CN117467648A - Application of yeast zymolyte in preparation of feed additive - Google Patents
Application of yeast zymolyte in preparation of feed additive Download PDFInfo
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- CN117467648A CN117467648A CN202311817924.6A CN202311817924A CN117467648A CN 117467648 A CN117467648 A CN 117467648A CN 202311817924 A CN202311817924 A CN 202311817924A CN 117467648 A CN117467648 A CN 117467648A
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- 239000003674 animal food additive Substances 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 101710130006 Beta-glucanase Proteins 0.000 claims abstract description 36
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- 102000004190 Enzymes Human genes 0.000 claims abstract description 27
- 241000287828 Gallus gallus Species 0.000 claims abstract description 20
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- 235000013330 chicken meat Nutrition 0.000 claims abstract description 14
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 12
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- 230000000593 degrading effect Effects 0.000 claims abstract description 8
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- 238000006243 chemical reaction Methods 0.000 claims description 3
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- 239000004480 active ingredient Substances 0.000 abstract 1
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- 241000235648 Pichia Species 0.000 description 2
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 229960001561 bleomycin Drugs 0.000 description 2
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 2
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- LUEWUZLMQUOBSB-GFVSVBBRSA-N mannan Chemical class O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-GFVSVBBRSA-N 0.000 description 2
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- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 description 1
- 101150102326 1.1 gene Proteins 0.000 description 1
- 108010025188 Alcohol oxidase Proteins 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 101001036083 Bacillus amyloliquefaciens Beta-glucanase Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 231100000678 Mycotoxin Toxicity 0.000 description 1
- 229920000392 Zymosan Polymers 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
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- 210000002966 serum Anatomy 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
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- 238000010254 subcutaneous injection Methods 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/244—Endo-1,3(4)-beta-glucanase (3.2.1.6)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/14—Pretreatment of feeding-stuffs with enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01006—Endo-1,3(4)-beta-glucanase (3.2.1.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- Organic Chemistry (AREA)
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- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
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Abstract
The invention provides an application of yeast zymolyte in preparing a feed additive, and belongs to the technical field of feed preparation. The recombinant pichia pastoris strain which simultaneously expresses the beta-glucanase and the beta-mannase mutant is constructed, the two enzymes are cooperated to carry out enzymolysis on the autologous cell wall, and the enzymolysis liquid is spray-dried to prepare the feed additive. The invention provides a group of yeast cell wall degrading enzymes, comprising a beta-glucanase mutant and a beta-mannase mutant. The two enzyme mutants have high enzyme activity at the optimal growth temperature of yeast, and have the function synergistic promotion effect when combined, and can fully hydrolyze insoluble glucan and mannan in the cell wall of the yeast into water-soluble oligosaccharides to release active ingredients of the yeast. The feed additive prepared from the yeast zymolyte has the beneficial effects of improving diarrhea symptoms of chickens, reducing feed-meat ratio of chickens and improving immune antibody titer of vaccine.
Description
Technical Field
The invention belongs to the technical field of feed preparation, and particularly relates to application of yeast zymolyte in preparation of a feed additive.
Background
The yeast cell wall is a green additive, is rich in various bioactive substances such as beta-glucan, mannan and the like, and has various physiological functions such as pathogen adsorption, mycotoxin adsorption, immunity enhancement, growth promotion, stress relief, nutrition supplement and the like. Mannans account for about 30% of the dry weight of the yeast cell wall, beta-glucans account for about 30%, and cross-linking of glucans and mannans plays an important role in the synthesis of the yeast cell wall. The traditional method for preparing the zymosan needs the steps of autolysis at high temperature of yeast, mechanical crushing treatment, cell wall degradation by adding cell wall degrading enzyme, and the like, the time for cracking the cell wall is long, and expensive equipment such as a high-pressure homogenizer, a glass bead grinding crusher and the like is needed, so that the problems of high energy consumption, low cell wall crushing efficiency and low water-soluble polysaccharide extraction efficiency exist.
