of test on a declining curve with salmonella quantity in 2 groups of cecal contents of test；Control group wean
After rise, be declined slightly when to 42d, but still be higher than other two groups.1 group is tested lower than 2 groups of test, but difference is not significant；It is low
In control group 35d significant difference (p < 0.05), 42d difference is extremely significant (p < 0.01).Test 2 groups compared with the control group, 35d is poor
Different significant (p < 0.05), 42d difference are extremely significant (p < 0.01).
2.2.3 to the influence of aerobic bacteria quantity
In test, 1 group of test increases with age in days with 2 groups of cecal content aerobic bacteria quantity of test and is reduced, but tests 1 group
42d increased compared with 35d, be higher than 2 groups of test；Control group increases with age in days and is increased, and 35d, 42d are above remaining two groups.Examination
1 group is tested compared with 2 groups of test, 2 groups of test, and significant difference (p < 0.05) are higher than when 42d；Test 1 group compared with the control group
The 42d time difference is heteropolar significant (p < 0.01)；Test 2 groups compared with the control group, the 42d time difference is heteropolar significant (p < 0.01), remaining does not show
Write
Influence of each processing group of table 5-9 to the total aerobic bacteria of piglet cecal content
Sheet3-9 Effect of different treatment on aerobic bacterial in cecum
of piglets
Find out from the above, harmful bacteria quantity can be reduced in 35d, 42d with 2 groups of yeast of test by testing 1 group in caecum,
The two effect is similar, and effect is better than ileum；And the effect of 2 groups of control aerobic bacteria quantity is tested better than 1 group of test.
2.2.4 to the influence of bifidobacteria
Influence of each processing group of table 5-10 to piglet cecal content Bifidobacterium
By table 5-10 it is found that cecal content Bifidobacterium number tests 1 group and 2 groups of test in rising with the increase of age in days
Trend, control group are declined slightly.During test, 2 groups of 1 group of test and test are consistently higher than control group；28 ages in days and 42 ages in days are poor
Different significant (p < 0.05)；It tests 1 group and is higher than 2 groups of test, but difference is not significant.
2.2.5 to the influence of Bacillus acidi lactici quantity
Influence of each processing group of table 5-11 to piglet cecal content Bacillus acidi lactici
By table 5-11 it is found that cecal content Bacillus acidi lactici number is presented with 2 groups of 1 group of the increase test and test of age in days
The trend of liter.During test, 2 groups of 1 group of test and test are consistently higher than control group, and 28 age in days differences are extremely significant, and 42 age in days differences are aobvious
It writes.It tests 1 group and is higher than 2 groups of test, but difference is not significant.
2.2.6. to the influence of anaerobic bacteria quantity
Influence of each processing group of table 5-12 to the total anaerobic bacteria of piglet cecal content
Find out from 5-12: 1 group of test and the test total anaerobism bacterium number of 2 groups of cecal contents are as the increase of age in days is in rising
Trend, control group are declined slightly, basic held stationary.It tests 1 group and 2 groups of test is consistently higher than control group, 28 age in days differences are aobvious
It writes (p < 0.05), 35-42 age in days difference is extremely significant (p < 0.01)；It tests 1 group and is higher than 2 groups of test, difference is not significant.
Find out from the above (table 5-10~5-12): C20140911 fermentation liquid and original yeast strain ferments liquid
Caecum beneficial bacterium quantity is effectively improved, illustrates that the two effect is similar, but C20140911 fermentation liquid effect is better than original saccharomycete
Strain fermentation liquid.
Influence of the 2.3 disconnected fermentation liquids to faecal flora fauna
2.3.1 to the influence of Escherichia coli quantity
As the increase of age in days is tested in 1 group and 2 groups of colonic contents of test, Escherichia coli quantity is on a declining curve, compares
Group 28d is declined, and 35d rises.During entire test, control group is above other two groups, the 42d compared with to 1 group of test
Significant difference (p < 0.05)；35d, 42d difference are significant (p < 0.05) compared with 2 groups of test；Test 1 group and 2 groups of differences of test
It is not significant
Influence of each processing group of table 5-13 to piglet colonic contents Escherichia coli
2.3.2 to the influence of salmonella quantity
Influence of each processing group of table 5-14 to colonic contents salmonella
From table 5-14 can be seen that test during test 1 group, test 2 groups of colonic contents Salmonella bacterium numbers be integrally under
Drop trend, 1 group of test slightly rises in 35d, but is lower than control group, and control group is in propradation after wean.Test 1 group
Not significant with 2 groups of differences of test, 42d significant difference (p < 0.05), remaining time is not significant compared with the control group.Test 2 groups
35d, 42d significant difference (p < 0.05) compared with the control group.
2.3.3 to the influence of aerobic bacteria quantity
It tests 1 group of colon aerobic bacteria slightly to rise after wean, change during entire test little；Test 2 groups 35d with
Preceding on a declining curve, lower than 1 group of test, 42d rises above 1 group of test；Control group increases with age in days and is risen, and omits to 42d
There is decline.Test 1 group with test 2 groups of test result differences it is not significant；It tests 1 group of 35d and is substantially less than control group (p < 0.05)；
It tests extremely significant lower than control group (p < 0.01) when 2 groups of 35d.
