CN106399135A

CN106399135A - Protein high-yielding Saccharomyces cerevisiae C20140911, and breeding and culturing method and application thereof

Info

Publication number: CN106399135A
Application number: CN201610755345.7A
Authority: CN
Inventors: 蒲万霞; 李春慧; 梁红雁
Original assignee: Lanzhou Institute of Animal Husbandry and Veterinary Medicine CAAS
Current assignee: Lanzhou Institute of Animal Husbandry and Veterinary Medicine CAAS
Priority date: 2016-08-29
Filing date: 2016-08-29
Publication date: 2017-02-15
Anticipated expiration: 2036-08-29
Also published as: CN106399135B

Abstract

The invention discloses a protein high-yielding Saccharomyces cerevisiae C20140911, and a screening method thereof, and also provides a breeding and culturing method and application of the Protein high-yielding Saccharomyces cerevisiae C20140911. The content and the kind quantity of proteins in a fermentation liquid of the protein high-yielding Saccharomyces cerevisiae C20140911 are far higher than those of original strains; 241 peptide segments are identified in the fermentation liquid of the strain disclosed in the invention, the peptide segment quantity is 254.4% higher than that in the original strains, 121 proteomes are identified in the fermentation liquid of the strain disclosed in the invention, and the proteome quantity is 157.4% higher than that in the original strains; the stain fermentation liquid has high protein content, participates in multiple metabolism pathways, and also participates in much carbohydrate metabolism and energy metabolism; and proteins only having electron carrier activity and transporter activity may have biological regulation and reacting and positioning process stimulation functions, and the protein functions and the participated metabolism ways of the strain fermentation liquid are far higher than those of the original strains. The strain disclosed in the invention can be used for preparing a feed additive and a micro-ecologic preparation, and has better effects than the original strains in improvement of the productivity of animals and reduction of diarrhea of weaned piglets.

Description

One plant height is laid eggs white yeast strain C20140911 and its seed selection, cultural method and application

Technical field

The present invention relates to animal feed additive technical field and in particular to a plant height is laid eggs white yeast strain S yeast (Saccharomyces cerevisiae) C20140911 and its seed selection, cultural method and application.

Background technology

Saccharomycete is a kind of eucaryon unicellular microorganism, has typical eucaryotic cell structure, has nucleus, endochylema film, cell Wall, cytoplasm and other inclusion etc..Because yeast be rich in good protein, amino acid, complete B family vitamin, with noeud vital Close several mineral materials and the function dietary fiber that state form exists, be widely used in produce reality.Yeast is that mankind's application is compared Early, it is also the microorganism being most widely used, people are frequently utilized that its fermentation manufactures various fermentation food and wine brewing, As brewer's yeast is applied to Beer Brewage；Selenium-rich, Zinc-rich saccharomyces cerevisiae, as the carrier of organic trace element, make trace element be more easy to inhale Receive etc.；In animal husbandry, yeast adds in feed mainly as single cell protein, to reduce the use of other protein raw materials.With When, also have and yeast culture (Yeast Culture, YC) is reported as the research of probiotics.Yeast culture is one The product of primary yeast culture, refers to process prepared product together with culture medium after the fermentation of strict control condition, its In main component be culture medium, saccharomycete and saccharomycetic metabolite.In yeast metabolism product, protein content is little, But it contains other " unknown somatomedin ", for adjusting the microecological balance of animal intestinal, improve the utilization rate of feed, increase Plus premunition and anti-stress ability, promote growth, thus improving the production performance of animal.Yeast culture answering in animal husbandry With having many advantages, because its both effect without interference with antibiotic, the use of antibiotic can be reduced, calling food simultaneously Product safety, limits today of antibiotic usage, and yeast product will have broader practice prospect.

Content of the invention

The purpose of the present invention is aiming at above-mentioned defect of the prior art, lays eggs white level to improve bacterial strain, in order to Prepare more preferable probiotics and feed addictive, to strengthening its Clinical practice effect, be food security and animal husbandry life Produce service, there is provided a plant height is laid eggs white yeast strain S yeast (Saccharomyces cerevisiae) C20140911 And its seed selection, cultural method and application.

To achieve these goals, the technical scheme of present invention offer is：One plant height is laid eggs white yeast strain S yeast (Saccharomyces cerevisiae) C20140911, it is in China Committee for Culture Collection of Microorganisms's commonly micro- life The deposit number at thing center is CGMCC No.9675, and the preservation time is September in 2014 18.

The rich protein yeast bacterial strain of above-mentioned high-biomass is in original saccharomyces cerevisiae Saccharomyces Gained is tamed through long-term on the basis of cerevisia.

Second object of the present invention there is provided an above-mentioned plant height and lays eggs white yeast strain (Saccharomyces Cerevisiae) the selection of C20140911, described selection be with 14g brewer's wort powdered medium, 1g peptone, 100ml distilled water is culture medium, and by culture medium autoclaving, it is 5.8 that gnotobasis adjusts initial incubation liquid pH, accesses 1ml initial Set out bacteria culture fluid, and temperature is 27.3 DEG C, rotating speed 120r/min, shakes 48-72H, and constant-temperature table is cultivated, and repeatedly passes on；From low The peptone of dosage 1% starts, and yeast strain (Saccharomyces cerevisiae) is carried out with growth tolerance domestication, progressively Increase peptone dosage to 8%, through multiple secondary screening with pass on test, filter out high yield protein yeast bacterial strain；

Third object of the present invention there is provided an above-mentioned plant height and lays eggs white yeast strain (Saccharomyces Cerevisiae) the cultural method of C20140911, is by malt extract medium 14.0g, peptone 8g, distilled water 100ml, high Pressure sterilizing；It is 5.8 that gnotobasis adjusts initial incubation liquid pH, accesses kind of a daughter bacteria 1ml, is 27.3 DEG C in temperature, rotating speed 120r/min, Constant-temperature table is cultivated.

Fourth object of the present invention there is provided an above-mentioned plant height and lays eggs white yeast strain (Saccharomyces Cerevisiae) application in preparation animal high-protein feed additive for the C20140911.