The heat generated by the high temperature autolysis or mechanical disruption of the yeast results in a decrease in the biological activity of a portion of the thermally unstable components. The yeast cell wall is insufficiently broken by simple autolysis or mechanical disruption, and the cell wall body formed by crosslinking glucan and mannan is still present in the precipitate in a water-insoluble form. These water insoluble components have low digestion availability and low bioactivity in fed animals. Only the water-insoluble glucan and the water-insoluble mannan in the cell wall are fully hydrolyzed into water-soluble oligosaccharides, the biological activity of the water-soluble oligosaccharides can be fully exerted, and the waste of useful resources is avoided. In the prior art, the cell wall degrading enzyme is added to degrade the cell wall of the yeast, the reaction conditions such as temperature, pH, salt ion concentration and the like are required to be strictly regulated, and the problem of low enzyme activity exists. Therefore, there is a need to develop enzymes capable of simply and efficiently degrading yeast cell walls under yeast physiological conditions for preparing a feed additive containing yeast hydrolysates.
Disclosure of Invention
The invention aims to provide an application of yeast zymolyte in preparing a feed additive, and belongs to the technical field of feed preparation.
In order to achieve the above purpose, the present invention provides the following technical solutions:
in one aspect, the invention provides a set of yeast cell wall degrading enzymes comprising a beta-glucanase mutant and a beta-mannanase mutant.
Further, the amino acid sequence of the beta-glucanase mutant is SEQ ID NO.1, and the amino acid sequence of the beta-mannase mutant is SEQ ID NO.2.
The invention also provides a feed additive containing a yeast zymolyte, wherein the yeast zymolyte is prepared by expressing beta-glucanase and beta-mannase through recombinant pichia pastoris and performing synergistic enzymolysis on an autologous cell wall.
Further, the amino acid sequence of the beta-glucanase is SEQ ID NO.1, and the amino acid sequence of the beta-mannase is SEQ ID NO.2.
Further, trp at amino acid positions 176 and 183 of the β -glucanase was mutated to Ala; the Trp at positions 196, 198 and 199 of the amino acid sequence of the beta-mannanase is mutated to Ala.
Further, the mass ratio of the addition amount of the feed additive in the feed is 1:1000.
Further, the feed additive can be used for improving diarrhea symptoms of chickens, reducing feed conversion ratio of chickens and improving immune antibody titer of vaccine.
The invention has the beneficial effects that the recombinant pichia pastoris strain which simultaneously expresses the beta-glucanase and the beta-mannase mutant is constructed, the beta-glucanase and the beta-mannase are controlled by a strict induction type alcohol oxidase promoter, the enzyme is not expressed in the enrichment fermentation stage, and the enzyme expression is started by methanol induction after high-density fermentation. The two enzyme mutants have higher activity than unmutated enzyme at the optimal growth temperature of the yeast, and are suitable for enzymolysis of the yeast cell wall under industrial fermentation conditions. The invention reduces the process of adding yeast cell wall degrading enzyme additionally and simplifies the regulation and control process of adding yeast cell wall degrading enzyme additionally on temperature, pH and salt ion concentration. The cell wall is subjected to enzymolysis autolysis from inside to outside, and the two enzymes are combined to perform function synergistic promotion, so that the water-insoluble glucan and the mannan are subjected to enzymolysis to form glucan oligosaccharide and oligomeric mannose with higher biological activity. The yeast zymolyte can be used for preparing feed additives, so that diarrhea symptoms of animals can be improved, vaccine immune antibody titer can be improved, and vaccine immune period can be prolonged.
Drawings
Fig. 1: and (3) a diagram of enzymolysis effects of cell wall polysaccharides of different Pichia strains under the condition of shake cultivation.
Fig. 2: and (3) an enzymolysis effect diagram of the cell wall polysaccharide of the recombinant pichia pastoris strain under the culture condition of the fermentation tank.
