CN116850267B - Yeast cell wall-antibacterial peptide A3 composite preparation and preparation method and application thereof - Google Patents
Yeast cell wall-antibacterial peptide A3 composite preparation and preparation method and application thereof Download PDFInfo
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- CN116850267B CN116850267B CN202310858098.3A CN202310858098A CN116850267B CN 116850267 B CN116850267 B CN 116850267B CN 202310858098 A CN202310858098 A CN 202310858098A CN 116850267 B CN116850267 B CN 116850267B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/062—Ascomycota
- A61K36/064—Saccharomycetales, e.g. baker's yeast
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
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- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/005—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor after treatment of microbial biomass not covered by C12N1/02 - C12N1/08
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
- C12N1/063—Lysis of microorganisms of yeast
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
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Abstract
The invention discloses a yeast cell wall-antibacterial peptide A3 composite preparation and a preparation method and application thereof, and belongs to the technical field of high-value utilization of yeast. The yeast cell wall-antibacterial peptide A3 composite preparation is prepared from yeast cell walls and antibacterial peptide A3 according to the mass ratio of (8-10): 1, compounding; the yeast cell wall is prepared by the following method: solid-liquid separation is carried out on the saccharomyces cerevisiae fermentation liquid, and bacterial precipitation is collected; diluting the bacterial precipitate to obtain bacterial liquid, regulating pH of the bacterial liquid to 6.0-7.0, adding papain, controlling temperature to 50-60 ℃, and treating for 12-36h; adding light calcium carbonate into the treated system, uniformly mixing, and drying to obtain the yeast cell wall. The yeast cell wall-antibacterial peptide A3 composite preparation can improve the antibacterial activity of the antibacterial peptide A3 after pepsin and trypsin act; and has synergistic effect in improving animal organism growth performance, antioxidant capacity, immunity index, etc.
Description
Technical Field
The invention relates to the technical field of high-value utilization of yeast, in particular to a yeast cell wall-antibacterial peptide A3 composite preparation, a preparation method and application thereof.
Background
In recent years, with the transformation of the breeding industry to intensification, scale and high density, the raised livestock and poultry are easy to suffer from various stress influences, mainly characterized by reduced immunity, easy morbidity and high death rate, and the quality of livestock products is influenced, so that the breeding benefit can be obviously reduced. The problems are solved by means of feed additives, and the method becomes a conventional way.
The yeast cell wall is a brand new natural green additive, and mainly comprises beta-glucan and mannan. The beta-glucan has an immunoregulation function, is non-specific immunostimulating polysaccharide, can be specifically combined with immune cell receptors of organisms, stimulates B cells, T cells and natural killer cells to generate a large number of macrophages, and further regulates and controls the organism immunity. The mannans can increase humoral immunity and cellular immunity of animals, regulate intestinal flora balance, combine and adsorb exogenous pathogenic bacteria, and have the functions of resisting radiation, resisting oxidation, resisting tumor and the like.
Since the nutrients of the yeast cells are surrounded by thick cell walls, it is important to study efficient wall breaking means in order to fully utilize the rich nutrient resources in the yeast cells. The breaking of the yeast wall is to break the yeast cell wall and release the nutrient substances therein. Common methods for breaking the wall of the yeast include an enzymatic method, an ultrasonic method, a high-pressure homogenizing method, an autolysis method and the like, but the existing methods for breaking the wall of the yeast still have the problems of long wall breaking time, low wall breaking rate, low dissolution rate of zymosan and the like.
Antimicrobial peptides, which are an important component of the natural defense system of most organisms against invading pathogens, have a unique mechanism of antimicrobial action, rapidly disinfect and are not prone to eliciting bacterial resistance. Antibacterial peptides act mainly by affecting the cytoplasmic membrane, so bacteria must change their membrane composition and structure to develop resistance to the antibacterial peptide, but this can also cause serious damage to the bacterial cells themselves. The outer layer of the bacterial cell membrane is rich in anionic phospholipids, and the zwitterionic lipid of the mammalian cell membrane is rich, which is the main reason that the antibacterial peptide can be antibacterial without damaging the mammalian cell. Therefore, the antibacterial peptide is a biological medicine with great development potential. The applicant screens a strain of bacillus polymyxa (CN 110484467B) for producing antibacterial peptide in earlier research, which can naturally and highly produce antibacterial peptide with small molecular weight (not more than 5 kDa), wherein the polypeptide with the molecular weight not more than 5kDa accounts for more than 65% of the volume of the supernatant of the bacillus polymyxa protein, the polypeptide with the molecular weight of 5kDa-3kDa accounts for approximately 20%, the yield (protein concentration) of the polypeptide with the molecular weight of 5kDa-3kDa obtained after ultrafiltration concentration is up to 2.73mg/mL, the purification multiple of the polypeptide is up to 2.12, and the activity recovery rate is up to 19.65%. However, the antibacterial activity of the produced antibacterial peptide is reduced by 20% after pepsin action, and the antibacterial activity is reduced by 18% after trypsin action, so that the oral application effect of the antibacterial peptide is still to be further improved.
