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

Abstract

The invention discloses a high-yield protein yeast strain (Saccharomyces cerevisiae) C20140911 and its screening method and application, and provides its breeding, cultivation method and application. The beneficial effects of the present invention are as follows: the protein content and type of the fermentation liquid of the yeast strain disclosed in the present invention are much higher than that of the starting bacteria, and a total of 241 peptides and 121 proteomes were identified from the fermentation liquid of the strain; 254.4% and 157.4%; the fermentation liquid of this strain not only has high protein content, but also participates in many metabolic pathways, and participates in more carbohydrate metabolism and energy metabolism; it has the only protein with electron carrier activity and transporter activity, which may exercise Functions such as biological regulation, stimulus response and positioning process, the protein function and involved metabolic pathways in the fermentation liquid of the bacterial strain of the invention are far more than those of the original bacteria, the bacterial strain of the present invention can be used to prepare feed additives and microecological preparations, improve animal production performance, The effect of reducing diarrhea in weaned piglets is better than that of starting bacteria.

Description

A high-yield protein yeast strain C20140911 and its selection, cultivation method and application

technical field

The invention relates to the technical field of animal feed additives, in particular to a high-yielding yeast strain Saccharomyces cerevisiae (Saccharomyces cerevisiae) C20140911 and its selection, cultivation method and application.

Background technique

Yeast is a eukaryotic unicellular microorganism with a typical cell structure, including nucleus, cytoplasmic membrane, cell wall, cytoplasm and other inclusions. Yeast is widely used in production because it is rich in high-quality protein, amino acids, complete B vitamins, various minerals in the form of life-combined forms, and functional dietary fiber. Yeast is the earliest and most widely used microbe used by humans. People often use its fermentation function to make various dough foods and brew wine. For example, beer yeast is used in beer production; selenium-rich and zinc-rich yeast are used as organic trace elements Carrier, making trace elements easier to absorb, etc.; in animal husbandry, yeast is mainly added to feed as single-cell protein to reduce the use of other protein raw materials. At the same time, there are also research reports on the use of yeast culture (Yeast Culture, YC) as a probiotic. Yeast culture is a product of yeast culture, which refers to the product processed together with the medium after fermentation under strictly controlled conditions. The main components are medium, yeast and yeast metabolites. The protein content in yeast metabolites is very small, but it contains other "unknown growth-promoting factors", which are used to regulate the micro-ecological balance of the intestinal tract of animals, improve feed utilization, increase disease resistance and stress resistance, and promote growth. Thereby improving animal performance. The application of yeast culture in animal husbandry has many advantages, because it will not interfere with the action of antibiotics, and at the same time can reduce the use of antibiotics. Today, when food safety is called for and the use of antibiotics is restricted, yeast products will have a broader application prospect .

Contents of the invention

The purpose of the present invention is to address the defects in the above-mentioned prior art, in order to improve the protein production level of bacterial strains, to prepare better microecological preparations and feed additives, in order to enhance its clinical use effect, and serve food safety and animal husbandry production. Provided is a high-yield protein yeast strain Saccharomyces cerevisiae C20140911 and its selection, cultivation methods and applications.

In order to achieve the above object, the technical solution provided by the present invention is: a high-yield protein yeast strain Saccharomyces cerevisiae (Saccharomyces cerevisiae) C20140911, its preservation number in the General Microbiology Center of China Microbiological Culture Collection Management Committee is CGMCC No.9675, and the preservation time is for September 18, 2014.

The above-mentioned protein-rich yeast strain with high biomass is obtained through long-term domestication on the basis of the original beer yeast Saccharomycescerevisia.

The second object of the present invention is to provide a breeding method for the above-mentioned high-yield protein yeast strain (Saccharomycescerevisiae) C20140911, said breeding method is to use 14g wort powdery medium, 1g peptone, 100ml double distilled water as the medium , the culture medium is autoclaved, and the pH of the initial culture solution is adjusted to 5.8 in a sterile environment, and 1ml of the initial starting bacteria culture solution is inserted, the temperature is 27.3°C, the speed is 120r/min, shaken for 48-72H, and the constant temperature shaking table is cultivated repeatedly. Subculture: start with a low dose of 1% peptone, grow tolerance and domesticate yeast strains (Saccharomyces cerevisiae), gradually increase the peptone dose to 8%, and screen out high protein-producing yeast strains after multiple re-screening and passage tests;

The third object of the present invention is to provide a culture method for the above-mentioned high-yield protein yeast strain (Saccharomycescerevisiae) C20140911, which is 14.0g of wort medium, 8g of peptone, 100ml of double distilled water, and autoclaving; The pH of the initial culture solution was 5.8, 1ml of the seed bacteria was inserted, and the temperature was 27.3°C, the rotation speed was 120r/min, and the culture was carried out on a constant temperature shaker.

The fourth object of the present invention is to provide the application of the above-mentioned high-yielding yeast strain (Saccharomycescerevisiae) C20140911 in the preparation of animal high-protein feed additives.

The high-yield protein yeast strain disclosed by the invention has a higher protein content and type in the fermented liquid than the original starting strain.

The protein content of the fermented liquid of the high-yielding yeast strain disclosed by the invention is 0.329 mg/ml, which is 8.2% higher than the 0.304 ml/mg of the starting bacterium fermented liquid.

A total of 241 peptides and 121 protein groups were identified in the fermentation liquid of the high-yielding yeast strain of the present invention; a total of 68 peptides and 47 protein groups were identified in the fermentation liquid of the original starting strain, which were 254.4% and 157.4% higher than .

A total of 109 annotated proteins were identified by the mass spectrometry of the present invention. 63 unique proteins have been identified in the fermentation broth of the high-yielding yeast strain, and 11 proteins are unique to the starting bacteria, which is nearly 6 times that of the starting bacteria; 35 proteins are shared with the starting bacteria. indivual.

The fermented liquid of a high-yielding yeast strain disclosed by the invention not only has high protein content, but also has far more protein functions and involved metabolic pathways than the starting bacteria. It is involved in more carbohydrate metabolism and energy metabolism, and it is the only protein with electron carrier activity and transporter activity, which may perform functions such as biological regulation, stimulus response and localization process.

The WEGO functional annotation clustering analysis of the specific proteins of the present invention shows that there are 63 specific proteins in the fermentation broth of the high-yielding yeast strain, 105 annotations of proteins involved in biological progress, and annotations of proteins involved in molecular functions (molecular function) 77 kinds, 41 kinds of protein annotations of cellular components. There are 12 unique proteins in the fermentation broth of the original strain, 12 annotations of proteins involved in biological progress, 7 annotations of proteins involved in molecular function, and 5 annotations of proteins in cellular components kind.