Fig. 3: influence of Yeast hydrolysate feed additive on chicken immunity.
Detailed Description
Example 1: enzyme structure modification and mutant design based on computer aided design
The bacillus amyloliquefaciens beta-glucanase GenBank accession number is: AAA87323.1, bacillus amyloliquefaciens beta-mannase GenBank accession number: ACX94042.1. The amino acid sequences of both enzymes were analyzed using Discovery Studio software. The active center of the beta-glucanase is located between 171-183 positions, the beta-glucanase is subjected to point mutation by reducing the steric hindrance of the active center of the enzyme protein, trp at 176-183 positions is mutated into Ala, and the amino acid sequence of the beta-glucanase mutant is SEQ ID NO.1; the active center of the beta-mannase is located between 193-201, the beta-mannase is subjected to point mutation by reducing the steric hindrance of the active center of the enzyme protein, trp at 196, 198 and 199 are mutated into Ala, and the amino acid sequence of the beta-mannase mutant is SEQ ID NO.2.
Example 2: recombinant pichia pastoris strain construction for simultaneously expressing beta-glucanase and beta-mannanase
1.1 Gene Synthesis
The DNA sequence SEQ ID NO.3 and the DNA sequence SEQ ID NO.4 of the beta-mannanase mutant are synthesized according to the codon preference of pichia pastoris. XhoI and NotI restriction enzyme sites are added at the upstream and downstream respectively, and the mixture is entrusted to the synthesis of biological engineering (Shanghai) stock, and cloned to pUC57 plasmid to obtain pUC57-bglA plasmid and pUC57-bman;
1.2 Construction of recombinant expression vector for beta-glucanase
(1) Double-restriction enzyme digestion is carried out on pUC57-bglA plasmid by using restriction enzymes XhoI and NotI, and bglA gene fragment is recovered;
(2) Double-enzyme cutting of an expression vector pPICZ alpha A by using restriction enzymes XhoI and NotI, and recovery of a gene fragment;
(3) And (3) connecting the nucleic acid fragments recovered by double digestion in the steps (1) and (2) by using T4 DNA ligase, then converting the nucleic acid fragments into E.coli DH5 alpha competent cells, extracting plasmids, and carrying out double digestion identification on XhoI and NotI to obtain a recombinant expression plasmid pPICZ alpha-bglA.
1.3 Construction of beta-mannanase recombinant expression vector
(1) Double-restriction enzyme digestion is carried out on pUC57-bman plasmid by using restriction enzymes XhoI and NotI, and bman gene fragments are recovered;
(2) Double-enzyme cutting of an expression vector pPICZ alpha A by using restriction enzymes XhoI and NotI, and recovery of a gene fragment;
(3) And (3) connecting the nucleic acid fragments recovered by double digestion in the steps (1) and (2) by using T4 DNA ligase, then converting the nucleic acid fragments into E.coli DH5 alpha competent cells, extracting plasmids, and carrying out double digestion identification on XhoI and NotI to obtain a recombinant expression plasmid pPICZ alpha-bman.