For the combined use of yeast cell walls and antibacterial peptides, patent CN 104782909A discloses an antibacterial peptide mold remover for feed, which is produced by constructing yeast engineering bacteria for fermentation, wherein a large amount of antibacterial peptides are expressed in the fermentation culture process, cell bodies of the yeast and expression products of the antibacterial peptides are simultaneously contained in the final fermentation culture products, and after autolysis wall breaking of the cell bodies, the yeast cell walls and the antibacterial peptides are separated, purified and collected to obtain mold remover products. However, this patent still uses the adsorption property of the yeast cell wall and the bactericidal function of the antibacterial peptide, and does not produce a synergistic effect; moreover, this patent only proposes a design concept, and the effect thereof is not verified.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a yeast cell wall-antibacterial peptide A3 composite preparation, and a preparation method and application thereof. The invention adopts a wall breaking method combining autolysis and enzymolysis to prepare the yeast cell wall; then the prepared yeast cell wall and the preservation number are CCTCC NO: the antibacterial peptide A3 produced by the bacillus polymyxa of M2019530 is compounded, so that the antibacterial activity of the antibacterial peptide A3 after pepsin and trypsin act can be improved; and has synergistic effect in improving animal organism growth performance, antioxidant capacity, immunity index, etc.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a yeast cell wall-antibacterial peptide A3 composite preparation, which is prepared by compounding a yeast cell wall and an antibacterial peptide A3 according to a mass ratio of (8-10): 1;
the yeast cell wall is prepared by the following method:
and (3) the preservation number CCTCC NO: inoculating Saccharomyces cerevisiae M2015209 into yeast liquid culture medium, shake culturing at 30deg.C for 24 hr to obtain yeast fermentation liquid, solid-liquid separating, diluting thallus precipitate to obtain bacterial liquid, regulating pH of bacterial liquid to 6.0-7.0, adding papain, controlling temperature to 50-60deg.C, and treating for 12-36 hr; adding light calcium carbonate into the treated system, uniformly mixing, and drying to obtain the yeast cell wall.
Wherein, the preservation number is CCTCC NO: saccharomyces cerevisiae of M2015209 is described in another patent of the applicant, CN 105062905B.
Preferably, the bacterial cell precipitate is diluted with water to a mass concentration of 25 to 35%. The dilution concentration of the bacterial precipitate is verified by fumbling, the dilution concentration is too high, bacterial liquid is too viscous, and the bacterial precipitate is not easy to stir and mix uniformly, so that the wall breaking rate is affected; the dilution concentration is too low, the moisture content is high, the bacteria content is low, the volume addition amount of papain is high, and the wall breaking cost is increased. The bacterial precipitate is diluted to 25-35% of mass concentration, and the wall breaking effect and the wall breaking cost can be comprehensively balanced.
Preferably, the addition amount of the papain is 0.5-2 per mill of the volume of the bacterial liquid.
Preferably, the addition amount of the light calcium carbonate is 5% of the volume of the bacterial liquid. The addition of light calcium carbonate can prevent the yeast cell wall from pasting tower during the spray drying process caused by high sugar content.
Preferably, the composition of the yeast liquid medium is as follows: glucose 2wt%, yeast extract 0.5wt%, peptone 1wt%, potassium dihydrogen phosphate 0.2wt% except water.
Preferably, the antibacterial peptide A3 is prepared by the following method:
the preservation number is CCTCC NO: inoculating M2019530 bacillus polymyxa into a bacillus liquid culture medium, and standing and culturing at 37 ℃ for 20 hours to obtain bacillus polymyxa fermentation broth; centrifuging the fermentation liquor of the bacillus polymyxa, collecting supernatant, respectively selecting ultrafiltration membranes with molecular retention of 5kDa and 3kDa for ultrafiltration, collecting components with molecular weight of 5kDa-3kDa, and freeze-drying to obtain the antibacterial peptide A3.
More preferably, the bacillus liquid medium comprises the following components: corn steep liquor 0.5wt%, peptone 5wt%, glucose 3wt%, calcium chloride 0.25wt% and water for the rest.
Wherein, the preservation number is CCTCC NO: m2019530 is described in another patent of the applicant, CN 110484467B.
Preferably, the yeast cell wall-antibacterial peptide A3 composite preparation is prepared by compounding yeast cell walls and antibacterial peptide A3 according to a mass ratio of 9:1.
In a second aspect of the present invention, there is provided a method for preparing the yeast cell wall-antimicrobial peptide A3 complex formulation, comprising the steps of:
and uniformly mixing the yeast cell wall prepared by the method and the antibacterial peptide A3 according to the mass ratio.
In a third aspect of the present invention, there is provided the use of the yeast cell wall-antimicrobial peptide A3 complex formulation as described above in (1) or (2) below:
(1) The activity retention rate of the antibacterial peptide A3 after the effect of pepsin and trypsin is improved;
(2) The product for improving the growth performance, oxidation resistance and immunity of the animal body is prepared.
The invention has the beneficial effects that:
(1) The invention adopts a wall breaking method of autolysis and enzymolysis simultaneous treatment, and utilizes endogenous enzymes contained in live yeast to hydrolyze macromolecular substances in the thallus; simultaneously, exogenous protease is added to enzymolysis of a protein layer of a yeast cell wall, so that a large amount of mannans and glucans are released, a proper condition for exciting enzyme activity is created, the wall breaking effect of the yeast cell wall is enhanced, and the yeast polysaccharide is fully utilized. Compared with the existing yeast wall breaking method, the method improves the wall breaking rate of the yeast cell wall and the dissolution rate of the yeast polysaccharide; in addition, compared with the yeast cell walls prepared by other wall breaking methods, the yeast cell walls prepared by the method have obvious effect of improving the growth immunity of animal organisms.
(2) The preservation number is CCTCC NO: after pepsin and trypsin act on the antibacterial peptide produced by the bacillus polymyxa of M2019530, the antibacterial activity of the antibacterial peptide is reduced, and the application of the antibacterial peptide as an oral product is affected. According to the invention, unexpected discovery is carried out after the yeast cell wall and the antibacterial peptide A3 are compounded, so that the activity retention rate of the antibacterial peptide A3 after pepsin and trypsin act can be improved.
