CN110959750B - Application of yeast cell wall in field of fishing antibacterial agent - Google Patents
Application of yeast cell wall in field of fishing antibacterial agent Download PDFInfo
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- CN110959750B CN110959750B CN201811161637.3A CN201811161637A CN110959750B CN 110959750 B CN110959750 B CN 110959750B CN 201811161637 A CN201811161637 A CN 201811161637A CN 110959750 B CN110959750 B CN 110959750B
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- cell wall
- yeast cell
- yeast
- use according
- precipitate
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Classifications
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- 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
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
- A01K61/13—Prevention or treatment of fish diseases
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K20/00—Accessory food factors for animal feeding-stuffs
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
- Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
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- General Engineering & Computer Science (AREA)
Abstract
The invention relates to the field of fishing antibacterial agents, in particular to application of a yeast cell wall in the field of fishing antibacterial agents. The yeast cell wall is obtained by enzymatic hydrolysis of yeast with alkaline protease, mannanase, beta-glucanase and cellulase. The yeast cell wall is derived from yeast, has a good antibacterial effect on aquatic pathogenic bacteria, and can effectively prevent and treat aquatic animal diseases and promote aquatic animal growth when applied to the field of fishing antibacterial agents. Compared with antibiotic additives, the yeast cell wall has the advantages of no toxicity, no residue and no drug resistance; compared with the Chinese herbal medicine additive, the yeast cell wall has definite components, can be industrially produced, and has stable quality.
Description
Technical Field
The invention relates to the field of fishing antibacterial agents, in particular to application of a yeast cell wall in the field of fishing antibacterial agents.
Background
The positive effects of the antibiotics are mainly reflected in the aspects of preventing and treating aquatic animal diseases, promoting growth, saving nutrient components and the like. In particular, the application of antibiotics effectively controls the occurrence of a plurality of aquatic diseases and promotes the development of the aquaculture industry. However, due to the continuous use of antibiotics, drug-resistant strains are easy to generate, drug residues exist and microecological balance of aquatic animals is easy to be destroyed, on one hand, the aquaculture diseases are more and more difficult to treat; on the other hand, the quality safety of the aquatic products cannot be guaranteed. Thus, for human health and sustainable development of the aquaculture industry, the side effects of antibiotics in aquaculture must be well recognized. The side effects of antibiotics are manifested collectively in several ways.
(1) Production of drug resistant strains. Some antibiotics that have been effective in production have reduced efficacy or completely disappeared after prolonged use at low doses, because the pathogenic bacteria are resistant or resistant to the antibiotics, i.e., resistant strains are produced. The production of drug-resistant strains makes the drug dosage in production larger and worse, thus not only increasing the production cost, but also increasing the control difficulty. The generation of drug-resistant strains also poses a threat to public health of humans.
(2) Drug residues are generated in the aquatic products. Antibiotics enter the blood circulation of aquatic animals after use, most of which are discharged outside the body, and few of which remain in tissues in the body and accumulate in the body with multiple uses. The residue of antibiotics affects the physical health of humans and also affects the development of aquaculture.
(3) Destroying the microecological balance. Water is the environment in which aquatic animals live, with many beneficial microorganisms such as photosynthetic bacteria, nitrifying bacteria, and the like; there are also a large number of beneficial microorganisms in the intestines of aquatic animals, such as lactobacillus, vibrio partalis, etc. They play a critical role in maintaining the stability of water environment and metabolic balance of aquatic animals, and become an important tissue component in the micro-ecological balance of the inside and outside of aquatic animals. The use of antibiotics inhibits or kills pathogenic microorganisms and also inhibits these beneficial microorganisms, so that the in-vitro and in-vivo microecological balance of the aquatic animals is destroyed, and the microorganisms are deteriorated or the digestion and absorption are impaired to cause new diseases.
(4) Has effect in inhibiting immune system. The effect of antibiotics on the immune system is mainly manifested by the inhibition of phagocytes. Firstly, antibiotics directly affect phagocyte function; secondly, the chemotaxis, uptake, killing and other functions of phagocytes on microorganisms are influenced by influencing the microorganisms.