The GO function annotation of the specific protein of the present invention shows that among the specific proteins of the fermentation broth of the high-yielding yeast strain, there are 33 protein annotations mainly involved in the cellular process (cellular process) in the classification of the biological process (Biological Process), and participate in the metabolic process ( There are 32 annotations for metabolic process proteins, 10 annotations for single-organism processes, 8 annotations for cellular component organization or biogenesis, and 5 annotations for the rest involved in localization. There are 2 types of response to stimulus, 6 types of biological regulation, etc. Participating in molecular function (Molecular Function) mainly includes 30 annotations of catalytic activity proteins, 27 annotations of binding proteins, 6 annotations of structural molecules, and 3 types of transporters in the rest , 1 protein binding transcription factor activity with transcription factor activity, 1 nucleic acid binding transcription factor activity with transcription activity, etc.; and 1 antioxidant activity (ntioxidant activity) annotation, electron carrier activity ( electron carrier activity) 1 type. Annotated proteins participating in the Cellular Component classification include 18 types of cells, 9 types of organelles, 9 types of macromolecular complexes, 4 types of extracellular regions, and 4 types of membranes. One type of membrane-enclosed lumen.

Among the proteins unique to the fermentation broth of the starting strain, there are 8 protein annotations in the Biological Process category that are mainly involved in the metabolic process, 5 protein annotations for the single-organism process, and 5 protein annotations for the cellular process (cellular process). ), there are 4 types of protein annotations, and the rest include 2 types involved in response to stimulus, 1 type in localization, and 1 type in cellular component organization or biogenesis, etc. In the category of Molecular Function, there are 6 kinds of annotated proteins involved in catalytic activity (catalytic activity), 6 kinds of binding protein (binding), and 1 kind of transport activity (transporter activity). In the classification of Cellular Component, there are 2 types of annotated proteins involved in the composition of cells, 1 type composed of organelles, 1 type composed of macromolecular complexes, and 1 type composed of extracellular regions. 1 type, 4 types of cell membrane (membrane) composition.

The KEGG pathway annotation of the present invention shows that the metabolic pathways of some of the matching proteins are obtained after comparison and analysis of the specific proteins of the fermentation broth of the high-yielding yeast strains in the KEGG database, and the matching information of the remaining proteins cannot be found due to limited research. The matching proteins participated in 24 metabolic pathways: 4 kinds of purine metabolism (Purine metabolism), 4 kinds of glycolysis (Glycolysis/Gluconeogenesis), 2 kinds of photosynthetic carbon fixation (Carbon fixation in photosynthetic organsisms), nitrogen metabolism (Nitrogen metabolism) 2 types, 2 types of alanine, asparatate and glutamate metabolism (Alanine, asparatate and glutamate metabolism), 2 types of prokaryotic carbon fixation (Carbon fixation pathways in prokaryotes), 1 type of pyruvate metabolism (Pyruvate metabolism) ,1 inositol phosphate metabolism,1 pantothenate and CoA biosynthesis,1 glyoxylate and dicarboxylate metabolism,citrate cycle (Citrate cycle (TCA cycle) 1 species, valine, leucine and isoleucine biosynthesis (Valine, leucine and isoleucine biosynthesis) 1 species, glycine, serine and threonine metabolism (Glycine, serine and threonine Metabolism) 1 species, amino sugar and nucleotide sugar metabolism (Amino sugar and nucleotide sugar metabolism) 1 species, cysteine and methionine metabolism (Cysteine and methionine metabolism) 1 species, cytochrome P450 drug metabolism (Drugmetabolism-cytochrome P450 ) 1 type, Fructose and mannose metabolism (Fructose and mannose metabolism) 1 type, arginine and proline metabolism (Arginine and proline metabolism) 1 type, cytochrome P450 exogenous chemical substance metabolism (Metabolism of xenobiotics by cytochrome P450 ) 1 species, naphthalene degradation (Napht halene degradation) 1 type, chloroalkane and chloroalkene degradation (Chloroalkane and chloroalkene degradation) 1 type, fatty acid degradation (Fatty acid degradation) 1 type, tyrosine metabolism (Tyrosine metabolism) 1 type, vitamin A metabolism (Retinol metabolism) 1 species.

The unique protein information of the fermentation broth of the original strain was compared and analyzed in the KEGG database, and a total of 8 metabolic pathways were involved: 1 kind of fructose and mannose metabolism (Fructose and mannose metabolism), 1 kind of purine metabolism (Purine metabolism), glycolysis ( Glycolysis/Gluconeogenesis) 1 species, riboflavin metabolism (Riboflavin metabolism) 1 species, methane metabolism (Methane metabolism) 1 species, pentose phosphate pathway (Pentose phosphate pathway) 1 species, aminobenzoate degradation (Aminobenzoate degradation) 1 species, photosynthetic solid Carbon effect (Carbonfixation in photosynthenic organisms) 1 kind).

The beneficial effects of the present invention are: the protein content and type of the fermented liquid of the yeast strain disclosed by the present invention are much higher than those of the starting bacteria. The protein content in the fermented liquid of the bacterial strain is 0.329 mg/ml, which is 8.2% higher than the 0.304 ml/mg of the fermented liquid of the starting bacterium. A total of 241 peptides and 121 proteomes were identified from the fermentation broth of the strain; 68 peptides and 47 proteomes were identified in the fermentation broth of the original strain, which were 254.4% and 157.4% higher than the original strain, respectively. Among the identified proteins, there are 63 unique proteins in the fermentation broth of the bacterium, 11 proteins unique to the starting bacterium, and 35 proteins shared with the starting bacterium. The fermentation liquid of this strain not only has high protein content, but also participates in many metabolic pathways, and participates in more carbohydrate metabolism and energy metabolism; it has the only protein with electron carrier activity and transporter activity, which may exercise biological regulation, stimulation response and Positioning process and other functions. The protein functions and involved metabolic pathways in the bacterial strain fermentation liquid of the invention are far more than those of the starting bacteria. The bacterial strain of the invention can be used to prepare feed additives and microecological preparations, improve animal production performance, and reduce diarrhea of weaned piglets, and the effect is better than that of the original bacteria.

Description of drawings

Figure 1 is a gel electrophoresis image in molecular biology identification.

Fig. 2 is the chromatogram result identified by ESI mass spectrometry.