(3) Compared with the single use of the yeast cell wall and the antibacterial peptide A3, the invention has the advantages of improving the growth of organisms, resisting oxidization, improving the immunity, inhibiting pathogenic bacteria and the like, and has synergistic effect.
Drawings
Fig. 1: microscopic examination diagrams of the wall breaking of the yeast autolysis experiments at different temperatures; in the figures, A-D are microscopic images of autolysis treatment at 30℃and 40℃and 50℃and 60℃respectively.
Fig. 2: microscopic examination diagrams of the wall breaking of the yeast autolysis experiments at different times; in the figures, A-G are microscopic images of autolysis treatment under the conditions of 0h, 24h, 48h, 72h, 96h, 120h and 144h respectively.
Fig. 3: microscopic examination images of the autolysis experiment wall breaking of yeasts with different pH values; in the figures, A-F are microscopic images of autolysis treatments at pH values of 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, respectively.
Fig. 4: microscopic examination diagrams of wall breaking of the yeast autolysis experiment of the inducer NaCl with different addition amounts; in the figures, A-E are microscopic images of autolysis treatment under the conditions that the addition amount of NaCl is 0%, 1%, 2%, 3% and 4% of the weight of the bacterial liquid respectively.
Fig. 5: microscopic images of wall breaking of yeasts by different enzyme preparations; in the figure: a is a microscopic image of high-temperature inactivation treatment; B-F are microscopic examination images of enzymolysis treatment under the conditions of adding 1 permillage neutral proteinase, 1 permillage alkaline proteinase, 1 permillage papain, 1 permillage beta-glucanase and 1 permillage mannanase into the high-temperature inactivated bacteria liquid volume respectively.
Fig. 6: wall breaking microscopic examination chart of autolysis and single wall breaking enzyme investigation experiment; in the figure, A-E are microscopic images of autolysis+1 mill neutral protease, autolysis+1 mill alkaline protease, autolysis+1 mill papain, autolysis+1 mill beta-glucanase and autolysis+1 mill mannanase.
Fig. 7: wall breaking microscopic examination chart of autolysis and composite wall breaking enzyme investigation experiment; in the figure, A-G are respectively autolyzed +1 permillage papain and autolyzed +1 permillage beta-glucanase; autolyzed + 1%o mannanase; autolysis + 0.5%o papain + 0.5%o beta-glucanase; autolysis + 0.5%o papain + 0.5%o mannanase; autolysis +0.5%o beta-glucanase +0.5%o mannanase; microscopic image of autolysis + enzymatic treatment under autolysis + 0.4%o papain + 0.3%o beta-glucanase + 0.3%o mannanase conditions.
Fig. 8: microscopic examination of wall breaking by autolysis and enzymolysis of yeast at different temperatures; in the figures, A-E are microscopic images of autolysis +1%o papain treatment at 25 ℃, 40 ℃, 50 ℃,60 ℃ and 70 ℃.
Fig. 9: microscopic examination images of autolysis and enzymolysis wall breaking of yeasts at different pH values; in the figures, A-F are microscopic images of the treatment with papain at pH original, 5.5, 6.0, 6.5, 7.0, and 7.5, respectively, autolyzed +1%o.
Fig. 10: microscopic examination images of autolysis and enzymolysis wall breaking of yeast at different treatment times; in the figures, A-E are microscopic images of autolyzed +1 permillage papain under the conditions of 0h, 12h, 24h, 36h and 48h respectively.
Fig. 11: microscopic examination pictures of wall breaking by autolysis and enzymolysis of yeasts with different papain addition amounts; in the figure, A-D are microscopic images of autolyzed +0.5 permillage papain, autolyzed +1 permillage papain, autolyzed +2 permillage papain and autolyzed +3 permillage papain respectively.
Fig. 12: microscopic examination graphs of the autolysis enzymolysis of 5 ton yeast fermentation tanks for different wall breaking time; in the figures, A-E are microscopic images of treatments 0h, 12h, 24h, 36h and 48h, respectively.
Fig. 13: the activity of the antibacterial peptide A3 after pepsin and trypsin are acted is kept to examine the result.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and are commercially available. Wherein:
yeast liquid medium: glucose 2wt%, yeast extract 0.5wt%, peptone 1wt%, potassium dihydrogen phosphate 0.2wt% except water.
Bacillus liquid medium: corn steep liquor 0.5wt%, peptone 5wt%, glucose 3wt%, calcium chloride 0.25wt% and water for the rest.
Papain (20U/g), neutral protease (20U/g), alkaline protease (20U/g), purchased from Nanning Donghenghua Biotechnology Inc.; beta-glucanase (20U/g), available from Sichuan green gallop Biotechnology Co., ltd; mannanase (20U/g) was purchased from Hebei Qiansheng biotechnology Co.
Example 1: optimization investigation of yeast wall breaking method
The preservation number is CCTCC NO: saccharomyces cerevisiae M2015209 is inoculated in a yeast liquid culture medium, and is subjected to shaking culture at 30 ℃ for 24 hours to obtain bacterial liquid. The bacterial liquid is subjected to wall breaking treatment by adopting an autolysis wall breaking method, an enzymolysis wall breaking method and an autolysis enzymolysis method. The wall breaking effect of the yeast is observed by adopting a gram staining method and microscopic examination, and the wall breaking rate of the yeast cells is measured according to the following method: and (3) collecting yeast liquid before and after wall breaking, diluting by proper times, and respectively performing cell count under an optical microscope by using a blood cell counting plate.