Although the antibiotics have better application effects in the prevention and treatment of aquaculture diseases, with the improvement of living standard of people, the quality safety awareness of aquatic products is continuously enhanced, the contradiction between the advantages and disadvantages of the antibiotics is increasingly stimulated, and the antibiotics have attracted general attention of people. Therefore, in order to produce pollution-free aquatic products and improve the quality of the aquatic products, the advantages and disadvantages of the use of antibiotics are scientifically and reasonably known, the antibiotics are carefully used, and the national forbidden antibiotic medicines such as chloramphenicol, erythromycin, bacitracin zinc, tylosin, ciprofloxacin and the like are strictly forbidden to ensure the safety and the sanitation of the cultured aquatic products.
At present, other antibacterial agents except antibiotics in the market mainly come from Chinese herbal medicines, and the quality of products is quite different due to undefined components.
Disclosure of Invention
The invention solves the prior art problems that: antibiotics are forbidden in aquatic feeds, cannot be used, are only used on site, and have the problems of drug resistance, residues and the like; the functions and the content of the components for enhancing immunity of the Chinese herbal medicine additive are not clear, and the consistency of the use effect of the feed produced in batches is difficult to ensure.
The invention aims to provide an application of a yeast cell wall in the field of fishing antibacterial agents, wherein the yeast cell wall is derived from yeast, has a good antibacterial effect, particularly can effectively prevent and treat aquatic animal diseases and promote the growth of aquatic animals. Compared with antibiotic additives, the yeast cell wall has the advantages of no toxicity, no residue and no drug resistance; compared with the Chinese herbal medicine additive, the yeast cell wall has definite components, can be industrially produced, and has stable quality.
Specifically, aiming at the defects in the prior art, the invention provides the following technical scheme:
use of a yeast cell wall obtained by enzymatic hydrolysis of yeast with alkaline protease, mannanase, beta-glucanase and cellulase in the field of antimicrobial agents for fishing.
Preferably, in the above application, the yeast cell wall is obtained by subjecting yeast to enzymolysis with alkaline protease, mannanase, beta-glucanase and cellulase one by one.
Preferably, in the above application, the yeast cell wall is added to a fish feed for feeding aquatic animals.
Preferably, in the above application, from 0.01% to 0.5% of yeast cell wall is added by weight of the fish feed.
Preferably, in the above application, the yeast cell wall is mixed with a carrier to prepare a premix for feeding aquatic animals, and preferably the carrier is one or more of corn flour, wheat flour, soybean flour, rice bran or zeolite powder.
Preferably, in the above application, the yeast cell wall is mixed with other feed additives to prepare premix for feeding aquatic animals.
Preferably, in the application, the yeast is Saccharomyces cerevisiae FX-2 (Saccharomyces cerevisiae FX-2), and the preservation number is CCTCC NO: M2016418.
Preferably, in the above application, the alkaline protease, mannanase, beta-glucanase and cellulase are added in amounts of 2.0 to 5.5%o, 2.0 to 5.0%o, 0.2 to 0.8%o and 0.3 to 0.8%o, respectively, based on the dry mass of the yeast cell wall.
Preferably, in the above application, the yeast cell wall is obtained by subjecting yeast to autolysis wall breaking, and then to enzymolysis with alkaline protease, mannanase, beta-glucanase and cellulase one by one.
Preferably, in the above application, the preparation of the yeast cell wall comprises the steps of:
(1) Autolyzing the yeast-containing material to break wall and separating to obtain yeast cell wall precipitate;
(2) And (3) carrying out enzymolysis on the yeast cell wall precipitate one by using alkaline protease, mannanase, beta-glucanase and cellulase to obtain the yeast cell wall.
Preferably, in the above application, in the step (1), the autolysis is performed at a salt concentration of 2 to 4.5%, a pH of 5.0 to 6.5 and a temperature of 55 to 75 ℃.
Preferably, in the above application, the yeast-containing raw material is obtained by fermentation treatment using Saccharomyces cerevisiae strain, molasses as carbon source and urea as nitrogen source.
Preferably, in the above application, the pH of the fermentation treatment is 4.0-6.0, the fermentation time is 16-24 hours, and the fermentation temperature is 28-30 ℃.