Among them, the upper figure is the chromatogram of the fermentation liquid of the original starting strain, and the lower figure is the chromatogram of the fermentation liquid of the strain of the present invention.

Figure 3 is a cluster analysis diagram of the consensus protein group WEGO functional annotation.

Figure 4 is a classification map of Biological Progress in the consensus histone GO functional annotation.

Figure 5 is the Molecular Function classification map in the consensus histone GO functional annotation.

Figure 6 is a classification diagram of Cell Component in the consensus histone GO functional annotation.

Figure 7 is a cluster analysis diagram of the WEGO functional annotation of the unique proteome.

Figure 8 is a classification map of Biological Progress in the GO functional annotation of specific proteins.

Figure 9 is the Molecular Function classification map in the GO functional annotation of specific proteins.

Figure 10 is a classification diagram of Cell Component in the GO functional annotation of specific proteins.

detailed description

Example 1:

Breeding method

1. A strain of protein-rich yeast (Saccharomyces cerevisiae) C20140911, its preservation number in the General Microbiology Center of China Microbiological Culture Collection Management Committee is CGMCC No.9675, and the preservation date is September 18, 2014.

The above-mentioned protein-rich yeast strain with high biomass is obtained through long-term domestication on the basis of the original beer yeast Saccharomycescerevisia.

The breeding method of the above-mentioned high-protein strains is to use 14.0g of wort powdery medium, 1g of peptone, and 100ml of double distilled water as the medium (autoclaved, the pH of the initial culture solution is adjusted to 5.8 in a sterile environment), and 1ml of the initial The starting bacterial culture solution was cultured on a constant temperature shaker at a temperature of 27.3°C, a rotation speed of 120r/min, shaking for 48-72H, and repeated passage.

In order to cultivate and domesticate high-protein strains, starting from a low dose of 1% peptone, the yeast strain Saccharomyces cerevisiae was acclimatized for growth and tolerance, and gradually increased the peptone dose to 8%. Quantitative protein-rich yeast strains.

Culture conditions: Wort medium (14g), peptone (8g), double distilled water (100ml), autoclaved; aseptic environment to adjust the pH of the initial culture solution to 5.8, constant temperature shaker culture: temperature 27.3 ℃, speed 120r /min.

Detection of survival conditions: culture at constant temperature for 72 hours, dilute 10 times with normal saline, and use a hemocytometer to detect the number of viable bacteria, which can reach 10 ⁸ /ml.

The bacteria in the culture are produced in the beer industry and are not animal or plant pathogens; the wort and peptone in the culture medium are non-toxic and harmless, and will not pollute the environment.

The above-mentioned protein-rich yeast strain (Saccharomyces cerevisiae) C20140911 is mainly used in the preparation of animal high-protein feed additives. (Can you describe this part in detail, how to add it, how much to add, and what beneficial effects it can bring)

Embodiment 2: the identification of strain

1. Morphological identification:

On the malt juice agar medium, the colonies are milky white, round in shape, with neat edges, shiny, and flat; when observed under a microscope, the cells of the strain are mostly oval or long oval, and the cells are budding, without hyphae, and without Throw spores and ascospores.

2. Physiological and biochemical identification:

Test method: Bacterial suspension method: take a test tube containing 2ml of sterile water, use an inoculation needle to pick a single colony that has been purified from the plate, and dilute it into sterile water to make a uniform bacterial suspension with a McFarland turbidity of 0.5, drop it into 1 drop in each micro-biochemical identification tube that needs to be tested. (Note: If the content is semi-solid or inclined, perform puncture inoculation)

test results:

Fermentation test, fermentation of glucose, galactose, sucrose, maltose, and raffinose; non-fermentation of: lactose, xylose, cellobiose, and trehalose.

Carbon assimilation test, assimilation: glucose, maltose, sucrose, galactose and mannose; dissimilation: cellobiose, D-xylose, melibiose, trehalose and L-arabinose.

Nitrogen assimilation test, assimilation: (NH ₄ ) ₂ SO ₄ ; assimilation: KNO ₃ .

Alcohol assimilation test, assimilation: ethanol; dissimilation: sorbitol.

The above experimental results showed that the strain was yeast.

3. Molecular biological identification:

3.1 Test method: Bacteria culture—DNA extraction—PCR—electrophoresis—sequencing—NCBI blast comparison.

① Bacterial liquid culture: Pick the preserved yeast strain on the slope, add it to 5ml of the liquid wort medium prepared in advance, and culture it on a shaking table at 23°C for 48 hours, and set it aside.

② DNA extraction: OMEGA BIO-TEK kit steps for extraction.

③PCR: Primer preparation: forward primer NL-1:5'-GCATAT CAA TAA GCG GAG GAAAAG-3', reverse primer NL-4:5'-GGT CCGTGTTTCAAGACGG-3', prepared by Shanghai Sangong.

Reaction system 25 μL: 2×Taq Master Mix 12.50 μL, NL-1 1.00 μL, NL-4 1.00 μL, DNA template 1.00 μL, ddH ₂ 0 9.50 μL, TOTAL 25.00 μL.

Reaction conditions (35cycles): pre-denaturation: 94°C, 4min; denaturation: 94°C, 30s; refolding: 58°C, 30s; T1: 72°C, 45s; T2: 72°C, 10min; storage: 15°C, ∞.

④ Electrophoresis conditions: 1.4% agarose gel for electrophoresis, spotting: 2.5 μL of 6×Loading Buffer (sampling buffer), 5 μL of DNA, electrophoresis: after spotting, set the electrophoresis condition to voltage 150V for 25 minutes.

⑤The products with brighter electrophoresis bands were sent to Shanghai Sangon Sequencing Department for sequencing.

⑥Enter the sequencing results into www.NCBI.nlm.nih.gov, and use BLAST software to compare the homology between the measured gene sequence and the sequence in the Genbank database.

3.2 Test results:

① The electrophoresis diagram is shown in Figure 1. (about 600bp in length, 1, 2 are original yeast strains, 3, 4 are domesticated protein-rich yeast strains)

② BLAST comparison results: Saccharomyces cerevisiae S288c, NC_001144.5, chromosomeXII.

The above-mentioned experimental results show that: the bacterial strain provided by the invention is a yeast strain.

Embodiment 3: Determination of strain protein content

1. Test method:

Sample group 1: fermentation liquid samples of original starting yeast strains; group 2: fermentation liquid samples of high protein-producing yeast strains.