Yeast cell wall breaking rate (%) = [ (M-N)/M ] ×100%
Wherein M is the number of intact yeast cells before wall breaking; n, number of intact yeast cells after wall breaking.
1. Autolysis wall breaking
(1) Autolysis temperature investigation
The bacterial solutions were subjected to autolysis treatment at 30℃and 40℃and 50℃and 60℃for 24 hours, and the cell wall-breaking rates of yeast cells at different autolysis temperatures were examined, and the results are shown in Table 1 and FIG. 1.
Table 1: wall breaking rate of yeast autolysis experiment at different temperatures
As can be seen from Table 1, the cell wall breaking rate of yeast was high at 60℃autolysis temperature, so that the next experiment was conducted at 60 ℃.
(2) Autolysis time investigation
The bacterial solutions are subjected to autolysis treatment for 0h, 24h, 48h, 72h, 96h, 120h and 144h respectively at 60 ℃, and the wall breaking rate of the yeast cells under different autolysis time conditions is detected, and the results are shown in Table 2 and figure 2.
Table 2: wall breaking rate of yeast autolysis experiment at different time
As can be seen from Table 2, the autolysis wall-breaking rate of yeast was increased with the increase of time, but the time was too long, and the autolysis time was once set to 72 hours, and the next experiment was performed.
(3) Autolysis pH investigation
The pH values of the bacterial solutions are respectively adjusted to be 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, then autolysis treatment is carried out for 72 hours at 60 ℃, and the wall breaking rate of the yeast cells under different autolysis pH values is detected, and the results are shown in Table 3 and figure 3.
Table 3: wall breaking rate of autolysis experiment of yeasts with different pH values
As can be seen from Table 3, when the pH value is 8.0, the autolysis wall-breaking effect of the yeast is optimal, and the pH value of 8.0 is selected for the next experiment.
(4) Investigation of inducer with different addition amounts
The pH value of the bacterial liquid is regulated to 8.0, different amounts of autolysis inducer NaCl are respectively added, the addition amounts of NaCl are respectively 0%, 1%, 2%, 3% and 4% of the weight of the bacterial liquid, then autolysis treatment is carried out for 72 hours at 60 ℃, and the wall breaking rate of the yeast cells under the autolysis condition of the inducer with different addition amounts is detected, and the results are shown in Table 4 and figure 4.
Table 4: wall breaking rate of inducer NaCl yeast autolysis experiment with different addition amounts
As can be seen from Table 4, the addition of inducer NaCl had no significant effect on the results of the autolysis wall-breaking rate of yeast, and was not added once.
From the above experimental results, the conditions for autolysis optimization of yeast are as follows: 60 ℃, pH8.0, no inducer is added, and autolysis is carried out for 72h; the wall breaking rate of yeast cells is about 45%, but the autolysis time is too long, and it is hoped that the time can be shortened by other wall breaking methods.
2. Enzymolysis wall breaking
After the yeast liquid is subjected to high-temperature inactivation treatment at 121 ℃,1 permillage neutral protease, 1 permillage alkaline protease, 1 permillage papain, 1 permillage beta-glucanase and 1 permillage mannanase which are respectively added into the volume of the yeast liquid, and shaking is carried out for 24 hours by a shaking table at 50 ℃, so that the wall breaking rate of the yeast cells is detected, and the results are shown in Table 5 and figure 5.
Table 5: influence of different enzyme preparations on wall breaking of Yeast
As can be seen from Table 5 and FIG. 5, the different proteases have good effect on breaking the wall of the yeast, the wall breaking effect of papain is optimal, and the wall breaking rate of the yeast cell is 57.69%. However, after the yeast is inactivated, endogenous enzymes contained in the live yeast cannot be utilized, so that microscopic examination results can show that the individual yeasts are obviously reduced, but cell walls cannot be completely broken, and the wall breaking rate cannot achieve an ideal effect. Therefore, the activity of the saccharomycetes is maintained, and the next experiment is carried out by adopting a wall breaking method combining autolysis and enzymolysis based on proper conditions of autolysis of the saccharomycetes.
3. Autolytic enzymatic binding
(1) Autolysis + single wall breaking enzyme investigation
1 per mill neutral proteinase, 1 per mill alkaline proteinase, 1 per mill papain, 1 per mill beta-glucanase and 1 per mill mannanase are respectively added into the saccharomycete liquid, and shaking table at 50 ℃ is carried out for 24 hours, so that the cell wall breaking rate of the saccharomycete is detected, and the results are shown in table 6 and figure 6.
Table 6: influence of autolysis+Single wall breaking enzyme on Yeast wall breaking
As can be seen from Table 6, the different proteases have good effect on breaking the wall of the yeast, the wall breaking effect of the papain is optimal, and the wall breaking rate of the yeast cells is highest and is 87.69% when papain is added in an amount of 1 per mill of the volume of the bacterial liquid.
(2) Autolysis and composite wall breaking enzyme investigation
1%o papain, 1%o beta-glucanase, 1%o mannanase, 0.5%o papain+0.5%o beta-glucanase, 0.5%o papain+0.5%o mannanase, 0.5%o beta-glucanase+0.5%o mannanase, 0.4%o papain+0.3%o beta-glucanase+0.3%o mannanase are added into yeast liquid respectively, and shaking is carried out at 50 ℃ for 24 hours, so that the wall breaking rate of yeast cells is detected, and the results are shown in table 7 and figure 7.