High yield protein yeast bacterial strain disclosed by the invention, is higher than original bacterium that sets out its zymotic fluid protein content and species 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 altogether in high yield protein yeast bacterial strain fermentation liquor of the present invention；Original Starting strain zymotic fluid identifies peptide fragment 68, protein groups 47 altogether, exceeds 254.4% and 157.4% respectively.

Mass Spectrometric Identification of the present invention amounts to 109 to annotated albumen, in described high yield protein yeast bacterial strain fermentation liquor Identify 63, peculiar albumen, the peculiar protein 11 of the bacterium that sets out, close to 6 times of the bacterium that sets out；Bacterium has 35, albumen with setting out.

A plant height disclosed by the invention is laid eggs that not only protein content is high for white yeast strain ferments liquid, protein function and participating in Metabolic pathway is far more than the bacterium that sets out.Participate in more carbohydrate metabolism and energetic supersession, have and uniquely there is electron carrier Activity and the protein of transhipment son activity, may exercise the functions such as bioelectric detecting, IR and localization.

The present invention peculiar albumen WEGO functional annotation cluster analysis shows, 63 in described high yield protein yeast bacterial strain fermentation liquor Plant peculiar albumen, participate in albumen totally 105 annotations of biological processes (biological progress), 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, participates in the albumen of biological processes (biological progress) totally 12 Bar annotates, and the albumen participating in molecular function (molecular function) annotates 7 kinds, cytology component (Cellular Component albumen note 5 kind).

The present invention peculiar Protein G O functional annotation shows, in the peculiar albumen of described high yield protein yeast bacterial strain fermentation liquor, It is primarily involved in the albumen of cell processes (cellular process) in biological processes (Biological Process) classification It is annotated with 33 kinds, participate in metabolic process (metabolic process) albumen and be annotated with 32 kinds, single creature process (single- Organism process) there are 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 Catalysis activity (catalytic activity) albumen that mainly has of (Molecular Function) annotates 30 kinds, associated proteins (binding) 7 kinds of note 2, molecular structure albumen (structural molecule) annotates 6 kinds, and remaining has transport protein (transporter) 3 kinds, there is associated proteins (the protein binding transcription of transcription factor activity Factor activity) a kind, there is nucleic acid (the nucleic acid binding transcription of transcriptional activity Factor activity) a kind wait annotation；And antioxidation activity (ntioxidant activity) note a kind, electron carrier 1 kind of active (electron carrier activity).Participating in annotation albumen in Cellular Component classification has cell (cell) 18 kinds, 9 kinds of organelle (organelle), 9 kinds of macromolecular compound (macromolecular complex), born of the same parents 4 kinds of outskirt (extracellular region), 4 kinds of cell membrane (membrane), membrane closure chamber (membrane-enclosed Lumen) a kind.

In the peculiar albumen of starting strain zymotic fluid, in biological processes (Biological Process) classification, it is primarily involved in generation The 8 kinds of albumen that have of journey of apologizing for having done sth. wrong (metabolic process) annotate, 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, and remaining has participation to answer Swash 2 kinds, a kind, the biosynthesis of cell of positioning (localization) of reaction (response to stimulus) 1 kind of (cellular component organization or biogenesis) etc..Molecular function (Molecular Function in) classifying, annotation albumen mainly has 6 kinds of participation catalysis activity (catalytic activity), associated proteins (binding) 6 kinds, a kind of transport activity (transporter activity).Cell composition (Cellular Component) point Annotation be made up of participation cell (cell) 2 kinds of albumen in class, form a kind of organelle (organelle), macromolecular compound (macromolecular complex) form a kind, extracellular region (extracellular region) forms a kind, cell membrane (membrane) 4 kinds forming.

KEGG path of the present invention annotation shows, the peculiar albumen of described high yield protein yeast bacterial strain fermentation liquor is in KEGG database In compare after, obtain some of them mate albumen metabolic pathway, residual protein because study limited, also cannot find The information of its coupling, coupling albumen participates in 24 metabolic pathways altogether：It is respectively 4 kinds of purine metabolism (Purine metabolism), 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, asparatate With 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 circulation (a kind of Citrate cycle (TCA cycle), valine, 1 kind of the biosynthesis (Valine, leucine and isoleucine biosynthesis) of leucine and isoleucine, sweet 1 kind of propylhomoserin, serine and threonine metabolism (Glycine, serine and threonine metabolism), amino sugar and 1 kind of nucleotide sugar metabolism (Amino sugar and nucleotide sugar metabolism), cysteine and methionine 1 kind of metabolism (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, arginine and a kind of Proline Metabolism (Arginine and proline metabolism), cell color 1 kind of plain P450 xenobiotics metabolism (Metabolism of xenobiotics by cytochrome P450), naphthalene 1 kind of degraded (Naphthalene degradation), degraded (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).

The peculiar Protein Information of original bacterial strain fermentation liquor compares in KEGG database, participates in 8 metabolism ways altogether Footpath：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 metabolism (Riboflavin Metabolism) a kind, a kind of methane metabolism (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).

Beneficial effects of the present invention are：Its zymotic fluid protein content of yeast strain disclosed by the invention and species are far above Set out bacterium.In this bacterial strain fermentation liquor, protein content is 0.329mg/ml, improves than the 0.304ml/mg of the fermented liquid that sets out 8.2%.Peptide fragment 241, protein group 121 is identified altogether from this bacterial strain fermentation liquor；The fermented liquid that sets out identifies peptide altogether Section 68, protein group 47, improve 254.4% and 157.4% than the bacterium that sets out respectively.In the albumen having identified, this bacterium sends out 63, the peculiar albumen of zymotic fluid, the peculiar protein 11 of the bacterium that sets out, bacterium has 35, albumen with setting out.This bacterial strain fermentation liquor not only albumen Content is high, and the metabolic pathway participating in is also many, participates in more carbohydrate metabolism and energetic supersession；Have and uniquely there is electricity Subcarrier activity and the protein of transhipment son activity, may exercise the functions such as bioelectric detecting, IR and localization.Should In the bacterial strain fermentation liquor of invention, the metabolic pathway of protein function and participation is far more than the bacterium that sets out.The bacterial strain of the present invention can be used for preparing Feed addictive and probiotics, improve breeding performonce fo animals, reduce diarrhea of weaned piglets, and effect is better than the bacterium that sets out.