1.1 Sample processing and SDS-PAGE:

Centrifuge 100ml of yeast fermentation broth at 3000rpm for 10min, take the supernatant, then centrifuge at 7830rpm at 4°C for 30min, take 15ml sample, dialyze overnight at 3KDa, and the dialysate is 100mM ammonium bicarbonate solution. After dialysis, the sample was ultrafiltered with a 3KDa ultrafiltration tube at 7830 rpm at 4°C until the volume was less than 2ml. The concentration was determined by Bradford method, 20 μl was taken for SDS-PAGE electrophoresis, and Coomassie brilliant blue staining.

1.2 Enzymatic hydrolysis in solution:

Take 50 μl samples from each group and add DTT to a final concentration of 10 mM. Add IAA to a final concentration of 50 mM, and react in the dark for 30 min. Add LysC and react at 37°C for 3 hours. Add 4 volumes of 25 mM ammonium bicarbonate solution. Add 4 μg Trypin to react overnight at 37°C, and acidify with 0.1% TFA to terminate the reaction. C18-SD Extraction Disk Cartridge desalted, vacuum freeze-dried. Add 35 μl 0.1% FA to re-dissolve the pellet.

1.3 Capillary high performance liquid chromatography:

The nanoliter flow rate HPLC liquid phase system Easy nLC was used for separation. Buffer solution: solution A is 0.1% formic acid aqueous solution, and solution B is 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 84%). The chromatographic column is equilibrated with 95% liquid A. The sample is loaded from the autosampler to the loading column Thermo scientific EASY column (2cm*100μm 5μm-C18), and then separated by the analytical column Thermoscientific EASY column (75μm*100mm 3μm-C18) at a flow rate of 300nl/min. The relevant liquid phase gradient is as follows: from 0 minutes to 50 minutes, the linear gradient of B solution is from 0% to 50%; from 50 minutes to 54 minutes, the linear gradient of B solution is from 50% to 100%; from 54 minutes to 60 minutes, the B solution is maintained at 100%.

1.4 ESI mass spectrometry identification:

The samples were separated by capillary high-performance liquid chromatography and then analyzed by Q-Exactive mass spectrometer (ThermoFinnigan). Analysis time: 60min, detection method: positive ion, precursor ion scanning range: 300-1800m/z, primary mass spectrometry resolution: 70,000at m/z 200, AGC target: 3e6, primary maximum IT: 10ms, Number of scanranges :1, Dynamic exclusion: 3.0s. The mass-to-charge ratio of polypeptides and polypeptide fragments is collected according to the following method: 10 fragment spectra (MS2scan) are collected after each full scan, MS2Activation Type: HCD, Isolation window: 2m/z, secondary mass spectrometry resolution: 17,500at m/z 200, Microscans: 1, secondary Maximum IT: 60ms, Normalized collision energy: 27eV, Underfill ratio: 0.1%.

1.5 Mass spectrometry data analysis

The original file (MGF file) was searched by Mascot software. The database is the database uniprot_saccharomyces_112806_20141020.fasta downloaded from uniprot (112806 sequences were included, and the download time was 2014-10-20). The search parameters were set as follows: enzyme was trypsin; missed cleavage was set to 2; static modification was set to Carbamidomethy C; dynamic modification was set to Oxidation M. Peptides tolerance is set to 20ppm, ms/mstolerance is set to 0.1Da, Ion score>20.

1.6 Bioinformatics analysis

Search through the internationally authoritative database UniProt knowledgebase (Swiss-Prot/TrEMBL, www.expasy.org) and Gene Ontology (GO) Database (http://www.geneontology.org/), and initially explore the identified differentially expressed proteins The subcellular localization, biological functions and cellular processes involved.

2. Test results

2.1 Protein quantitative results:

SDS-PAGE electrophoresis proved that there were abundant proteins in the fermentation broths of the two groups, and the quantitative results of the samples were shown in Table 3-1.

Table 3-1

sample 1 2 Protein concentration (μg/μl) 0.304 0.329

2.2.2 ESI mass spectrometry identification results:

The statistics of the sample protein identification results are shown in Table 3-2.

Table 3-2

sample Proteome individual peptides 1 41 68 2 121 241

From the two chromatograms in Figure 2, it can be seen that the protein chromatographic absorption peaks of the two groups of samples are richer, which proves that the protein content and types are more, and through comparative analysis, it can be seen that the protein absorption peak area of the 2 groups is larger than that of the 1 group , and there were more absorption peaks, preliminarily indicating that the protein content in the samples of the two groups was relatively rich.

2.3 Bioinformatics analysis - GO analysis and preliminary analysis of KEGG metabolic pathways:

The identified proteins were compared with the protein sequences in the NCBI nr database using the localized sequence alignment software NCBI BLAST+ (ncbi-blast-2.2.28+-win32.ext). According to the principle of similarity, the obtained functional information of homologous proteins can be used for functional annotation of target proteins. We only keep the top 10 aligned sequences with Evalue≤1e-3 for subsequent analysis. The resulting alignment similarities ranged from 50-100%, with most of the target protein sequences having an alignment similarity of 98% or above.

A total of 109 annotated proteins were identified by mass spectrometry in the original data, including 11 proteins unique to group 1, 63 proteins unique to group 2, and 35 proteins shared by both groups. The identified protein sequence information was extracted in batches from the UniProtKB database (http://www.uniprot.org) (version number: Release 2014_10) and saved in FASTA format.

In addition, by searching the database UniProt knowledgebase (Swiss-Prot/TrEMBL, www.expasy.org) and Gene Ontology (GO) Database (http://www.geneontology.org/), the cellular functions involved in these three groups of proteins The processes and the biological cellular functions involved were initially analyzed.

2.3.1 Analysis of common proteins in two groups of fermentation broths:

Using the Mapping function in Blast2GO (Version 2.8.0) to extract the GO function entries associated with the aligned sequences of all identified proteins, a total of 126 GO functions related to 27 protein sequences (77.14%) were extracted Items, in this project, a total of 25 protein sequences were annotated by 78 GO function items, with an average GO level of 5.41. After supplementary annotation, the final statistical result of annotation is: a total of 27 protein sequences were annotated by 91 GO functional entries.

Consensus histone WEGO functional annotation cluster analysis:

Among the 35 proteins in the total group, according to the clustering analysis of protein WEGO functional annotations, the annotations of proteins involved in biological process (biological progress) accounted for 51%, the annotations of proteins of molecular function (molecular function) accounted for 28%, and the annotations of cytological components Analysis (cell component) annotations account for 20%. Using WEGO software to complete the GO functional enrichment calculation and graphical representation of the main functional categories of the identified proteins, the results are shown in Figure 3.