Table 7: influence of autolysis and composite wall breaking enzyme on wall breaking of yeast
As can be seen from table 7, the wall breaking effect of single papain is better than that of complex enzyme, so papain was selected for the next experiment.
(3) Investigation of different temperatures
Adding papain 1%of the volume of the bacterial liquid into the yeast liquid, respectively placing into 25 ℃, 40 ℃, 50 ℃,60 ℃ and 70 ℃ shaking table for 24 hours, and detecting the wall breaking rate of the yeast cells, wherein the results are shown in Table 8 and figure 8.
Table 8: influence of different temperatures on autolysis and enzymolysis wall breaking of yeast
As can be seen from Table 8, the yeast at 60℃has the highest enzymolysis wall breaking rate and the best wall breaking effect, and the next experiment is carried out at 60 ℃.
(4) Investigation of different pH values
The pH values of the yeast liquid are respectively adjusted to 5.5, 6.0, 6.5, 7.0 and 7.5, papain with the volume of 1%o of the bacterial liquid is added into the bacterial liquid, and the bacterial liquid is put into a shaking table at 60 ℃ for shaking for 24 hours, so that the wall breaking rate of the yeast cells is detected, and the results are shown in Table 9 and figure 9.
Table 9: influence of different pH values on autolysis and enzymolysis wall breaking of yeast
As can be seen from Table 9, the yeast cell wall-breaking rate was highest at pH6.5, and pH6.5 was selected for the next experiment.
(5) Investigation of different treatment times
Regulating pH value of yeast liquid to 6.5, adding papain 1%of the volume of the liquid into the liquid, respectively shaking in a shaker at 60deg.C for 0h, 12h, 24h, 36h, and 48h, and detecting yeast cell wall breaking rate, wherein the results are shown in Table 10 and figure 10.
Table 10: influence of different treatment times on autolysis and enzymolysis wall breaking of yeast
As can be seen from Table 10, the wall breaking rate of the yeast cells can reach 77.08% at 12 hours, 95.19% at 24 hours, 96.77% at 36 hours, 98.67% at 48 hours, and the autolysis and enzymolysis wall breaking time is selected to be 24 hours.
(6) Investigation of the different papain addition levels
Regulating pH value of yeast liquid to 6.5, adding papain 0.5%o, 1%o, 2%o, and 3%o of the volume of the bacterial liquid into the bacterial liquid, respectively, shaking at 60deg.C for 24 hr, and detecting yeast cell wall breaking rate, wherein the results are shown in Table 11 and figure 11.
Table 11: influence of papain on autolysis and enzymolysis wall breaking of yeast
As can be seen from Table 11, the wall breaking rate of papain at 60 ℃ for 24 hours and pH6.5 can reach 90.48% at 0.5%o, and the wall breaking rate of papain at 1%o can reach 94.23%. In view of cost consideration, the conditions for determining the autolysis and enzymolysis wall breaking of the yeast are as follows: 0.5 per mill papain, 24h,60 ℃ and pH6.5, and the wall breaking rate can reach 90.48 percent.
Example 2: autolysis enzymolysis amplification test of saccharomycetes
1. Test method
The preservation number is CCTCC NO: the Saccharomyces cerevisiae activating solution of M2015209 is inoculated into a 5 ton fermentation tank filled with a yeast liquid culture medium according to the volume ratio of 5 percent, and is fermented and cultured for 24 hours at the temperature of 30 ℃ to obtain fermentation liquor. And (3) carrying out solid-liquid separation on the fermentation liquid, collecting bacterial precipitate, diluting the bacterial precipitate with sterile water to obtain bacterial liquid with the mass concentration of 30%, regulating the pH value of the bacterial liquid to 6.5, adding papain with the bacterial liquid volume of 0.5 per mill into the bacterial liquid, controlling the temperature to 60 ℃, stirring once every 12 hours, sampling once, and carrying out microscopic examination on the bacterial liquid to determine the wall breaking rate of the yeast cells. And calculating the yeast polysaccharide dissolution rate by using a test method of the yeast polysaccharide dissolution rate.
The method for testing the dissolution rate of the zymosan is as follows:
(1) Drawing a standard curve: accurately weighing 0.01g of anhydrous glucose which is dried to constant weight by an oven at 80 ℃, dissolving the glucose in a small amount of distilled water, and transferring the glucose into a 100mL volumetric flask for constant volume. This is a glucose standard, wherein the glucose content is 0.1mg/mL. Glucose standard solutions 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6mL are sequentially taken and respectively placed in 9 test tubes with plugs, and distilled water of 2.0, 1.8, 1.6, 1.4, 1.2, 1.0, 0.8, 0.6 and 0.4mL is sequentially added into each test tube to form a 2mL solution system. 1.0mL of 6.25% phenol solution is added into each test tube, 5.0mL of concentrated sulfuric acid is rapidly added dropwise after shaking, shaking is carried out, standing is carried out for 5min at room temperature, boiling water bath is carried out for 25min, and the mixture is taken out and cooled to room temperature. The mixture was placed in a constant quartz cuvette and absorbance was measured at 490nm using an ultraviolet spectrophotometer. The glucose standard curve equation is: y=15.2x+0.077 (R 2 = 0.9901), in which: y, D 490nm A value; x, glucose concentration (mg/mL).
(2) Sample measurement: centrifuging the bacterial liquid sample at 4000r/min for 15min, collecting supernatant as zymosan dissolution liquid, diluting the zymosan dissolution liquid by a proper multiple, sucking 2.0mL of dilution liquid, placing the dilution liquid in a test tube with a plug, and drawing the subsequent test method by the same standard curve.