Preferably, in the above application, in step (2), the yeast cell wall precipitate is diluted to a suspension having a concentration of 10 to 20% based on dry matter of the yeast cell wall precipitate before subjecting the yeast cell wall precipitate to enzymatic hydrolysis with alkaline protease, mannanase, beta-glucanase and cellulase.
Preferably, in the above application, the enzymolysis temperature of the alkaline protease is 40-60 ℃, the pH is 6.0-8.5, and the hydrolysis time is 7-10 hours;
the enzymolysis temperature of the mannase is 40-60 ℃, the pH is 6.0-8.0, and the hydrolysis time is 10-15 hours;
The enzymolysis temperature of the beta-glucanase is 55-60 ℃, the pH is 5.0-6.0, and the hydrolysis time is 5-8 hours;
the enzymolysis temperature of the cellulase is 50-65 ℃, the pH is 4.0-5.5, and the hydrolysis time is 8-10 hours.
The beneficial effects obtained by the invention are as follows: the yeast cell wall is derived from yeast, has a good antibacterial effect on aquatic pathogenic bacteria, and can effectively prevent and treat aquatic animal diseases and promote aquatic animal growth when applied to the field of fishing antibacterial agents. Compared with antibiotic additives, the yeast cell wall has the advantages of no toxicity, no residue and no drug resistance; compared with the Chinese herbal medicine additive, the yeast cell wall has definite components, can be industrially produced, and has stable quality.
Strain preservation information and acquisition of identification information
The strain Saccharomyces cerevisiae FX-2 (Saccharomyces cerevisiae FX-2) used in the invention is preserved in China Center for Type Culture Collection (CCTCC) at 8-1 of 2016, and the preservation number is CCTCC: m2016418, deposit address: eight paths of 299 number Wuhan university school in Wuhan district of Wuhan, hubei province, postal code: 430072; telephone: (027) -68754052. Saccharomyces cerevisiae FX-2 is described in patent document 201611141122.8, "Saccharomyces cerevisiae high density culture method and pH control method".
The saccharomyces cerevisiae FX-2 used in the invention is from fermented dough, the fermented dough contains a plurality of wild bacteria, the fermented dough is taken as a sample, a dough leaching solution is prepared, pure strain is obtained by separating through a dilution coating flat plate separation method, and the strain belongs to the saccharomyces cerevisiae through identification.
The strain identification method comprises the steps of carrying out homology analysis on the sequence of the D1/D2 region of 26S rDNA of the strain and the sequence in a GenBank nucleic acid database, and determining that the strain obtained by separation of the invention belongs to Saccharomyces cerevisiae (Saccharomyces cerevisiae) and is biologically classified as Saccharomyces cerevisiae FX-2 (Saccharomyces cerevisiae).
Detailed Description
In view of the lack of better antibacterial agents with antibacterial effect in the prior aquatic feed: antibiotics are forbidden in aquatic feeds, cannot be used, are only used on site, and have the problems of drug resistance, residues and the like; the functions and the content of the components for enhancing immunity of the Chinese herbal medicine additive are not clear, and the consistency of the use effect of the feed produced in batches is difficult to ensure. The invention provides an application of a yeast cell wall in the field of fishing antibacterial agents, wherein the yeast cell wall is obtained by enzymolysis of yeast through alkaline protease, mannanase, beta-glucanase and cellulase. The yeast cell wall is derived from yeast, has a good antibacterial effect, and particularly can effectively prevent and treat aquatic animal diseases and promote the growth of aquatic animals for aquatic pathogenic bacteria.
In a preferred embodiment of the invention, the invention provides the use of a yeast cell wall in the field of antimicrobial agents for fishing, said yeast cell wall being obtained by subjecting yeast to autolysis to wall breaking and then to enzymatic hydrolysis one by one with alkaline protease, mannanase, beta-glucanase and cellulase. The autolyzed salt, preferably sodium chloride, is treated in a meta-acidic environment to maintain the aseptic environment, excite the endogenous enzyme activity of the yeast and promote the cell lysis of the yeast, so as to remove the content in the yeast cells and make the residual cell wall precipitate of the yeast contain higher polysaccharide components. The step-by-step enzymolysis means that enzymolysis is carried out sequentially, namely, enzymolysis treatment of alkaline protease, mannanase, beta-glucanase and cellulase is carried out sequentially.