Consensus histone GO functional annotation

Among them, as shown in Figure 4, in the Biological Progress graph classification, there are 20 protein annotations involved in the metabolic process, accounting for 39.2% of the total BP, and 15 protein annotations in the cellular process, accounting for the total BP 29.4% of them, and the rest are involved in biological regulation, signaling, multiple biological processes (single-organism process/multi-organism process), cellular component organization or biogenesis ), stress response (response to stimulus) and other processes; in the Molecular Function classification map (Figure 5), there are 18 proteins with catalytic activity (catalytic activity), accounting for 64.3% of the total MF, and the remaining proteins are involved in In the process of binding protein (binding), molecular structure activity (structural molecule activity), molecular transport activity (molecular transducer activity), etc.; as shown in Figure 6, in the Cell Component classification, there are 7 kinds of proteins in the cell component (cell) Sequences account for 35% of the total CC, and the rest of the proteins are involved in processes such as extracellular regions, organelles, membranes, and macromolecular complexes.

KEGG pathway annotation:

These proteins are queried and compared in the KEGG database, and the metabolisms involved include Glycolysis/Gluconeogenesis (4), Carbon fixation in photosynthetic organisms (3), Starch and sucrose metabolism of starch and sucrose metabolism (2), Methane metabolism Methanemetabolism (1).

2.3.2 The specific protein analysis of the two groups of fermentation broth:

Using the Mapping function in Blast2GO (Version 2.8.0) to extract the GO function entries associated with the aligned sequences of all identified proteins, a total of 296 GO functions related to 56 protein sequences (75.68%) were extracted entry. In this project, a total of 54 protein sequences were annotated by 182 GO functional entries, with an average GO level of 6.775. After supplementary annotation, the final statistical result of annotation is: a total of 59 protein sequences were annotated by 225 GO functional entries.

WEGO functional annotation cluster analysis of specific proteins:

The clustering analysis of WEGO functional annotation of unique proteins is shown in Figure 7: Among them, there are 11 kinds of unique proteins in a group, and 12 annotations of proteins involved in biological progress (biological progress), accounting for 50.0%, proteins involved in molecular function (molecular function) Annotated 7 kinds, accounting for 29.2%, 5 kinds of protein annotations of cell component, accounting for 20.8%; 63 kinds of unique proteins in 2 groups, 105 annotations of proteins involved in biological progress, accounting for 46.1% , 77 proteins involved in molecular function were annotated, accounting for 33.8%, and 41 proteins were annotated in cell component, accounting for 17.9%.

Functional annotation of specific protein GO:

As shown in Figure 8-10, among the 1 group of unique proteins, there are 8 protein annotations that are mainly involved in the metabolic process (metabolic process) in the Biological Process classification, accounting for 47.1% of the total BP, and the single biological process (single- Organism process) has 5 protein annotations, accounting for 29.4% of BP, cellular process (cellular process) has 4 protein annotations, accounting for 23.5% of BP, and the rest have stress response (response to stimulus) (2), localization (localization) (1), cell biosynthesis (cellular component organization orbiogenesis) (1), etc.; Molecular Function classification mainly includes catalytic activity (catalytic activity) (6), binding protein (binding) (6), transporter activity (transporter activity) (1) Protein annotation; CellularComponent classification includes cell (2), organelle (1), macromolecular complex (1), extracellular region (1), Cell membrane (membrane) (4).

Among the two groups of unique proteins, there are 33 annotations of cellular process proteins in the Biological Process classification, accounting for 34.7% of the total BP, and 32 annotations of metabolic process proteins, accounting for 10% of the total BP. 33.7%, single-organism process has 10 kinds of protein annotations, cell biosynthesis (cellular component organization or biogenesis) has 8 kinds, and the rest have localization (localization) (5), stress response (response to stimulus) (2), biological regulation (biological regulation) (6), etc.; in the Molecular Function classification, there are mainly 30 annotations of catalytic activity proteins, accounting for 45.5% of the total MF, and 27 annotations of binding proteins, Accounting for 40.9% of MF, the rest include molecular structural protein (structural molecule) (6), transporter (transporter) (3), binding protein with transcription factor activity (protein binding transcription factor activity) (1), transcriptional activity Nucleic acid binding transcription factor activity (1) and other annotations; and antioxidant activity (antioxidant activity) (1), electron carrier activity (electron carrier activity) (1); the annotation protein in the CellularComponent classification has cell (cell) 18 species, accounting for 40.9% of the total CC, 9 organelles, accounting for 20.5% of the CC, macromolecular complex (9), extracellular region (4), cell membrane (membrane ) (4), membrane-enclosed lumen (membrane-enclosed lumen) (1).

KEGG pathway annotation:

A group of unique protein information was compared and analyzed in the KEGG database, and the metabolic pathways of some of the matching proteins were obtained. Due to limited research, the matching information of the remaining proteins could not be found. This group of proteins participated in 8 metabolic pathways: fructomannose Fructose and mannose metabolism(1), Purine metabolism(1), Glycolysis/Gluconeogenesis(1), Riboflavin metabolism(1), Methane metabolism(1), Pentose phosphate pathway Phosphate pathway(1), Aminobenzoate degradation(1), Carbon fixation in photosynthenicorganisms(1).

After comparing and analyzing the two groups of unique proteins in the KEGG database, the matching proteins participated in 24 metabolic pathways: Purine metabolism (4), glycolysis/Glycolysis/Gluconeogenesis (4), and photosynthetic carbon fixation (Carbon fixation in photosynthetic organisms ( 2), nitrogen metabolism Nitrogen metabolism (2), alanine, aspartate and glutamate metabolism Alanine, asparatate and glutamate metabolism (2), prokaryotic carbon fixation Carbon fixation pathways in prokaryotes (2), pyruvate metabolism Pyruvate metabolism (1), inositol phosphate metabolism (1), Pantothenate and CoA biosynthesis (1), glyoxylate and dicarboxylate pathway Glyoxylate and dicarboxylate metabolism (1), citrate Citrate cycle (TCA cycle) (1), Valine, 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 mannose metabolism Fructose and mannose metabolism (1), arginine and proline metabolism Arginine and proline metabolism (1), cytochrome P450 metabolism of exogenous chemical substances Metabolism of xenobiotics by cytochrome P450 (1), naphthalene Degradation of Naphthalene degradation(1), Chloroalkane and chloroalkene degradation(1), Fatty acid degradation tty acid degradation (1), tyrosine metabolism Tyrosine metabolism (1), vitamin A metabolism Retinol metabolism (1).