Yeast polysaccharide dissolution rate (mg/g) =polysaccharide concentration (mg/mL) ×polysaccharide dissolution volume (mL)/corresponding yeast mud mass (g).
2. Test results
The results of the detection of the wall breaking rate of the yeast cells and the dissolution rate of the yeast polysaccharide are shown in Table 12; the mirror image is shown in fig. 12.
Table 12:5 ton fermentation tank autolysis enzymolysis yeast cell wall breaking rate and yeast polysaccharide dissolution rate at different times
As can be seen from Table 12, the autolysis and enzymolysis are combined to break the wall for 12 hours, the dissolution rate of the zymosan is 151.291mg/g, and the wall breaking rate of the yeast cell is 73.18%; breaking cell wall for 24h, wherein the dissolution rate of zymosan is 160.852mg/g, and the wall breaking rate of yeast cell is 95.61%; breaking wall for 36h, wherein the dissolution rate of zymosan is 179.935mg/g, and the wall breaking rate of yeast cells is 97.68%; the wall is broken for 48 hours, the dissolution rate of the zymosan is 183.419mg/g, and the wall breaking rate of the yeast cell is 98.97%.
Example 3: preparation of Yeast cell wall
The preservation number is CCTCC NO: the Saccharomyces cerevisiae M2015209 is inoculated into a yeast liquid culture medium, the inoculum size is 5 percent of the volume of the yeast liquid culture medium, and fermentation culture is carried out for 24 hours at 30 ℃ to obtain fermentation liquor. Separating solid and liquid of the fermentation liquid, collecting bacterial precipitate, diluting the bacterial precipitate with sterilized water to obtain bacterial liquid with mass concentration of 30%, regulating pH value of the bacterial liquid to 6.5, adding papain with bacterial liquid volume of 0.5%o, controlling temperature at 60deg.C, and treating for 24 hr; adding light calcium carbonate (light calcium) into the treated system, wherein the addition amount of the light calcium carbonate is 5% of the volume of the bacterial liquid, uniformly mixing, and spray drying to prepare the yeast cell wall.
Example 4: preparation of antibacterial peptide A3
Inoculating the bacillus polymyxa with the preservation number of CCTCC No. M2019530 into a bacillus liquid culture medium, wherein the inoculum size is 5% of the volume of the bacillus liquid culture medium, and performing stationary culture for 20h at 37 ℃ to obtain bacillus polymyxa fermentation broth; centrifuging the fermentation liquor of the bacillus polymyxa, collecting supernatant, respectively selecting ultrafiltration membranes with molecular retention of 5kDa and 3kDa for ultrafiltration, collecting components with molecular weight of 5kDa-3kDa, and freeze-drying to obtain the antibacterial peptide A3.
Example 5: preparation of yeast cell wall-antibacterial peptide A3 composite preparation
The yeast cell wall prepared in example 3 and the antibacterial peptide A3 prepared in example 4 are uniformly mixed according to the mass ratio of 9:1 to prepare the yeast cell wall-antibacterial peptide A3 composite preparation.
Comparative example 1: preparation of Yeast cell wall by enzymatic hydrolysis alone
The preservation number is CCTCC NO: the Saccharomyces cerevisiae M2015209 is inoculated into a yeast liquid culture medium, the inoculum size is 5 percent of the volume of the yeast culture medium, and fermentation culture is carried out for 24 hours at 30 ℃ to obtain fermentation liquor. Separating solid and liquid of fermentation liquid, collecting bacterial precipitate, diluting bacterial precipitate with sterilized water to obtain bacterial liquid with mass concentration of 30%, adjusting pH value of the bacterial liquid to 6.5, sterilizing at high temperature, cooling, adding papain with bacterial liquid volume of 0.5%o, controlling temperature at 60deg.C, and treating for 24 hr; adding light calcium carbonate into the treated system, wherein the addition amount of the light calcium carbonate is 5% of the volume of the bacterial liquid, uniformly mixing, and spray-drying to prepare the yeast cell wall.
Comparative example 2: preparation of Yeast cell wall by autolysis alone
The preservation number is CCTCC NO: the Saccharomyces cerevisiae M2015209 is inoculated into a yeast liquid culture medium, the inoculum size is 5 percent of the volume of the yeast liquid culture medium, and fermentation culture is carried out for 24 hours at 30 ℃ to obtain fermentation liquor. Separating solid and liquid of the fermentation liquid, collecting bacterial precipitate, diluting the bacterial precipitate with sterilized water to obtain bacterial liquid with mass concentration of 30%, adjusting pH value of the bacterial liquid to 6.5, controlling temperature to 60 ℃, and treating for 24h; adding light calcium carbonate into the treated system, wherein the addition amount of the light calcium carbonate is 5% of the volume of the bacterial liquid, uniformly mixing, and spray-drying to prepare the yeast cell wall.
The wall breaking rate of the yeast cell walls prepared in example 3 and comparative examples 1 to 2 was measured by the method of example 1, and the results are shown in Table 13.
Table 13: wall breaking rate of yeast cell walls prepared by different methods
From the results in table 13, it can be seen that: the invention adopts the wall breaking method combining autolysis and enzymolysis, and has remarkable synergy in the aspect of improving the wall breaking rate of yeast cells.