Influence of each processing group of table 5-15 to colonic contents aerobic bacteria
Find out from the above (table 5-14--5-15): colon test 1 group with test 2 groups can have in 35d, 42d
Effect control harmful bacteria quantity illustrates that the two effect is similar.
2.3.4 to the influence of bifidobacteria
Influence of each processing group of table 5-16 to piglet colonic contents Bifidobacterium
By table 5-16 it is found that Bifidobacterium number is as the increase each group of age in days is in the trend risen in colonic contents.?
2 groups of 28 ages in days and 35 ages in days, 1 group of test and test are significantly higher than control group (p < 0.05)；42 ages in days are tested 1 group and are significantly higher than
Control group (p < 0.05), 2 groups of test be not significant with control group difference.1 group is tested during experiment and is higher than 2 groups of test, but difference is not
Significantly.
2.3.5 to the influence of Bacillus acidi lactici quantity
By table 5-17 it is found that testing 1 group with Bacillus acidi lactici number in 2 groups of colonic contents of test as the increase of age in days is presented
The trend of liter, control group are declined.28 ages in days, test 1 group and test 2 groups it is extremely significant be higher than control group (p < 0.01), 35~
2 groups of 42 ages in days, 1 group of test and test are significantly higher than control group (p < 0.05)；It is 1 group of test high in 35-42 age in days during experiment
In testing 2 groups, but difference is not significant.
Influence of each processing group of table 5-17 to piglet colonic contents Bacillus acidi lactici
2.3.6. to the influence of anaerobic bacteria quantity
Influence of each processing group of table 5-18 to the total anaerobic bacteria of piglet colonic contents
By table 5-18 it is found that the total anaerobism bacterium number of colonic contents is with the increase of age in days, 2 groups of 1 group of test and test are presented
The trend of liter, on a declining curve before 35 age in days of control group, 42 ages in days are restored；It tests 1 group and is consistently higher than test 2 groups and right
According to group.It tests 1 group to be significantly higher than control group (p < 0.05) in 35-42 age in days, 2 groups of test is significantly higher than control in 35 ages in days
Group.It is not significant to test difference between 1 group and 2 groups of test.
(table 5-16~3-18) in summary, C20140911 bacterial strain fermentation liquor and original yeast strain ferments liquid may not be used
Increase colon beneficial bacterium quantity with degree, C20140911 bacterial strain fermentation liquor effect is more preferable.
Influence of the embodiment 6:C20140911 bacterial strain fermentation liquor to weaned piglets
1. test method
Processing grouping is same as Example 5, respectively at 21d, 28d, 35d, 42d weighing, record feed intake.
Influence of the table 6-1 C20140911 bacterial strain fermentation liquor to weaned piglets
Note: in table data using average ± standard error (X ± SE) indicate, with a line internal ratio compared with.
By table 6-1 it is found that 1 group of test is remarkably improved the daily gain of weanling pig compared with control group with 2 groups of test, day adopts
Appetite promotes weanling pig growth, wherein testing 1 group of relatively 2 groups of daily gain of test improves 16.90%.Test 1 group of pig weight
Compared with the control group, heteropolar significant (p < 0.01) in the last method of double differences of 42d test in 35d significant difference (p < 0.05), daily gain
Also extremely significant higher than control group (p < 0.01), the trend that feed intake is improved with daily ingestion amount.And test 2 groups of weight with compare
Group is compared, and the 2 weeks trend being significantly increased, difference is not significant after ablactation；Daily gain and daily ingestion amount are extremely significant to be higher than pair
According to group (p < 0.01).Feed-weight ratio is the result shows that 1 group of test is lower than test 2 groups and control group.
C20140911 bacterial strain fermentation liquor and original yeast strain ferments liquid can be improved production performance, but C20140911
Bacterial strain fermentation liquor group effect is better than original yeast strain ferments liquid.
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Claims (3)
- White yeast strain S yeast (Saccharomyces cerevisiae) C20140911 1. a plant height is laid eggs, in The deposit number of state's Microbiological Culture Collection administration committee common micro-organisms center is CGMCC No. 9675, and the preservation time is On September 18th, 2014.
- The white yeast strain S yeast (Saccharomyces 2. a plant height according to claim 1 is laid eggs Cerevisiae) the cultural method of C20140911, which is characterized in that it is the peptone 8g by malt extract medium 14.0g, it is double Steam water 100ml, high pressure sterilization；Gnotobasis tune initial incubation liquid pH is 5.8, access kind daughter bacteria 1ml, is 27.3 DEG C in temperature, Revolving speed 120r/min, constant-temperature table culture.
- The white yeast strain S yeast (Saccharomyces 3. a plant height according to claim 1 is laid eggs Cerevisiae) application of the C20140911 in preparation animal high-protein feed additive.
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