3 Conclusions:

A total of 68 peptides and 47 proteins were identified in group 1; 241 peptides and 121 proteins were identified in group 2. A total of 109 annotated proteins were identified in the original data, including 11 proteins unique to group 1, 63 proteins unique to group 2, and 35 proteins shared by both groups. It shows that there is abundant protein in the fermented liquids of the two groups, but the protein content and types in the fermented liquids of the high-yielding yeast strains in this invention are higher than those of the starting bacteria.

Compared with group 1, the fermented liquid of group 2 not only has richer protein content, but also participates in more metabolic pathways. At the same time, high carbohydrate metabolism and energy metabolism generally exist in the fermentation broth, and they also participate in and affect a small number of biological pathways. The two groups have the only protein with electron carrier activity and transporter activity, which may exercise biological regulation and stimulate response. And positioning process and other functions. The protein functions and involved metabolic pathways in the bacterial strain fermentation liquid of the invention are far more than those of the starting bacteria.

Embodiment 4: Optimization of culture conditions by response surface methodology

1. Selection of factor levels for response surface analysis

Table 4-1 shows the factors and levels for response surface analysis.

Table 4-1

3.2 Response surface test design and results

Box-Behnken test design and results are shown in Table 4-2. Using Design-Expert software to carry out multiple regression analysis on the test data in Table 2, the quadratic regression model of the number of yeast colonies and each factor variable 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 number A B C Preparation colony count/(10 ⁸ CFU/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

Use the software to analyze the variance of the model, and the results are shown in Table 4-3.

Table 4-3

3.3 Verification experiment of the model equation:

Use the software Design-Expert.V8.0.6 to find the extreme point of the regression model, that is, the best culture conditions: the initial pH is 5.8, the culture temperature is 27.26°C, the liquid volume is 100.00mL, and the theoretically predicted maximum number of yeast colonies It is 2.89*10 ⁸ CFU/mL. In order to test the reliability of the response surface analysis method, the best culture conditions were selected for the verification experiment. Considering the convenience of practical operation, the best culture conditions were adjusted as follows: the initial pH was 5.8, the culture temperature was 27.30 °C, and the liquid volume was 100.00 mL. Under these conditions, three parallel experiments were carried out, and the number of yeast colonies was 3.00*10 ⁸ CFU/mL. It is close to the theoretical prediction value, which shows that the model is credible, and the response surface analysis method is feasible for the optimization of yeast preparation culture conditions.

The optimal culture conditions are: the initial pH is 5.8, the culture temperature is 27.30°C, and the liquid volume is 100.00mL

Example 5: Effect of C20140911 Strain Fermentation Broth on the Intestinal Microflora of Piglets

1 Materials and methods

1.1 Materials

1.1.1 Reagents and instruments

C20140911 fermentation broth; original yeast fermentation broth; MacConkey (Hangzhou Microbial Reagent Co., Ltd.); SS agar (Hangzhou Microbial Reagent Co., Ltd.); MRS (OXOID, ENGLAND); blood plate (Lanzhou Rongchang Biological Products Co., Ltd.).

1.1.2 Experimental animals

The experiment was carried out in a pig farm in Yuzhong. Nine litters of healthy 20-day piglets were selected, with six pigs in each litter. A total of 54 piglets (Duroc×York×Landrace three-way hybrid pigs) were randomly divided into 3 groups, with 3 litters in each group. The litter was 1 replicate. During the test period, the animals were managed according to the routine, free to eat and drink water, and the diarrhea was not treated with drugs during the test period.

1.2. Experimental design

The test was 3 treatments and 3 repeated single factor experiments. The 3 treatments were the first group (C20140911 fermentation broth group), the second group (the original yeast strain fermentation broth group) and the third group (blank control group). Start to observe continuously for 3 days, if there is no abnormality, start to take the fermentation liquid orally, continue for 5 days, wean on 28d, and randomly select 1 head in each treatment repetition on 23d, 28d, 35d, and 42d for dissection. The end is ligated, and the intestinal contents are aseptically collected at a certain site. The contents are stored anaerobically at -20°C for microbial flora detection.

1.3 Test method

1.3.1 Separation and counting by conventional methods

Take about 0.2g of ileal content, add about 1.8ml of normal saline, and shake well; about 0.5g of cecum and colon content, add about 4.5ml of normal saline, and shake well. Then the normal saline is diluted 10 times, take 0.1 mL was added to different plates for classification and counting. Each sample was performed twice in parallel, and the number of culture dishes with a colony number (CFU) between 30 and 300 was selected for counting, and the average value was calculated to calculate the number of bacteria per gram of content, and the logarithmic representation. These include the determination of Lactobacillus (MRS), Bifidobacterium (self-made culture medium), Escherichia coli (McConkey), Salmonella (SS) and the total number of aerobic bacteria and anaerobic bacteria (fresh blood plate).

2 results

2.1 Effect of fermentation broth on ileum microflora

2.1.1 Effects on the number of E. coli

Table 5-1 Effects of each treatment group on Escherichia coli in piglet ileum

Note: 1) The unit of the data in the table is lgCFU/g wet content; 2) The data in the table is the mean ± standard error; 3) The letters on the shoulder indicate the comparison of the same intestinal segment and the same age; the same below.

The number of Escherichia coli in the ileum of the test group 1 and test 2 decreased with the increase of age, while that of the control group increased with the increase of age. There was a significant difference between the test group 1, the test group 2 and the control group at 42 days of age (p<0.05) (p<0.01). The difference between the test group 1 and the test group 2 was not significant.

2.1.2 Effect on the number of Salmonella

During the test, the number of Salmonella in the ileum of the test group 1 and test 2 decreased with the increase of age, and the number of Salmonella in the control group increased after weaning, and was always higher than that of the other two groups. Compared with the control group, the difference between the two test groups was not significant at 28d and 35d, and the difference was extremely significant at 42d (p<0.01), but there was no significant difference between the two groups.

Table 5-2 Effect of each treatment group on Salmonella in piglet ileum content

2.1.3 Effect on the number of aerobic bacteria

Table 5-3 The effect of each treatment group on the total aerobic bacteria in piglet ileum

During the test, the number of aerobic bacteria in the ileum of the test 1 and test 2 groups showed an overall downward trend, while that of the control group increased with the increase of age. There was no significant difference between the test group 1 and the test group 2; compared with the control group, the difference was not significant at 28d and 35d, but the difference was significant at 42d (p<0.05); compared with the control group, the difference was not significant at 28d and 35d. Significantly, the 42d difference was significant (p<0.05).