Separating the autolyzed substances, removing yeast autolyzed substances to obtain yeast cell wall precipitate, and carrying out composite enzymolysis on the yeast cell wall precipitate by adopting a plurality of enzymes, wherein the enzymes comprise alkaline protease, mannanase, beta-glucanase and cellulase, and the enzymolysis treatment is carried out sequentially. The structure of the yeast cell wall is sequentially phosphorylated mannans, proteins and glucans from the outer layer to the inner layer, so that the inventor finds that the structure of the yeast cell wall can be destroyed by adopting alkaline protease treatment to destroy the yeast cell wall and degrade the proteins to expose the mannans and the glucans and then adopting mannanase treatment to destroy the structure of the yeast cell wall so as to separate mannans; then adopting beta-glucanase to process, degrading glucan in the cell wall to change the glucan into a small molecular fragment; finally, cellulase is adopted for treatment, so that glucan in the yeast cell wall is further changed into a small molecular fragment, and thus the functional site is exposed.
In a preferred embodiment of the invention, the yeast cell wall is added to a fish feed to feed an aquatic animal, with 0.01% -0.5% yeast cell wall by weight of the fish feed.
In a preferred embodiment of the present invention, the yeast cell wall is mixed with a carrier to prepare a premix for feeding aquatic animals, preferably one or more of corn flour, wheat flour, soybean flour, rice bran or zeolite powder.
In a preferred embodiment of the invention, the yeast cell wall is mixed with other feed additives to form a premix for feeding the aquatic animals.
In a preferred embodiment of the present invention there is provided the use of a yeast cell wall in the field of antimicrobial agents for fishing, the preparation of said yeast cell wall comprising the steps of:
(1) Autolysis wall breaking
Autolyzing a yeast-containing raw material under the conditions that the salt mass concentration is 2-4.5%, the pH value is 5.0-6.5 and the temperature is 55-75 ℃, and separating to obtain a yeast cell wall precipitate;
(2) Enzymolysis treatment
Diluting yeast cell wall precipitate with water to obtain dry matter with mass concentration of 10-20%, adding alkaline protease with mass concentration of 2.0-5.5%o (the enzyme content is the same as that of yeast cell wall dry matter), controlling temperature to 40-60deg.C, pH to 6.0-8.5, and hydrolyzing for 7-10 hr; then adding mannase with mass concentration of 2.0-5.0%o, controlling the temperature to 40-60 ℃ and the pH to 6.0-8.0, and hydrolyzing for 10-15 hours; then adjusting the pH value to 5.0-6.0, adding 0.2-0.8%o beta-glucanase, controlling the temperature to 55-60 ℃ and hydrolyzing for 5-8 hours; finally, the temperature is regulated to 50-65 ℃, and 0.3-0.8 per mill of cellulase is added under the condition of pH value of 4-5.5, and the hydrolysis time is 8-10 hours.
(3) Centrifugal drying
And (3) heating the enzymolysis product obtained in the step (2) to 90 ℃, preserving heat for 30min-1h, and drying to obtain the yeast cell wall.
In a further preferred embodiment of the invention, prior to the autolysis treatment, saccharomyces cerevisiae is used, molasses is used as a carbon source, urea is used as a nitrogen source, fermentation pH is 4.0-6.0, temperature is 28-30 ℃, fermentation is carried out for 16-24 hours, and the yeast raw material with the optimal yeast cell wall polysaccharide content is obtained. Preferably, the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae FX-2 (Saccharomyces cerevisiae FX-2) accession number: cctccc No. M2016418.
The raw materials and equipment manufacturers used in this example, as well as the equipment and analysis methods used in the analysis of the products, are described below, wherein the chemicals are not identified as chemically pure grades of conventional reagents. Among them, information of the raw materials used in examples and comparative examples is shown in the following table.