1.4 Recombinant pichia pastoris strain construction for simultaneously expressing beta-glucanase and beta-mannanase
(1) Respectively enzyme-cutting plasmids pPICZ alpha-bman and pPICZ alpha-bglA by using PmeI enzyme, and recovering linearization plasmids by ethanol precipitation;
(2) The linearized plasmid pPICZ alpha-bman and pPICZ alpha-bglA are mixed and then are electrically transformed into pichia pastoris X-33 competent cells; YPD plates containing 1000ug/ml bleomycin were screened for resistant single colonies. The single colonies screened were detected with two pairs of bmanf/bmanr and bglAf/bglaar primers. The colony which is positive only in the bmarf/bmark detection is a recombinant yeast strain which only expresses beta-mannanase and is named as X33/bman; the colony which is positive in the bglAf/bglAr detection is a recombinant yeast strain which only expresses beta-glucanase and is named as X33/bglA; the colony which is positive in the detection of both pairs of primers is a recombinant Pichia pastoris strain which simultaneously expresses beta-glucanase and beta-mannanase, and is named as X33/bman-bglA. Primer sequence:
bmanf：CTCGAGATGCTCAAAAGGTTAGCAGTC，SEQ ID NO.5；
bmanr：GCGGCCGCTTATTCCGTGATCGGCGTCAAGG，SEQ ID NO.6；
bglAf：CTCGAGATGAAACGAGTGTTGCTAATTC，SEQ ID NO.7；
bglAr：GCGGCCGCTTATTTTTTTCTATAGCGCATCC，SEQ ID NO.8；
1.5 Inducible expression of recombinant Pichia pastoris strain and enzymolysis of cell wall polysaccharide
(1) Respectively picking up recombinant Pichia strains X33/bman, X33/bglA and X33/bman-bglA, inoculating 50ml centrifuge tubes containing 5ml YPD culture medium, and placing the centrifuge tubes in a shaking table at 30 ℃ and 250rpm for overnight culture;
(2) Inoculating 2ml of seed culture solution into 1000ml of shake flask with 200ml of BMGY culture medium, and culturing at 30 ℃ and 250rpm for 24h;
(3) Adding 0.5% methanol into shake flask, placing in shake flask at 30deg.C, and performing induced fermentation culture at 250rpm for 24 hr to express beta-glucanase and beta-mannase, and performing enzymolysis on cell wall polysaccharide. Samples were taken at intervals of 6h during the induction fermentation, centrifuged at 5000rpm/min for 3min, and the wet weight of the cells was measured.
As can be seen from FIG. 1, the wet weight of the cells cultured for 24 hours by adding methanol to the blank X33 yeast is continuously increased from 192g/L to 333g/L; the wet weight of the thalli grows 6 hours before the X33/bman yeast is added with methanol for culture, and then the thalli is reduced until 24 hours is reduced to 143g/L; the wet weight of the thallus is increased for 6 hours before the X33/bglA yeast is added with methanol for culture, and then the thallus is reduced to 130g/L after 24 hours; the wet weight of the thallus is increased for 6 hours before the X33/bman-bglA yeast is added with methanol for culture, then the thallus is reduced to 47g/L after 18 hours, and the thallus is not reduced substantially after 24 hours.
The recombinant Pichia pastoris strain expressing the beta-glucanase and the beta-mannase simultaneously induces for 24 hours, so that the wet weight of the insoluble components of the thallus is reduced by 64.62 percent compared with the strain which expresses the beta-glucanase only, and is reduced by 67.83 percent compared with the strain which expresses the beta-mannase only. The two enzyme combinations have the function synergistic promotion effect, and can fully hydrolyze the water-insoluble glucan and mannan in the cell wall into water-soluble oligosaccharide.
Example 3: fermentation method for preparing yeast zymolyte by using fermentation tank
(1) Seed culture
(1) Drawing lines on YPD plates containing 1000ug/ml bleomycin by frozen engineering bacteria X33/bman-bglA at-80 ℃, culturing for about 72 hours at 28 ℃, picking up a monoclonal in 5ml BMGY culture medium, culturing for 16-24 hours at 30 ℃ under 250r/min in a shaking way;
(2) 200 μl of the seed solution was inoculated into 200ml BMGY medium (1000 ml conical flask) and 5 flasks were used as seed solutions for upper tank at 30℃for 250r/min for 12-14 h until OD600 = 2-6.