From the above results, it can be seen that the difference of each treatment group on the harmful bacteria in the ileum was not significant at 28 days and 35 days, and the number of harmful bacteria in the test group 1 and test group 2 was significantly lower than that of the control group at 42 days (p<0.05), while the test group There was no significant difference between group 1 and group 2. This shows that the fermented liquid of the bacterial strain of the present invention is similar to the effect of the original yeast fermented liquid, and can effectively reduce the number of harmful bacteria

2.1.4 Effect on the number of bifidobacteria

Table 5-4 The effect of each treatment group on the content of bifidobacteria in piglet ileum

As can be seen from Table 5-4, the number of bifidobacteria in the ileum content was significantly higher than that of the control group (p<0.05) in the 28-day-old test group 1, and the difference between the other groups was not significant; Increasing each group showed an upward trend

2.1.5 Effect on the number of Lactobacillus

Table 5-5 The effect of each treatment group on the content of Lactobacillus in the ileum of piglets

It can be seen from Table 5-5 that the number of Lactobacillus in the ileum content increased with the increase of age, and the test group 1 and test 2 showed an upward trend. During the whole test period, the experimental group 1 and the experimental group 2 were significantly higher than the control group (p<0.05); the difference between the experimental group 1 and the experimental group 2 was not significant.

2.1.6. Effect on the number of anaerobic bacteria

Table 5-6 The effect of each treatment group on the total anaerobic bacteria in piglet ileum content

It can be seen from Table 5-6 that during the whole test process, the total anaerobic bacteria count in the ileum content increased with the age, and the test group 1 and test 2 showed an upward trend, while the control group remained stable. The total number of anaerobic bacteria in the test group 1 and the test group 2 was always higher than that in the control group, but there was no significant difference between the groups.

2.2 Effect of fermentation broth on cecum microflora

2.2.1 Effects on the number of E. coli

Table 5-7 Effects of each treatment group on Escherichia coli in piglet cecum contents

In the cecum, with the increase of age, the number of E. coli in the test group 1 and the test group 2 decreased, the control group decreased at 28 days, and then began to rise, and the control group was higher than the test group 1 and test 2 during the whole test period . The difference between the test group 1 and the test group 2 was not significant, but it was significantly different from the control group at 35 days (p<0.05), and the difference at 42 days was extremely significant (p<0.01); the difference between test group 2 and the control group at 35 days and 42 days was very significant (p<0.05) 0.01).

2.2.2 Effect on the number of Salmonella

Table 5-8 Effect of each treatment group on Salmonella in cecal contents

In the test, the number of Salmonella in the cecal contents of the test group 1 and the test group 2 showed a downward trend; the control group increased after weaning, and decreased slightly at 42 days, but was still higher than the other two groups. The test group 1 was lower than the test group 2, but the difference was not significant; the test group was lower than the control group at 35 days with a significant difference (p<0.05), and at 42 days with a very significant difference (p<0.01). Compared with the control group, there was a significant difference at 35 days (p<0.05), and a very significant difference at 42 days (p<0.01).

2.2.3 Effect on the number of aerobic bacteria

In the test, the number of aerobic bacteria in the cecum content of the test group 1 and the test group 2 decreased with age, but the number of test group 1 increased at 42 days compared with 35 days, which was higher than that of test group 2; the control group increased with age , 35d, 42d were higher than the other two groups. Compared with the test group 2, the test group 1 was higher than the test group 2 at 42 days, and the difference was significant (p<0.05); the test group 1 was significantly different from the control group at 42 days (p<0.01); Compared with the control group, the difference was extremely significant at 42 days (p<0.01), and the rest were not significant.

Table 5-9 Effect of each treatment group on the total aerobic bacteria in piglet cecum contents

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

From the above results, it can be seen that the yeast in the cecum test group 1 and the test group 2 can reduce the number of harmful bacteria at 35 days and 42 days, and the effects of the two are similar, and the effect is better than that of the ileum; and the effect of controlling the number of aerobic bacteria in the test group 2 is better than that of the ileum Test 1 group.

2.2.4 Effect on the number of bifidobacteria

Table 5-10 Effects of each treatment group on bifidobacteria in piglet cecum contents

It can be seen from Table 5-10 that the number of bifidobacteria in the cecal contents increased with the increase of the age in the test group 1 and test 2, and decreased slightly in the control group. During the test, the test group 1 and the test group 2 were always higher than the control group; the difference between the age of 28 days and the age of 42 days was significant (p<0.05); the test group 1 was higher than the test group 2, but the difference was not significant.

2.2.5 Effect on the number of Lactobacillus

Table 5-11 The effect of each treatment group on the content of lactobacilli in piglet cecum

It can be seen from Table 5-11 that the number of Lactobacillus in the cecum content increased with the increase of the age in the test group 1 and the test group 2. During the test, the test 1 and test 2 groups were always higher than the control group, the difference was extremely significant at 28 days, and the difference at 42 days was significant. Test 1 group was higher than test 2 group, but the difference was not significant.

2.2.6. Effect on the number of anaerobic bacteria

Table 5-12 Effects of each treatment group on the total anaerobic bacteria in piglet cecal contents

From 5-12, it can be seen that the total anaerobic bacteria count in the cecal contents of the test group 1 and test group 2 showed an upward trend with the increase of the age, while the control group decreased slightly and basically remained stable. The test group 1 and test group 2 were always higher than the control group, the difference was significant at 28 days (p<0.05), and the difference at 35-42 days was extremely significant (p<0.01); the test group 1 was higher than test group 2, the difference was not significant .

It can be seen from the above results (Table 5-10~5-12): Both the C20140911 fermentation broth and the original yeast strain fermentation broth can effectively increase the number of beneficial bacteria in the cecum, indicating that the two have similar effects, but the C20140911 fermentation broth is better than the original Yeast strain fermentation broth.

2.3 The effect of broken fermentation broth on the colonic microflora

2.3.1 Effects on the number of E. coli

The number of Escherichia coli in the colon contents of the test group 1 and the test group 2 showed a downward trend with the increase of the age, the control group decreased at 28 days, and increased at 35 days. Throughout the test process, the control group was higher than the other two groups, and compared with the test 1 group, the 42d difference was significant (p<0.05); compared with the test 2 group, the 35d and 42d differences were significant (p<0.05); the test 1 There was no significant difference between the group and the test 2 groups

Table 5-13 Effects of each treatment group on Escherichia coli in piglet colon contents

2.3.2 Effect on the number of Salmonella

Table 5-14 Effects of each treatment group on Salmonella in colonic contents

It can be seen from the figure above that during the test period, the number of Salmonella in the colon contents of the test group 1 and the test group 2 showed an overall downward trend, and the test group 1 increased slightly at 35 days, but was lower than the control group, and the control group was on the rise after weaning. The difference between test 1 group and test 2 group was not significant, compared with the control group, the difference was significant at 42 days (p<0.05), and the rest of the time was not significant. Compared with the control group, there was a significant difference in the 35d and 42d of the test group 2 (p<0.05).