Brief description

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 zymotic fluid chromatogram, and figure below is bacterial strain fermentation liquor chromatogram of the present invention.

Fig. 3 is total protein groups WEGO functional annotation cluster analysis figure.

Fig. 4 is the Biological Progress classification chart in total histone GO functional annotation.

Fig. 5 is the Molecular Function classification chart in total histone GO functional annotation.

Fig. 6 is the Cell Component classification chart in total histone GO functional annotation.

Fig. 7 is peculiar protein groups WEGO functional annotation cluster analysis 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：

Culture selection

1st, one plant of rich protein yeast bacterial strain (Saccharomyces cerevisiae) C20140911, it is in Chinese micro- life The deposit number of thing culture presevation administration committee common micro-organisms center is CGMCC No.9675, and preservation date is 2014 9 The moon 18.

The selection of above-mentioned high protein bacterial strain, is with 14.0g brewer's wort powdered medium, 1g peptone, the double steaming of 100ml Water is culture medium (autoclaving, it is 5.8 that gnotobasis adjusts initial incubation liquid pH), accesses 1ml and initially sets out bacteria culture fluid, constant temperature Shaking table culture, temperature is 27.3 DEG C, rotating speed 120r/min, shakes 48-72H, repeatedly passes on.

Produce the bacterial strain of high protein for culture domestication, from the beginning of the peptone of low dosage 1%, to yeast strain Saccharomyces cerevisiae carries out growth tolerance domestication, is stepped up peptone dosage to 8%, through multiple secondary screening With pass on test, filter out high-biomass richness protein yeast bacterial strain.

Condition of culture：Malt extract medium (14g), peptone (8g), distilled water (100ml), autoclaving；Gnotobasis Initial incubation liquid pH is adjusted to be 5.8, constant-temperature table is cultivated：Temperature is 27.3 DEG C, rotating speed 120r/min.

Detection viable conditions：Incubated 72h, after 10 times of normal saline dilution, detects viable bacteria with blood cell counting plate Number, can reach 10⁸/ml.

In this culture, bacterial classification produces bacterial classification for brewing industry, is not animals and plants cause of disease；Brewer's wort, peptone in culture medium All nontoxic, do not pollute the environment.

Above-mentioned one plant rich 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 add, addition is how many, can carry Carried out which kind of beneficial effect lamp)

Embodiment 2：The identification of bacterial classification

1. Morphological Identification：

On wort agar culture medium, bacterium colony is milky, and form is circle, and neat in edge is glossy, flat；Micro- During sem observation, the many ovalizes of strain cell or long avette, cell is budding, does not form the sub- spore of mycelia, no ballistopore and ascus Son.

2. Physiology and biochemistry identification：

Test method：Bacteria suspension method：Take in one and contain 2ml sterilized water test tube, picking has purified list from flat board with transfer needle Individual bacterium colony dilutes the homogeneous bacterial suspension making 0.5 Maxwell turbidity to sterilized water, instills the micro biochemical identification needing test Guan Zhong, often pipe 1.(note：If content is semi-solid or inclined-plane, then carry out percutaneous puncture-inoculation)

Result of the test：

Fermentation test, glucose fermentation, galactolipin, sucrose, maltose and gossypose；Azymic：Lactose, wood sugar, fiber Disaccharides and trehalose.

Carbon assimilation is tested, assimilation：Glucose, maltose, sucrose, galactolipin and mannose；Do not assimilate：Cellobiose, D- Wood sugar, melibiose, trehalose and L-arabinose.

Nitrogen assimilation is tested, assimilation：(NH₄)₂SO₄；Do not assimilate：KNO₃.

Alcohols assimilation experiments, assimilation：Ethanol；Do not assimilate：Sorbierite.

Above-mentioned test result indicate that, this bacterial strain be saccharomycete.

3. molecular biology identification：

3.1 test method：Bacterium solution culture DNA extracts PCR electrophoresis sequencing NCBI blast Compare.

1. bacterium solution culture：On picking inclined-plane, yeast preserves strain, adds in cut-and-dried liquid malt extract medium 5ml, 23 DEG C of culture 48h of shaking table, standby.

2. DNA extracts：OMEGA BIO-TEK kit step is extracted.

③PCR：Prepared by primer：Forward primer NL-1:5'-GCATAT CAA TAA GCG GAG GAAAAG-3', reversely Primer NL-4:5'-GGT CCGTGTTTCAAGACGG-3', by Shanghai raw work preparation.

Reaction system 25 μ L：2 × Taq Master Mix12.50 μ L, NL-1 1.00 μ L, NL-4 1.00 μ L, DNA profiling L.00 μ L, ddH₂0 9.50 μ L, TOTAL25.00 μ L.

Reaction condition (35cycles):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；Preserve：15 DEG C, ∞.

4. deposition condition：1.4% Ago-Gel carries out electrophoresis, point sample：(point sample buffers 6 × Loading Buffer Liquid) 2.5 μ L, DNA5 μ L, electrophoresis：Deposition condition is set as voltage 150V 25min after point sample.

5. the brighter product of electrophoretic band is delivered to the raw work sequencing portion in Shanghai and is sequenced.

6. sequencing result is inputted www.NCBI.nlm.nih.gov, using BLAST software, by the gene order recording with The sequence of Genbank database carries out tetraploid rice.

3.2 result of the test:

1. electrophoretogram is as shown in Figure 1.(length about 600bp, 1,2 is yeast original strain, and 3,4 is the rich protein yeast of domestication Bacterial strain)

2. BLAST comparing result:Saccharomyces cerevisiae S288c,NC_001144.5,chromosome XII.

Above-mentioned test result indicate that：The bacterial strain that this invention is provided is 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 treatment and SDS-PAGE：

Yeast fermentation broth 100ml is centrifuged 10min through 3000rpm, takes supernatant, then through 7830rpm, after 4 DEG C of centrifugation 30min Take 15ml sample, 3KDa dialyzed overnight, dislysate is 100mM ammonium bicarbonate soln.Sample 3KDa super filter tube after dialysis, 7830rpm, 4 DEG C of ultrafiltration are less than 2ml to volume.Bradford method measures concentration, takes 20 μ l to carry out SDS-PAGE electrophoresis, coomassie Light blue dyes.