TABLE 1 information on raw materials used in the present invention
| Raw materials | Manufacturer' s |
| Alkaline protease (100 u/g) | NANNING PANGBO BIOLOGICAL ENGINEERING Co.,Ltd. |
| Mannanase (10000 u/g) | Mountain east and west Tang Biotechnology Co.Ltd |
| Beta-glucanase (20 u/g) | Indonesia Germany |
| Cellulase (2000 u/g) | NANNING PANGBO BIOLOGICAL ENGINEERING Co.,Ltd. |
| Molasses | Xinjiang Yilite sugar industry Co.Ltd |
| Urea | Hua Lu constant rise in Shandong provinceChemical Co Ltd |
| Pseudomonas fluorescens | Aquatic organism institute of national academy of sciences of China |
| Aeromonas comfort | Aquatic organism institute of national academy of sciences of China |
| Aeromonas hydrophila | Aquatic organism institute of national academy of sciences of China |
| Aeromonas veronii (Willd) | Aquatic organism institute of national academy of sciences of China |
| Aeromonas frog killing | Aquatic organism institute of national academy of sciences of China |
| Streptococcus agalactiae | Aquatic organism institute of national academy of sciences of China |
| Flavomycin | Nanjing Corp Biotechnology Co., ltd |
Example 1
Preparation of Yeast cell wall
The yeast cell wall is prepared by the following method:
(1) Saccharomyces cerevisiae FX-2 (Saccharomyces cerevisiae) accession number was used as the Saccharomyces cerevisiae strain: the CCTCC NO is M2016418, the carbon source is molasses, the nitrogen source is urea, the pH value is 4.5, the culture temperature is 30 ℃, and the fermentation period is 16 hours, so as to obtain the yeast raw material.
The specific culture medium comprises the following components in percentage by weight:
(2) Autolysis treatment is carried out on the yeast raw material, sodium chloride is added to ensure that the mass concentration of the sodium chloride is 2%, the pH value is 5.5, the temperature is 55 ℃, after autolysis is carried out for 20 hours, the yeast cell wall precipitate is obtained after centrifugal separation.
(3) Enzymolysis:
a. Diluting the precipitate with water to 15%, adding alkaline protease (calculated as yeast cell wall dry matter, the same shall apply below) with mass concentration of 2%, controlling temperature to 55deg.C, pH value to 8.5, and hydrolyzing for 7 hr;
b. adding mannase with mass concentration of 2.5%o, controlling the temperature to 55 ℃, the pH value to 8.0, and controlling the hydrolysis time to 15 hours;
c. continuously adding beta-glucanase with the mass concentration of 0.2 per mill, controlling the temperature to 55 ℃, adjusting the pH value to 6.0, and controlling the hydrolysis time to 5 hours;
d. adjusting the temperature to 65 ℃, adding cellulase with the mass concentration of 0.3 per mill, adjusting the pH value to 4.0, and controlling the hydrolysis time to 8.5 hours;
(4) After the reaction is finished, the temperature is raised to 90 ℃ and kept for 1h, and the yeast cell wall is obtained after drying.
(II) in vitro bacteriostasis test of Yeast cell wall
Preparing a yeast cell wall sample prepared in the step (I) into a solution, taking 500mL to 1000mL, centrifuging for 10 to 20min at 5000 to 6000g, taking a supernatant, evaporating and concentrating the supernatant by a rotary evaporator at 75 to 80 ℃ until the concentration of the yeast cell wall dry matter is about 40% (namely, 1mL of water contains 0.4g of yeast cell wall dry matter, namely, the concentration of the yeast cell wall is 0.4 g/mL), and using the solution for a subsequent bacteriostasis experiment. The specific experimental process and experimental results are as follows:
1. Preparation of bacteriostasis test sample
Preparing the yeast cell wall sample prepared in the step (one) into a solution with the dry matter concentration of 10%, taking 500mL, centrifuging for 20min at 5000g, taking supernatant, evaporating and concentrating at 80 ℃ by using a rotary evaporator until the dry matter concentration is 40%, and using the supernatant for a subsequent bacteriostasis test.
2. Antibacterial test protocol
The difference of the antibacterial effect of the yeast cell wall prepared by the invention is checked from the size of the antibacterial circle. The experimental process comprises preparation of a culture medium, strain culture and bacteriostasis circle test.
2.1 preparation of the Medium.