2.3.3 Effect on the number of aerobic bacteria

Colonic aerobic bacteria in test group 1 increased slightly after weaning, and did not change much during the whole test period; test group 2 showed a downward trend before 35 days, which was lower than test group 1, and increased higher than test group 1 at 42 days; Increase and rise, to 42d a slight decline. There was no significant difference in test results between test group 1 and test group 2; test group 1 was significantly lower than control group at 35 days (p<0.05); test group 2 was extremely significantly lower than control group at 35 days (p<0.01).

Table 5-15 Effects of each treatment group on aerobic bacteria in colon contents

It can be seen from the above results (Table 5-14--5-15): In colon test group 1 and test group 2, the number of harmful bacteria can be effectively controlled at 35 days and 42 days, which shows that the effects of the two are similar.

2.3.4 Effect on the number of bifidobacteria

Table 5-16 Effects of each treatment group on bifidobacteria in colon content of piglets

It can be seen from Table 5-16 that the number of bifidobacteria in the colon content increases with the increase of age in each group. At the age of 28 days and 35 days, the test group 1 and the test group 2 were significantly higher than the control group (p<0.05); at the age of 42 days, the test group 1 was significantly higher than the control group (p<0.05), and the test group 2 was significantly higher than the control group (p<0.05). The difference was not significant. During the experiment, the test 1 group was higher than the test 2 group, but the difference was not significant.

2.3.5 Effect on the number of Lactobacillus

It can be seen from Table 5-17 that the number of lactobacilli in the colonic contents of test group 1 and test group 2 increased with the increase of age, while that of the control group decreased. At the age of 28 days, the test group 1 and the test group 2 were significantly higher than the control group (p<0.01), at the age of 35 to 42 days, the test group 1 and the test group 2 were significantly higher than the control group (p<0.05); during the experiment, Test 1 group was higher than test 2 group at 35-42 days old, but the difference was not significant.

Table 5-17 Effects of each treatment group on lactobacilli in piglet colon contents

2.3.6. Effect on the number of anaerobic bacteria

Table 5-18 The effect of each treatment group on the total anaerobic bacteria in piglet colon content

It can be seen from Table 5-18 that the total anaerobic bacteria count in the colon content increases with the increase of age, the test group 1 and the test group 2 showed an upward trend, the control group showed a downward trend before the age of 35 days, and recovered at the age of 42 days; The experimental group 1 was always higher than the experimental group 2 and the control group. The test group 1 was significantly higher than the control group at the age of 35-42 days (p<0.05), and the test group 2 was significantly higher than the control group at the age of 35 days. There was no significant difference between test 1 group and test 2 group.

In summary (Table 5-16-3-18), the fermentation broth of C20140911 strain and the original yeast strain can increase the number of beneficial bacteria in the colon to varying degrees, and the fermentation broth of C20140911 strain is better.

Example 6: Effect of C20140911 strain fermentation broth on piglet production performance

1. Test method

The treatment groups were the same as in Example 5, and the animals were weighed and feed intake recorded on 21d, 28d, 35d, and 42d respectively.

Table 6-1 Effect of fermentation broth of C20140911 strain on piglet performance

Note: The data in the table are expressed as mean ± standard error (X±SE), and are compared within the same row.

It can be seen from Table 6-1 that compared with the control group, the test group 1 and the test group 2 can significantly increase the daily gain and daily feed intake of the weaned piglets, and promote the growth of the weaned piglets, and the test group 1 has a higher daily gain than the test group 2 16.90%. Compared with the control group, the weight of the piglets in the test group 1 was significantly different at 35 days (p<0.05), and at the end of the test at 42 days, the difference was extremely significant (p<0.01), and the daily weight gain and daily feed intake were also significantly higher than those of the control group (p<0.01), the feed intake tended to increase. Compared with the control group, the body weight of the test group 2 had a tendency to increase significantly at 2 weeks after weaning, and the difference was not significant; the daily weight gain and daily feed intake were significantly higher than those of the control group (p<0.01). The results of material to weight ratio showed that the test group 1 was lower than the test group 2 and the control group.

The fermentation broth of C20140911 strain and the fermentation broth of the original yeast strain can both improve the production performance, but the effect of the fermentation broth of the C20140911 strain is better than that of the original yeast strain.

Finally, it should be noted that: the above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it still The technical solutions recorded in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (4)

1. A high-yield protein yeast strain Saccharomyces cerevisiae C20140911, its preservation number in the General Microbiology Center of China Committee for Microbial Culture Collection is CGMCC No. 9675, and the preservation time is September 18, 2014. 2. The breeding method of a strain of high-yield protein yeast strain Saccharomycescerevisiae (Saccharomycescerevisiae) C20140911 according to claim 1, characterized in that, the breeding method is based on 14g wort powder medium, 1g peptone, 100ml double distilled Water is used as the culture medium, and the culture medium is autoclaved, and the pH of the initial culture solution is adjusted to 5.8 in a sterile environment, and 1ml of the initial starting bacteria culture solution is added, the temperature is 27.3°C, the speed is 120r/min, shake for 48-72H, and shake at a constant temperature Bed culture, repeated subculture; starting from a low dose of 1% peptone, the yeast strain (Saccharomyces cerevisiae) was acclimated to growth tolerance, and gradually increased the peptone dose to 8%. After multiple re-screening and passage tests, high-yield protein yeast was screened out strain. 3. The method for cultivating a high-yield protein yeast strain Saccharomycescerevisiae C20140911 according to claim 1, characterized in that 14.0 g of wort medium, 8 g of peptone, 100 ml of double distilled water, and autoclaved; Adjust the pH of the initial culture solution to 5.8 in a sterile environment, insert 1ml of seed bacteria, and cultivate at a temperature of 27.3°C and a rotation speed of 120r/min in a constant temperature shaker. 4. The application of a high-yield protein yeast strain Saccharomyces cerevisiae (Saccharomycescerevisiae) C20140911 according to claim 1 in the preparation of animal high-protein feed additives.