Enzymolysis in 1.2 solution：

Every group takes 50 μ l samples, adds DTT to final concentration of 10mM.Add IAA to final concentration of 50mM, lucifuge is reacted 30min.37 DEG C of LysC is added to react 3 hours.Add 4 times of volume 25mM ammonium bicarbonate solns.Add 37 DEG C of 4 μ g Trypin Reaction overnight, 0.1%TFA is acidified terminating reaction.C18-SD Extraction Disk Cartridge desalting processing, vacuum is frozen Dry.Plus 35 μ l 0.1%FA redissolve precipitation.

1.3 capillary high performance liquid chromatography：

Carry out separating using a nanoliter flow velocity HPLC liquid phase systems Easy nLC.Buffer solution：A liquid is 0.1% aqueous formic acid, B liquid is 0.1% formic acid acetonitrile solution (acetonitrile is 84%).Chromatographic column is balanced with 95% A liquid.Sample is by automatic sampler Sample to loading post Thermo scientific EASY column (2cm*100 μm of 5 μm of-C18), then through analytical column Thermo Scientific EASY column (3 μm of-C18 of 75 μm of * 100mm) separates, and flow velocity is 300nl/min.Related fluid phase gradient is such as Under：0 minute -50 minutes, B linear gradient was from 0% to 50%；50 minutes -54 minutes, B linear gradient from 50% to 100%；54 minutes -60 minutes, B liquid maintained 100%.

1.4 ESI Mass Spectrometric Identifications：

Sample is carried out with Q-Exactive mass spectrograph (ThermoFinnigan) after separating through capillary high performance liquid chromatography Mass spectral analysis.Analysis duration：60min, detection mode：Cation, precursor scans scope：300-1800m/z, first mass spectrometric divides Resolution：70,000at m/z 200, AGC target：3e6, one-level Maximum IT:10ms, Number of scan ranges:1, Dynamic exclusion:3.0s.The mass-charge ratio of the fragment of polypeptide and polypeptide gathers in following manner： Full scan (full scan) gathers 10 fragment patterns stored (MS2scan), MS2Activation Type afterwards every time:HCD, Isolation window:2m/z, second order mses resolution ratio：17,500at m/z 200, Microscans:1, two grades Maximum IT:60ms, 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 (accession sequence 112806, download time is database uniprot_saccharomyces_112806_20141020.fasta 2014-10-20).Search storehouse parameter setting as follows:Enzyme is trypsin；Missed cleavage is set to 2；Static modifying sets Determine Carbamidomethy C；Dynamic embellishment sets Oxidation M.Peptides tolerance is set to 20ppm, ms/ms Tolerance is set to 0.1Da, Ion score>20.

1.6 bioinformatic analysis

By the database UniProt knowledgebase of internal authority (Swiss-Prot/TrEMBL, ) and Gene Ontology (GO) Database (http www.expasy.org://www.geneontology.org/) carry out Retrieval, tentatively probe into the differentially expressed protein identifying Subcellular Localization situation, play biological function and participation thin Born of the same parents' process.

2. result of the test

2.1 quantification of protein results：

Through SDS-PAGE electrophoresis it was demonstrated that there is abundant albumen in two groups of zymotic fluids, sample protein matter quantitative result such as table 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 counts as shown in table 3-2.

Table 3-2

Sample	Protein group	Individually peptide fragment
			1	41	68
2	121	241

From the two width chromatograms of Fig. 2 as can be seen that the protein chromatography absworption peak of two groups of samples all relatively abundant it was demonstrated that albumen Content and species are more, and pass through comparative analysis, and 2 groups of absorbing proteins peak area is big compared with 1 group, and absworption peak is also more, just Protein content in step 2 groups of samples of explanation is abundanter.

2.3 bioinformatic analysis GO analyses and KEGG metabolic pathway initial analysis：

To be identified using localization sequence alignment program NCBI BLAST+ (ncbi-blast-2.2.28+-win32.ext) To protein compare with the protein sequence in NCBI nr database.According to similarity principle, the homologous protein of gained Function information can be used for the functional annotation of target protein.We only retain the comparison of first 10 and Evalue≤1e-3 of ranking Sequence carries out follow-up analysis.The comparison similarity ranges of gained are 50-100%, the ratio of wherein most target protein sequence It is 98% or more to similitude.

In initial data, Mass Spectrometric Identification amounts to 109 to annotated albumen, wherein 1 group peculiar protein 11, and 2 groups peculiar 63, albumen, two groups of total 35, albumen.The protein sequence information batch extracting being identified is from UniProtKB database (http://www.uniprot.org) (version number：Release 2014_10), preserved with FASTA form.

Additionally, by searching database UniProt knowledgebase (Swiss-Prot/TrEMBL, ) and Gene Ontology (GO) Database (http www.expasy.org://www.geneontology.org/), to this The cell function process that three histone matter are participated in and the biocytology function being related to have carried out initial analysis.

2.3.1 two groups of zymotic fluids have analysis of protein：

Using the comparison sequence to all albumen identifying for the Mapping function in Blast2GO (Version 2.8.0) The associated GO function entry of row is extracted, and extracts 126 GOs related to wherein 27 protein sequences (77.14%) altogether Function entry, in this project, totally 25 protein sequences are annotated by 78 GO function entries, and average GO level is 5.41.Through Complementary annotations, final annotation statistics is：Totally 27 protein sequences are annotated by 91 GO function entries.

Total histone WEGO functional annotation cluster analysis：

In total group of 35 protein, according to albumen WEGO functional annotation cluster analysis, 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 accounts for 20%.Using WEGO software to the albumen identifying It is as shown in Figure 3 with graphic result that the major function classification of matter completes the calculating of GO function enrichment.