(2) Glycerol enrichment culture
(1) Adjusting equipment: calibrating a pH electrode and an oxygen dissolving electrode of the fermentation tank, and calibrating the flow of the peristaltic pump;
(2) preparing 60L BMGY culture medium, adding into a 100L fermentation tank, a 121 ℃ high-pressure sterilization culture medium for 30min, a fermentation tank and a pipeline;
(3) when the culture solution in the fermentation tank is cooled to 30 ℃, 28% ammonia water is used for regulating the pH value of the culture medium to 5.0, and then PTM1 microelements are added according to the proportion of 4.0ml PTM1/L BMGY;
(4) adding the shake flask culture strain into a fermentation tank, starting fermentation tank culture, wherein the first stage is glycerol culture amplification thallus, the parameters of the fermentation tank are respectively set to be 500-800 r/min, the pressure in the tank is 9psi, the temperature is 30 ℃, the DO value (dissolved oxygen) is set to be more than 20%, and the fermentation tank is controlled in a P-I-D mode;
(5) when the DO value rises to near 100% (about 30 h); indicating that the glycerol in the culture solution is depleted, and switching to a glycerol supplementing stage to further increase the density of the thalli, and measuring the wet weight of the thalli, wherein the wet weight of the thalli is about 150 g/L;
(6) adding PTM1 microelements into 50% glycerol after autoclaving according to the proportion of 12ml PTM1 microelements per liter, mixing uniformly, adding into a fermentation tank at the rate of 18.0ml/h/L until the thallus wet weight reaches 380-400 g/L;
(7) after stopping adding glycerol, observing that DO value rises to nearly 100%, continuously maintaining glycerol starvation state for 60min, and transferring to methanol induction expression stage;
(3) Methanol-induced expression of beta-glucanase and beta-mannanase
(1) Methanol was added to the fermenter at a rate of 3.0ml/h/L to induce expression, and this low rate was maintained for 3h to adapt the yeast to an environment where methanol was the sole carbon source. During the period, DO becomes unstable and fluctuates greatly, and after the yeast adapts to the environment, DO remains stable and the low-rate methanol is continuously added for 2 hours;
(2) the rate of adding methanol is increased to 8.0ml/h/L, and the final concentration of the methanol is maintained to be 0.5% at the rate, and the adding of the methanol is stopped;
(3) after the induction expression is started, sampling is carried out for 1 time every 4 hours, the wet weight of thalli is measured, and the growth state of saccharomycetes is analyzed;
(4) and (3) carrying out induced fermentation for 24 hours, wherein the wet weight of the thalli is reduced to about 100g/L, and compared with 20 hours, the thalli is not obviously reduced, and the fermentation is ended. A small amount of supernatant was left and stored at 4 ℃ for later detection. After fermentation, the fermentation broth is dried by a spray dryer to be processed into powder, and the powder is packaged in plastic bags to prepare the yeast zymolyte feed additive.
As can be seen from FIG. 2, the wet weight of the cells continued to increase for 8 hours before the X33/bman-bglA yeast was cultured with methanol, and then decreased to 104g/L for 20 hours and not substantially decreased for 99g/L for 24 hours. The zymolysis is induced for 20 hours to fully hydrolyze the saccharomycete cell wall polysaccharide into water-soluble oligosaccharide.
Example 4: effect of Yeast hydrolysate feed additive on Chicken growth Property
The beta-glucanase and beta-mannase mutant yeast zymolyte feed additive is respectively added into basic feed according to different proportions and fully and uniformly stirred. The weight of the feed additive is as follows: three feeds are prepared according to the weight of the basic feed, namely 1:100, 1:1000 and 1:10000. And adding unmutated beta-glucanase, unmutated beta-mannanase yeast zymolyte feed additive group and basic feed control group in the same proportion.
700 healthy white feather broilers at 1 day old are randomly divided into 7 groups, 100 broilers/group, and are kept separately;
8 per day: 00 and 16:00 feeding is carried out once, free feeding and drinking are carried out, the test period is 40 days, the feeding period is according to the conventional immunization program of white feather broilers, immunization and feeding management are carried out, the growth condition of the tested chickens is observed every day, and the feed-meat ratio of the chickens is counted after the experiment.