Test example 1: stability study of antibacterial peptide A3
1. Test method
The stability of the antimicrobial peptide A3 after pepsin and trypsin action was studied by the method described in patent CN 110484467a, and the specific method is as follows:
under the water bath condition of 37 ℃, pepsin and trypsin with the reaction concentration of 1mg/mL are respectively used for treating the antibacterial peptide A3 prepared in the example 4 and the yeast cell wall-antibacterial peptide A3 composite preparation prepared in the example 5 for 30min, and the antibacterial activity of various enzyme treatment liquids on escherichia coli CVCC1556 is measured. The antibacterial peptide A3 in the yeast cell wall-antibacterial peptide A3 composite preparation is consistent with the antibacterial peptide A3 alone in content by taking the antibacterial peptide A3 without enzyme treatment and the yeast cell wall-antibacterial peptide A3 composite preparation as a control, and the antibacterial activity is 100%.
2. Test results
As shown in FIG. 13, the activity of the antibacterial peptide A3 is reduced by 21% after pepsin action, and the activity of the antibacterial peptide A3 is reduced by 19% after trypsin action; the activity of the yeast cell wall-antibacterial peptide A3 composite preparation is reduced by 5% after pepsin action, and the activity of the yeast cell wall-antibacterial peptide A3 composite preparation is reduced by 3% after trypsin action.
The above results indicate that: the activity retention rate of the antibacterial peptide A3 after pepsin and trypsin are acted can be improved by compounding the yeast cell wall and the antibacterial peptide A3.
Test example 2: mouse test
1. Materials and methods
1.1 laboratory animals and groupings: male Kunming mice 126 (24 g.+ -. 2 g) were divided into 7 groups of 18 (3 parallel groups). The specific experimental groupings are shown in Table 14.
Table 14: experimental group of influence of yeast wall-broken powder and antibacterial peptide A3 on mouse immune function
Wherein: the active saccharomyces cerevisiae powder adopts commercial saccharomyces cerevisiae powder with the volume of 200 hundred million/g; the autolyzed wall-broken yeast powder adopts the yeast cell wall prepared in comparative example 2; the enzymolysis wall-broken yeast powder adopts the yeast cell wall prepared in comparative example 1; the autolysis and enzymolysis wall-broken yeast powder adopts the yeast cell wall prepared in the example 3; the antibacterial peptide A3 is the antibacterial peptide prepared in example 4; the autolyzed yeast cell wall + antibacterial peptide A3 adopts the yeast cell wall-antibacterial peptide A3 composite preparation prepared in example 5.
Mice of each treatment group were fed a basal diet (basal diet is a laboratory mouse feed purchased from experimental animals breeding limited company, atanga, songyue) during the test period, and after pre-feeding for 7d, the mice were fed 1 time per day, continuously fed for 21d, and tested for 28d in total, according to the amounts shown in table 14.
1.2 test methods
1.2.1 grouping of animals and dosing regimen: the 126 mice were assigned into 7 groups, namely CK group (as blank control), active saccharomyces cerevisiae group (as positive control), autolyzed yeast cell wall group, enzymatically hydrolyzed yeast cell wall group, autolyzed yeast cell wall group, antibacterial peptide A3 group, autolyzed yeast cell wall+antibacterial peptide A3 group; there were 18 mice per group (3 parallel groups). Mice were fed basal diet during the test period, pre-fed for 1 week, weighed and then subjected to the lavage test (amounts shown in table 14) for 3 weeks, weighing once a week. The mice were free to eat, drink water, and change litter every three days. The mice were sacrificed for a period of 24 hours before slaughtering, weighed after the end of the test on day 28, collected with blood by the eyeball removal method, sacrificed and dissected to collect thymus, spleen and liver and weighed, and the intestinal contents were taken and stored at-20 ℃ for later use. Placing the blood sample in a centrifuge tube for about 1h, peeling off the coagulated blood and the tube wall, separating out serum sufficiently, centrifuging for 10min at 3000r/min, collecting serum, and preserving at-20deg.C.
1.2.2 measurement of antioxidant index: the concentration of superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) in serum is detected by using a kit of Nanjing institute of biological engineering.
1.2.3 determination of immune index: detecting the concentration of interleukin 2 (IL-2) and immunoglobulin G (IgG) in serum by using an Shanghai enzyme-linked biological mouse ELISA kit; the concentration of secretory immunoglobulin (sIgA) in the intestinal contents was measured using an Shanghai enzyme-linked biological mouse ELISA kit.
1.2.5 determination of intestinal flora: taking 0.1g of each intestinal tract content, shaking up in 100mL of sterilized normal saline, taking 1mL of the intestinal tract content into a test tube, adding 9mL of sterilized 0.9% normal saline, shaking and mixing uniformly, and sequentially diluting according to 10 times of equal ratio. Wherein lactobacillus is inoculated on a lactobacillus selective medium (LBS) for culture, and colony counting is carried out after culturing for 48 hours in a biochemical incubator at 37 ℃; coli was inoculated on eosin-methylene blue agar medium and cultured in a biochemical incubator at 37℃for 24 hours, followed by colony counting. The number of flora is expressed as the logarithm of the total number of bacterial communities per gram of intestinal content [ (lg cfu/g) ].
2. Test results
2.1 Effect of Yeast cell wall and antibacterial peptide A3 on mouse body weight
Table 15: influence of Yeast cell wall and antibacterial peptide A3 on mouse body weight (g)
Note that: different lower case letters indicate significant differences in results (P < 0.05), the same letters indicate insignificant differences in results (P > 0.05). The table below is the same.