Total histone GO functional annotation

Wherein, as shown in figure 4, in Biological Progress figure classification, participating in metabolic process (metabolic Process the 20 kinds of albumen that have) annotate, and account for total BP quantity 39.2%, cell processes (cellular process) have 15 hatching eggs White annotate, account for the 29.4% of BP sum, remaining participate in biological regulation (biological regulation), signal path (signaling), multiple biological processes (single-organism process/multi-organism process), cell Original paper component biosynthesis pathway (cellular component organization or biogenesis), stress reaction During (response to stimulus) etc.；In Molecular Function classification chart (Fig. 5), there is catalysis activity (catalytic activity) has 18 kinds of albumen annotations, accounts for the 64.3% of MF sum, residual protein participates in combination respectively Albumen (binding), molecular structure activity (structural molecule activity), molecular transport activity During (molecular transducer activity) etc.；As shown in fig. 6, in Cell Component classification, cell 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 path annotates：

These albumen pass through inquiry in KEGG database and compare, and the metabolism participating in has glycolytic pathway Glycolysis/Gluconeogenesis (4), photosynthetic carbon fixation acts on Carbon fixation in photosynthetic Organisms (3), starch Sucrose Metabolism Starch and sucrose metabolism (2), methane metabolism Methane metabolism(1).

2.3.2 the peculiar analysis of protein of two groups of zymotic fluids：

Using the comparison sequence to all albumen identifying for the Mapping function in Blast2GO (Version 2.8.0) The associated GO function entry of row is extracted, and extracts 296 GOs related to wherein 56 protein sequences (75.68%) altogether Function entry.In this project, totally 54 protein sequences are annotated by 182 GO function entries, and average GO level is 6.775.Through Complementary annotations, final annotation statistics is：Totally 59 protein sequences are annotated by 225 GO function entries.

Peculiar albumen WEGO functional annotation cluster analysis：

Peculiar albumen WEGO functional annotation cluster analysis is as shown in Figure 7：Wherein, totally 11 kinds of 1 group of peculiar albumen, participates in biological The albumen of process (biological progress) totally 12 annotations, account for 50.0%, participate in molecular function (molecular Function albumen) annotates 7 kinds, accounts for 29.2%, and the albumen note 5 kind of cytology component (cell component) accounts for 20.8%；63 kinds of 2 groups of peculiar albumen, participates in albumen totally 105 annotations of biological processes (biological progress), accounts for 46.1%, the albumen 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, in Biological Process classification, it is primarily involved in generation The 8 kinds of albumen that have of journey of apologizing for having done sth. wrong (metabolic process) annotate, and account 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 catalysis activity (catalytic activity) in Molecular Function classification (6), associated proteins (binding) (6), the albumen such as transport activity (transporter activity) (1) annotates；Cellular In Component classification, albumen has cell (cell) (2), organelle (organelle) (1), macromolecular compound (macromolecular complex) (1), extracellular region (extracellular region) (1), cell membrane (membrane) (4).

In 2 groups of peculiar albumen, in Biological Process classification, mainly there is cell processes (cellular Process albumen) is annotated with 33 kinds, accounts for the 34.7% of BP total amount, and metabolic process (metabolic process) albumen annotates There are 32 kinds, account for the 33.7% of BP total amount, single creature process (single-organism process) has 10 kinds of albumen annotations, (the cellular component organization or biogenesis) of cell biological synthesis has 8 kinds, and remaining has fixed Position (localization) (5), stress reaction (response to stimulus) (2), biological regulation (biogical Regulation) (6) etc.；Mainly there is catalysis activity (catalytic activity) egg in Molecular Function classification 30 kinds of white annotation, accounts for the 45.5% of MF total amount, and 7 kinds of associated proteins (binding) note 2 accounts for the 40.9% of MF, remaining has molecule Structural proteins (structural molecule) (6), transport protein (transporter) (3), there is transcription factor activity Associated proteins (protein binding transcription factor activity) (1), have the nucleic acid of transcriptional activity (nucleic acid binding transcription factor activity) (1) etc. annotates；And antioxidation activity (antioxidant activity) (1), electron carrier activity (electron carrier activity) (1)；Cellular In Component classification, annotation albumen has 18 kinds of cell (cell), 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), membrane closure chamber (membrane-enclosed lumen)(1).

KEGG path annotates：

1 group of peculiar Protein Information compares in KEGG database, obtains the metabolism that some of them mate albumen Approach, residual protein is limited because studying, and also cannot find the information of its coupling, and this histone participates in 8 metabolic pathways altogether：Fructose Sweet dew glycometabolism Fructose and mannose metabolism (1), purine metabolism Purine metabolism (1), sugar Glycolysis Glycolysis/Gluconeogenesis (1), riboflavin metabolism Riboflavin metabolism (1), methane metabolism Methane metabolism (1), pentose phosphate pathway Pentose phosphate pathway (1), aminobenzoic acid degradation Aminobenzoate degradation (1), photosynthetic carbon fixation acts on Carbon fixation in photosynthenic organisms(1).

After comparing in 2 groups of peculiar albumen KEGG databases, coupling albumen participates in 24 metabolic pathways altogether：Purine Metabolism Purine metabolism (4), glycolysis Glycolysis/Gluconeogenesis (4), photosynthetic carbon fixation acts on 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 are biological to be closed Become Pantothenate and CoA biosynthesis (1), glyoxylate and dicarboxylic acids approach Glyoxylate and Dicarboxylate metabolism (1), citrate circulation Citrate cycle (TCA cycle) (1), valine, bright The biosynthesis Valine of propylhomoserin and isoleucine, leucine and isoleucine biosynthesis (1), glycine, Serine and threonine metabolism Glycine, serine and threonine metabolism (1), amino sugar and nucleotide sugar Metabolism 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), Cytochrome P450 xenobiotics metabolism Metabolism of xenobiotics by cytochrome P450 (1), naphthalene degraded Naphthalene degradation (1), degraded Chloroalkane and chloroalkene degradation (1) of alkyl chloride and chloro-alkenes, aliphatic acid drops Solution Fatty acid degradation (1), tyrosine metabolism Tyrosine metabolism (1), Vitamin A Metabolism Retinol metabolism(1).

3 conclusions：

Peptide fragment 68, protein groups 47 are identified altogether in 1 group；2 groups identify peptide fragment 241, protein groups 121 altogether.Former Identify annotated albumen in beginning data and amount to 109, wherein 1 group peculiar protein 11,63,2 groups of peculiar albumen, two groups 35, total albumen.Illustrate all to exist rich in protein in two groups of zymotic fluids, but the high yield protein yeast bacterial strain in this invention In zymotic fluid, protein content and species are above the bacterium that sets out.