TABLE 1 Effect of different proportions of Yeast enzymatic hydrolysate feed additive feeding on white feather broiler growth
As can be seen from Table 1, the feed additive of the beta-glucanase and beta-mannase mutant yeast zymolyte prepared by the invention is added into basic feed according to the proportion of 1:100, 1:1000 and 1:10000 to feed white feather broilers, can obviously reduce the feed-meat ratio, and has obviously better effect than that of the feed additive of the non-mutated beta-glucanase and non-mutated beta-mannase yeast zymolyte. The yeast zymolyte feed additive of the beta-glucanase and the beta-mannase mutant has equivalent addition effect according to the proportion of 1:100 to 1:1000, so that the mass ratio of the addition amount of the yeast zymolyte feed additive in the feed is 1:1000, and the broiler raising benefit can be increased by adding the feed additive. And the diarrhea symptoms of the chicks can be improved by feeding the chicks with the mixture according to the dosage of 1:1000 or more, thereby being beneficial to the health of the chicks.
Example 5: effect of Yeast hydrolysate feed additive on chicken immunity
(1) The mass ratio of the addition amount of the beta-glucanase to the beta-mannase mutant yeast zymolyte feed additive in the feed is 1:1000, and the feed additive is added into the basic feed and fully and uniformly stirred. And adding unmutated beta-glucanase, unmutated beta-mannanase yeast zymolyte feed additive group and basic feed control group in the same proportion.
(2) Randomly dividing 60 SPF chickens with 1 day age into 3 groups, and raising 20 SPF chickens per group in isolation; the Newcastle disease inactivated vaccine is injected into the neck by subcutaneous injection, and each dose is 0.3ml (1 feather fraction). The serum was isolated from each of the groups collected 15 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months after immunization, HI antibodies were detected, and geometric mean of HI antibodies for each group was calculated.
(3) 8 per day: 00 and 16:00 feeding is carried out once, the food is taken and drunk freely, and the test period is 6 months.
As can be seen from FIG. 3, the beta-glucanase and beta-mannase mutant yeast zymolyte feed additive prepared by the invention is added into basic feed according to the mass ratio of 1:1000 to feed SPF chickens, the geometric average value of HI antibody after immunization of the Newcastle disease inactivated vaccine is 1.8 titer higher than that of a basic feed control group, and 1 titer higher than that of the non-mutated beta-glucanase and non-mutated beta-mannase yeast zymolyte feed additive group. The peak value of HI antibody was significantly higher in the enzyme mutant yeast zymolyte group than in the other two groups 1 month after immunization. 6 months after immunization, the control group HI antibody had decayed to 3.6, the unmutated beta-glucanase, unmutated beta-mannanase yeast enzyme diet supplement group HI antibody had decayed to 5.1, and the enzyme mutant yeast enzyme group still maintained a higher HI antibody of 6.4. Therefore, the yeast zymolyte feed additive can improve the immune antibody titer of the vaccine by mixing the feed additive according to the mass ratio of 1:1000.
Claims (2)
1. The yeast cell wall degrading enzyme is characterized by comprising a beta-glucanase mutant and a beta-mannanase mutant, wherein the amino acid sequence of the beta-glucanase mutant is SEQ ID NO.1, and the amino acid sequence of the beta-mannanase mutant is SEQ ID NO.2.
2. A feed additive containing yeast zymolyte is characterized in that the yeast zymolyte is prepared by expressing beta-glucanase and beta-mannase by recombinant pichia pastoris and performing synergistic enzymolysis on autologous cell walls; the amino acid sequence of the beta-glucanase is SEQ ID NO.1, and the amino acid sequence of the beta-mannase is SEQ ID NO.2; the Trp at positions 176 and 183 of the amino acid sequence of the beta-glucanase is mutated to Ala; the Trp at positions 196, 198 and 199 of the amino acid sequence of the beta-mannanase is mutated to Ala; the mass ratio of the addition amount of the feed additive in the feed is 1:1000; the feed additive can be used for improving diarrhea symptoms of chickens, reducing feed conversion ratio of chickens and improving vaccine immune antibody titer.
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