As can be seen from table 15, the weights of the groups tested were different from the gastric lavage 7d, and the weights of the 6 groups were significantly higher than those of the CK group, the 1 group, the 2 group, the 3 group, and the 5 group (P < 0.05); the stomach 14d,4 and 6 groups are significantly higher than the CK, 1, 2 and 5 groups (P < 0.05); the lavage 21d,4 and 6 groups were significantly higher than the other groups, and the 2 and 3 groups were significantly higher than the blank group (P < 0.05). It can be seen that the addition of the yeast cell wall can significantly improve the weight of animals, the influence of the independently added antibacterial peptide on the weight is small, and the effect of the added yeast cell wall and antibacterial peptide A3 composite preparation is optimal.
2.2 Effect of Yeast cell wall and antibacterial peptide A3 on antioxidant Capacity in mice
Table 16: influence of Yeast cell wall and antibacterial peptide A3 on the antioxidant Capacity of mice (U/mL)
As can be seen from table 16, 6 groups of SOD were significantly higher than the other groups (P < 0.05) after 21d of mice lavaged; group 6T-AOCs were significantly higher than the other groups (P < 0.05); groups 4, 5, 6 had significantly higher CAT than group CK, group 1 (P < 0.05). It can be seen that the addition of the yeast cell wall can obviously improve the oxidation resistance of organisms, and the antibacterial peptide has no obvious effect on the oxidation resistance index, but is better than a blank control group, and the effect of the combination preparation group of the yeast cell wall and the antibacterial peptide A3 is optimal.
2.3 Effect of Yeast cell wall and antibacterial peptide A3 on mouse immune index
Table 17: influence of Yeast cell wall and antimicrobial peptide A3 on mouse immune index
As can be seen from Table 17, after 21d of intragastric administration, the IL-2 content of groups 5 and 6 was significantly higher than that of the other groups (P < 0.05); the IgG content and sIgA content of the 6 groups are obviously higher than those of the other groups (P < 0.05). It can be seen that the addition of the antibacterial peptide alone can significantly improve the content of the anti-inflammatory factor IL-2, but the addition of the yeast cell wall and the antibacterial peptide A3 compound preparation has the best effect. The addition of the yeast cell wall and the antibacterial peptide A3 composite preparation can obviously improve the immune index IgG content and sIgA content, is superior to the addition of a yeast cell wall group and an antibacterial peptide group alone, and has obvious synergistic effect.
2.4 Effect of Yeast cell wall and antibacterial peptide A3 on the number of Lactobacillus and E.coli in the intestinal tract of mice
Table 18: effect of Yeast cell wall and antibacterial peptide A3 on the number of Lactobacillus and E.coli in the mouse intestinal tract (lg cfu/g)
As can be seen from Table 18, after 21d of intragastric administration, the number of Lactobacillus in groups 4, 5 and 6 was significantly higher than that in the other groups (P < 0.05); the number of coliforms in groups 5 and 6 is significantly lower than that in the other groups. The yeast cell wall has obvious effect on lactobacillus proliferation, the antibacterial peptide has obvious effect on escherichia coli, and the effect of adding the yeast cell wall and the antibacterial peptide A3 composite preparation is optimal.
The test of mice shows that the yeast cell wall can obviously improve the weight, the antioxidant capacity and the immune index of mice, the antibacterial peptide can obviously improve the immune index of mice, especially the content of anti-inflammatory factor IL-2, and can also obviously inhibit escherichia coli. The combination of the yeast cell wall and the antibacterial peptide A3 is superior to that of the single addition of the weight, the antioxidant index and the immune index, and has a synergistic effect.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims (5)
1. The application of the yeast cell wall-antibacterial peptide A3 composite preparation in improving the activity retention rate of the antibacterial peptide A3 after pepsin and trypsin act;
the yeast cell wall-antibacterial peptide A3 composite preparation is prepared from yeast cell walls and antibacterial peptide A3 according to the mass ratio of (8-10): 1, compounding;
the yeast cell wall is prepared by the following method:
the preservation number is CCTCC NO: inoculating Saccharomyces cerevisiae M2015209 into yeast liquid culture medium, shake culturing to obtain yeast fermentation liquid, separating solid from liquid, diluting thallus precipitate to obtain bacterial liquid, adjusting pH of bacterial liquid to 6.0-7.0, adding papain, controlling temperature to 50-60deg.C, and treating for 12-36 hr; adding light calcium carbonate into the treated system, uniformly mixing, and drying to prepare a yeast cell wall;
the antibacterial peptide A3 is prepared by the following method:
the preservation number is CCTCC NO: inoculating M2019530 bacillus polymyxa into a bacillus liquid culture medium, and standing and culturing at 37 ℃ for 20 hours to obtain bacillus polymyxa fermentation broth; centrifuging the fermentation broth of bacillus polymyxa, collecting supernatant, respectively selecting ultrafiltration membranes with molecular retention of 5kDa and 3kDa for ultrafiltration, collecting components with molecular weight of 5kDa-3kDa, and freeze-drying to obtain antibacterial peptide A3;
the bacillus liquid culture medium comprises the following components: corn steep liquor 0.5wt%, peptone 5wt%, glucose 3wt%, calcium chloride 0.25wt% and water for the rest.
2. The use according to claim 1, wherein in the preparation of the yeast cell wall, the cell pellet is diluted with water to a mass concentration of 25-35%.
3. The use according to claim 1, wherein the papain is added in an amount of 0.5-2% by volume of the bacterial liquid in the preparation of the yeast cell wall.
4. The use according to claim 1, wherein the light calcium carbonate is added in an amount of 5% by volume of the bacterial liquid in the preparation of the yeast cell wall.
5. The use according to claim 1, wherein the yeast cell wall-antimicrobial peptide A3 complex formulation is prepared by compounding yeast cell wall and antimicrobial peptide A3 in a mass ratio of 9:1.
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