2 groups of zymotic fluids compared with 1 group in not only protein content abundanter, and participate in metabolic pathway also more.Meanwhile, zymotic fluid The higher carbohydrate metabolism of middle generally existing and energetic supersession, also participate in have impact on a small amount of biological pathways etc., 2 groups have only One protein with electron carrier activity and transhipment son activity, may exercise bioelectric detecting, IR and localization Etc. function.In the bacterial strain fermentation liquor of this invention, the metabolic pathway of protein function and participation is far more than the bacterium that sets out.

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

3.2 response surface experiments designs and result

Box-Behnken experimental design and result are as shown in table 4-2.Number is tested to table 2 using Design-Expert software According to carrying out multiple regression analysis, the quadratic regression model obtaining saccharomycete clump count with each variable factors is：Y=2.87+ 0.13A-0.085B+0.015C+0.025AB+5.000E-003AC+0.000BC+2.000E-003A2-0.29B2+2.000E- 003C2.

Table 4-2

Test sequence 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

With software, variance analysis is carried out to model, result is as shown in table 4-3.

Table 4-3

The confirmatory experiment of 3.3 model equations：

Obtain the extreme point of regression model by software Design-Expert.V8.0.6, i.e. optimal culture condition：Initially PH is 5.8, and cultivation temperature is 27.26 DEG C, and liquid amount is 100.00mL, and saccharomycete clump count theoretical prediction maximum is 2.89* 10⁸CFU/mL.For checking the reliability of Responds Surface Methodology, select optimal culture condition confirmatory experiment it is contemplated that practical operation Convenient, adjustment optimal culture condition is：Initial pH is 5.8, and cultivation temperature is 27.30 DEG C, and liquid amount is 100.00mL.At this Under part, carry out 3 parallel tests, saccharomycete clump count is 3.00*10⁸CFU/mL.Close with theoretical expectation values, this model is described It is believable, the optimization that Responds Surface Methodology is used for yeast bacteria preparation condition of culture is feasible.

Optimal culture condition is：Initial pH is 5.8, and cultivation temperature is 27.30 DEG C, and liquid amount is 100.00mL

Embodiment 5：The microbiotic impact on intestine of young pigs of C20140911 bacterial strain fermentation liquor

1 materials and methods

1.1 material

1.1.1 reagent and instrument

C20140911 zymotic fluid；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 is carried out on Yuzhong pig farm, chooses 20d piglet 9 nest of health, 6, every nest, totally 54 (Durocs × York × long white tri-crossbreeding), it is randomly divided into 3 groups, every group of 3 nests, every nest is 1 repetition.During test, animal is routinely managed, Free choice feeding, free water, during test, diarrhoea takes drugless medication.

1.2. experimental design

Test for 3 process 3 repetition single factor experiments, 3 process be 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 starts Continuous Observation 3 days from 20d, without exception Then start oral zymotic fluid, continuous 5 days, 28d weaned, select 1 respectively at 23d, 28d, 35d, 42d at random in each process in repetition Carry out cuing open killing, to ileum, caecum, the ligation of colon two ends, take intestinal contents in certain site sterile.- 20 DEG C of content is detested Oxygen preserves, for microbiota detection.

1.3 test method

1.3.1 conventional method separates counting

Fetch intestinal contents about 0.2g respectively, add in about 1.8ml physiological saline, shake up；Caecum and colonic contents are about 0.5g, adds in 4.5ml physiological saline, shakes up. and then 10 times of gradient dilutions of physiological saline, take 0.1ml to be added to different flat boards Differential counting. every part of sample is parallel to be done twice, chooses culture dish between 30～300 for the clump count (CFU) and counts, calculates average Value, is calculated the bacterial population of every gram of content, and is represented with its logarithm value.Including lactobacillus (MRS), Bifidobacterium (training The self-control of foster base), the survey of Escherichia coli (Mai Kangkai), salmonella (SS) and aerobic bacteria sum and anaerobic bacteria sum (blood flat board) Fixed.

2 results

The microbiotic impact on ileum of 2.1 zymotic fluids

2.1.1 the impact to Escherichia coli quantity

The colibacillary impact on piglet ileal contents of each treatment group of table 5-1

Note：1) in table, data unit is the wet content of lgCFU/g；2) in table, data is mean value ± standard error；3) shoulder note word Matrix shows identical intestinal segment, the comparison of identical age in days；Similarly hereinafter.

Test 1 group and 2 groups of ileal contents coliform counts of test decrease with age in days increase, control group increases with age in days Plus raise.Significant difference (p when testing 1 group, 2 groups of test and control group 42 age in days<0.05)(p<0.01) 1 group of test and test 2 Group difference is not notable.

2.1.2 the impact to salmonella quantity

In process of the test, 1 group of test is increased with age in days with 2 groups of ileal contents Salmonella bacterium numbers of test and reduces, right Increase according to salmonella quantity after group wean, and be consistently higher than other two groups., compared with control group, 28d, 35d are poor for two test group Different not notable, the extremely notable (p of 42d difference<0.01) indifference conspicuousness, but between the two.

The impact to piglet ileal contents salmonella for each treatment group of table 5-2

2.1.3 the impact to aerobic bacteria quantity

The impact to the total aerobic bacteria of piglet ileal contents for each treatment group of table 5-3

In process of the test, 1 group of test and 2 groups of ileum aerobic bacteria quantity of test are integrally on a declining curve, and control group is then Increase with age in days and rise.Test 1 group and 2 groups of test no significant differences；Test 1 group compared with control group, 28d, 35d difference is not Significantly, 42d significant difference (p<0.05)；Test 2 groups compared with control group, 28d, 35d difference is not notable, 42d significant difference (p< 0.05).

From the above it can be seen that：Each treatment group is not notable on the difference of ileum harmful bacteria impact in 28d, 35d, 1 group, 2 groups of harmful bacteria quantity of test substantially less than control group (p is tested during 42d<0.05) poor between, testing 1 group and testing 2 groups Different not notable.Thus illustrate that bacterial strain fermentation liquor of the present invention is similar to the effect of original yeast fermentation broth, all can effectively reduce harmful Bacterium number amount

2.1.4 the impact to bifidobacteria

The impact to piglet ileal contents Bifidobacterium for each treatment group of table 5-4

From table 5-4, ileal contents Bifidobacterium number, test 1 group in 28 ages in days and be significantly higher than control group (p< 0.05), between other age in days each groups, difference is not notable；But the increase each group with age in days is in rising trend

2.1.5 the impact to Bacillus acidi lactici quantity

The impact to piglet ileal contents Bacillus acidi lactici for each treatment group of table 5-5

From table 5-5, ileal contents Bacillus acidi lactici number, with the increase of age in days, is tested 1 group and 2 groups of test is presented The trend of liter.Test 1 group and 2 groups of test is significantly higher than control group (p during whole test<0.05)；Test 1 group and 2 groups of test Difference is not notable.

2.1.6. the impact to anaerobic bacteria quantity

The impact to the total anaerobic bacteria of piglet ileal contents for each treatment group of table 5-6

From table 5-6, in whole process of the test, ileal contents total anaerobism bacterium number, with the increase of age in days, is tested 1 group and 2 groups of in rising trend, control group held stationaries of test.Test 1 group and 2 groups of total anaerobism bacterium numbers of test are consistently higher than comparison Group, but between each group, difference is not notable.

The impact to cecum microorganisms fauna for 2.2 zymotic fluids

2.2.1 the impact to Escherichia coli quantity

The colibacillary impact on piglet cecal content of each treatment group of table 5-7

In caecum, increase with age in days, 1 group of test and 2 groups of Escherichia coli quantity of test have all declined, and control group exists Decline during 28d, then begin to rise, during whole test, control group is above testing 1 group and 2 groups of test.Test 1 group and test Between 2 groups, difference is not notable, with control group 35d significant difference (p<0.05), the extremely notable (p of 42d difference<0.01)；Test 2 groups with Extremely notable (the p of control group 35d, 42d difference<0.01).

2.2.2 the impact to salmonella quantity

The impact to salmonella in cecal content for each treatment group of table 5-8

In test, 1 group of test is on a declining curve with salmonella quantity in 2 groups of cecal contents of test；Control group weans After rise, be declined slightly during to 42d, but still be higher than other two groups.Test 1 group and be less than 2 groups of test, but difference is not notable；Low In control group 35d significant difference (p<0.05), the extremely notable (p of 42d difference<0.01).Test 2 groups compared with control group, 35d difference Significantly (p<0.05), the extremely notable (p of 42d difference<0.01).

2.2.3 the impact to aerobic bacteria quantity

In test, 1 group of test is increased with age in days with 2 groups of cecal content aerobic bacteria quantity of test and reduces, but 1 group of test 42d increased compared with 35d, higher than testing 2 groups；Control group increases with age in days and raises, and 35d, 42d are above remaining two groups.Examination Test 1 group compared with testing 2 groups, be higher than 2 groups of test during 42d, and significant difference (p<0.05)；Test 1 group of 42d compared with control group Notable (the p of time difference heteropole<0.01)；Test 2 groups compared with control group, the notable (p of 42d time difference heteropole<0.01), remaining is not notable.

The impact to the total aerobic bacteria of piglet cecal content for each treatment group of table 5-9

Sheet3-9 Effect of different treatment on aerobic bacterial in cecum of piglets

Find out from the above, test 1 group in caecum and all can reduce harmful bacteria quantity with 2 groups of yeast of test in 35d, 42d, The two effect is similar, and effect is better than ileum；And test the effect of 2 groups of control aerobic bacteria quantity better than 1 group of test.

2.2.4 the impact to bifidobacteria

The impact to piglet cecal content Bifidobacterium for each treatment group of table 5-10

From table 5-10, cecal content Bifidobacterium number is tested 1 group and is tested 2 groups in rising with the increase of age in days Trend, control group is declined slightly.During test, 2 groups with test of 1 group of test is consistently higher than control group；28 ages in days and 42 ages in days are poor Different notable (p<0.05)；Testing 1 group is higher than 2 groups of test, but difference is not notable.

2.2.5 the impact to Bacillus acidi lactici quantity

The impact to piglet cecal content Bacillus acidi lactici for each treatment group of table 5-11

From table 5-11, cecal content Bacillus acidi lactici number is presented with 1 group of the increase test of age in days and 2 groups of test The trend of liter.During test, 1 group of test and 2 groups of test are consistently higher than control group, and 28 age in days differences are extremely notable, and 42 age in days differences show Write.Testing 1 group is higher than 2 groups of test, but difference is not notable.

2.2.6. the impact to anaerobic bacteria quantity

The impact to the total anaerobic bacteria of piglet cecal content for each treatment group of table 5-12

Find out from 5-12：Testing 1 group and the total anaerobism bacterium number of 2 groups of cecal contents of test is in rise with the increase of age in days Trend, control group is declined slightly, basic held stationary.Test 1 group and 2 groups of test is consistently higher than control group, 28 age in days differences show Write (p<0.05), the extremely notable (p of 35-42 age in days difference<0.01)；Testing 1 group is higher than 2 groups of test, and difference is not notable.

Find out (table 5-10～5-12) from the above：C20140911 zymotic fluid and original yeast strain ferments liquid Effectively improve caecum beneficial bacterium quantity, illustrate that the two effect is similar, but C20140911 zymotic fluid effect is better than original yeast strain Zymotic fluid.

The 2.3 disconnected impacts to faecal flora fauna for the zymotic fluid

2.3.1 the impact to Escherichia coli quantity

1 group of, comparison on a declining curve with Escherichia coli quantity in 2 groups of colonic contentses of test is tested in increase with age in days Group 28d has declined, and 35d rises.In whole process of the test, control group is above other two groups, and to 42d compared with testing 1 group Significant difference (p<0.05)；All notable (the p of 35d, 42d difference compared with testing 2 groups<0.05)；Test 1 group and 2 groups of differences of test Not notable

The colibacillary impact on piglet colonic contents of each treatment group of table 5-13

2.3.2 the impact to salmonella quantity

The impact to colonic contents salmonella for each treatment group of table 5-14

From the graph as can be seen that testing 1 group during test, testing 2 groups of colonic contents Salmonella bacterium numbers integrally in decline Trend, 1 group of test slightly rises in 35d, but is less than control group, and control group is in propradation after wean.Test 1 group and examination Test 2 groups of differences not notable, 42d significant difference (p compared with control group<0.05), remaining time is not notable.Test 2 groups with compare Group compares 35d, 42d significant difference (p<0.05).

2.3.3 the impact to aerobic bacteria quantity

Test 1 group of colon aerobic bacteria slightly to rise after wean, change less between whole experimental period；Test 2 groups 35d with Front on a declining curve, less than testing 1 group, 42d rises above 1 group of test；Control group increases with age in days and rises, to 42d slightly Decline.Test 1 group with test 2 groups of result of the test differences not notable；Test 1 group of 35d and be substantially less than control group (p<0.05)；Test During 2 groups of 35d, pole is substantially less than control group (p<0.01).

The impact to colonic contents aerobic bacteria for each treatment group of table 5-15

Find out (table 5-14--5-15) from the above：Colon test 1 group with test 2 groups can have in 35d, 42d Effect controls harmful bacteria quantity, illustrates that the two effect is similar.

2.3.4 the impact to bifidobacteria

The impact to piglet colonic contents Bifidobacterium for each treatment group of table 5-16

From table 5-16, in colonic contents, Bifidobacterium number is in the trend rising with the increase each group of age in days.? 28 ages in days and 35 ages in days, 1 group of test and 2 groups of test are significantly higher than control group (p<0.05)；It is right that 42 1 group of ages in days tests are significantly higher than According to group (p<0.05), 2 groups of test is not notable with control group difference.Testing 1 group during experiment is higher than 2 groups of test, but difference does not show Write.

2.3.5 the impact to Bacillus acidi lactici quantity

From table 5-17, test 1 group and present with the increase of age in days with Bacillus acidi lactici number in 2 groups of colonic contentses of test The trend of liter, control group has declined.28 ages in days, 1 group of test and 2 groups of poles of test are significantly higher than control group (p<0.01), 35～42 Age in days, 1 group of test and 2 groups of test are significantly higher than control group (p<0.05)；During experiment, 1 group of test in 35-42 age in days is higher than Test 2 groups, but difference is not notable.

The impact to piglet colonic contents Bacillus acidi lactici for each treatment group of table 5-17

2.3.6. the impact to anaerobic bacteria quantity

The impact to the total anaerobic bacteria of piglet colonic contents for each treatment group of table 5-18

From table 5-18, colonic contents total anaerobism bacterium number, with the increase of age in days, is tested 1 group and 2 groups of test is presented The trend of liter, on a declining curve before control group 35 age in days, 42 ages in days have recovered；Test 1 group and be consistently higher than test 2 groups and comparison Group.Test 1 group and be significantly higher than control group (p in 35-42 age in days<0.05), 2 groups of test is significantly higher than control group in 35 ages in days.Examination Between testing 1 group and testing 2 groups, difference is not notable.

In sum (table 5-16～3-18), C20140911 bacterial strain fermentation liquor and original yeast strain ferments liquid all may not be used Increase colon beneficial bacterium quantity with degree, C20140911 bacterial strain fermentation liquor effect is more preferable.

Embodiment 6：The impact to weaned piglets for the C20140911 bacterial strain fermentation liquor

1. test method

Process packet same as Example 5, weigh, record feed intake respectively at 21d, 28d, 35d, 42d.

The impact to weaned piglets for the table 6-1 C20140911 bacterial strain fermentation liquor

Note：In table, data adopts average ± standard error (X ± SE) to represent, with a line internal ratio relatively.

From table 6-1, test 1 group and 2 groups of test is all remarkably improved the daily gain of weanling pig compared with control group, day adopts Appetite, promotes weanling pig growth, wherein tests 1 group and relatively tests 2 groups of daily gains raisings 16.90%.Test 1 group of pig weight with Control group is compared, and in 35d significant difference (p ＜ 0.05), tests last method of double differences heteropole in 42d notable (p ＜ 0.01), daily gain and day Also pole is significantly higher than control group (p ＜ 0.01), the trend that feed intake is improved to feed intake.And test 2 groups of body weight and control group phase Than, the trend that 2 weeks are significantly increased after ablactation, difference is not notable；Daily gain and daily ingestion amount all pole are significantly higher than control group (p ＜ 0.01).Feed-weight ratio result shows that testing 1 group is less than test 2 groups and control group.

C20140911 bacterial strain fermentation liquor and original yeast strain ferments liquid all can improve 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 the preferred embodiments of the present invention, be not limited to the present invention, Although being described in detail to the present invention with reference to the foregoing embodiments, for a person skilled in the art, it still may be used To modify to the technical scheme described in foregoing embodiments, or equivalent is carried out to wherein some technical characteristics. All any modification, equivalent substitution and improvement within the spirit and principles in the present invention, made etc., should be included in the present invention's Within protection domain.

Claims

1. a plant height is laid eggs white yeast strain S yeast（Saccharomyces cerevisiae）C20140911, its 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.

2. a plant height according to claim 1 is laid eggs white yeast strain S yeast（Saccharomyces cerevisiae）The selection of C20140911 is it is characterised in that described selection is with the culture of 14g brewer's wort powdery Base, 1g peptone, 100ml distilled water are culture medium, and by culture medium autoclaving, it is 5.8 that gnotobasis adjusts initial incubation liquid pH, Access 1ml initially to set out bacteria culture fluid, temperature is 27.3 DEG C, rotating speed 120r/min, shake 48-72H, constant-temperature table is cultivated, instead Pass on again；From the beginning of the peptone of low dosage 1%, to yeast strain（Saccharomyces cerevisiae）Carry out growing resistance to Tamed, be stepped up peptone dosage to 8%, through multiple secondary screening with pass on test, filtered out high yield protein yeast bacterial strain.

3. a plant height according to claim 1 is laid eggs white yeast strain S yeast（Saccharomyces cerevisiae）The cultural method of C20140911 is it is characterised in that being by malt extract medium 14.0g, peptone 8g, double Steam water 100ml, autoclaving；It is 5.8 that gnotobasis adjusts initial incubation liquid pH, accesses kind of a daughter bacteria 1ml, is 27.3 DEG C in temperature, Rotating speed 120r/min, constant-temperature table is cultivated.

4. a plant height according to claim 1 is laid eggs white yeast strain S yeast (Saccharomyces Cerevisiae) application in preparation animal high-protein feed additive for the C20140911.