MH broth, MH agar medium 121 ℃, autoclaved for 20min and MH agar plates were prepared for use.
2.2 Strain culture
Inoculating preserved Pseudomonas fluorescens, aeromonas comfort, aeromonas hydrophila, aeromonas veronii, aeromonas frog-killing and Streptococcus agalactiae into MH broth, and incubating at 37deg.C for 12-14 hr. The recovered Pseudomonas fluorescens, aeromonas comfort, aeromonas hydrophila, aeromonas veronii, aeromonas frog killing and Streptococcus agalactiae are inoculated with MH broth again, and the culture is carried out for about 16 to 20 hours. The OD value is between 0.4 and 0.7 as detected by a spectrophotometer. The viable count is about 10 10 CFU/mL (CFU/mL indicates the total number of bacterial colonies contained in each mL of sample).
2.3 test of zone of inhibition
The bacteria inhibition zone experiment is carried out by adopting an oxford cup method, the measurement principle is that a culture medium is sterilized, then, an oxford cup (a round small tube with the inner diameter of 6mm, the outer diameter of 8mm and the height of 10mm, and the two ends of the tube are smooth, a glass tube and a porcelain tube can also be used) is directly and vertically placed on the surface of the culture medium by aseptic operation, the oxford cup is lightly pressurized to be in contact with the culture medium without a gap, a sample to be detected is added into the oxford cup, and the oxford cup can be filled with 240 microliters generally without overflowing. After filling, culturing for a certain time at a certain temperature, and observing to find that the oxford cup is taken as a center to form a transparent circle. This is because, in the course of the culture, the test bacteria start to grow on the one hand, and on the other hand, the sample to be measured is spherically diffused, and the closer to the cup, the greater the concentration of the sample to be measured, the further from the cup, the smaller the concentration of the sample to be measured. As the concentration of the sample to be measured is reduced, there is a zone of minimum inhibitory concentration within which bacteria cannot grow, but rather take the form of a transparent circle, known as a "zone of inhibition". The higher the concentration of the sample to be measured is, the larger the inhibition zone is. The size of the inhibition zone can be directly measured by a ruler.
2.3.1 preparation of the bacteria-containing Petri dish
Diluting the pure culture bacterial liquid in the step 2.2 by 1000 times by using normal saline (sterilized at 121 ℃ for 20min under high pressure) to prepare bacterial suspension;
pouring the sterilized MH agar into plates, and dripping 200 mu L of bacterial suspension into each plate after the MH plates are solidified;
and (3) using the sterilized glass rod to rapidly and uniformly coat the bacterial liquid on the whole dish to prepare the bacteria-containing dish.
2.3.2 the prepared yeast cell wall samples were adjusted to the same concentration.
2.3.3 placing oxford cup into prepared bacteria-containing plate.
2.3.4 Yeast cell wall samples were added to oxford cups (about 250. Mu.L each) and the liquid level was leveled with the oxford cup.
2.3.5 the plate of 2.3.4 was placed in a constant temperature incubator and incubated at 37℃for about 14 hours, and the results were observed.
In the experimental process, the yeast cell wall prepared in the step (one) is used as a sample to be tested, the flavomycin is used as a positive reference substance, and the normal saline is used as a negative reference substance. Among them, flavomycin is a phosphorylated polysaccharide antibiotic whose antibacterial action mechanism is to inhibit bacterial reproduction by interfering with the biosynthesis of peptidoglycan, a structural substance of the cell wall. The results are shown in Table 1.
TABLE 1 in vitro bacteriostatic effects of fishing antibacterial agents on common pathogenic bacteria of aquatic products
The results show that: the yeast cell wall prepared in the step (one) has obvious antibacterial effects on pseudomonas fluorescens, aeromonas comfort, aeromonas hydrophila, aeromonas verrucosa, aeromonas froglena and streptococcus agalactiae, and the in-vitro antibacterial effect of the yeast cell wall is close to that of flavomycin.
(III) application effect of yeast cell wall on carassius auratus gibelio
About 150g of healthy carassius auratus gibelio (provided by Shangshagang Zhenda aquaculture farm in Zhenyang county of Jiangsu province, the same applies below) is selected and divided into three groups at random, and a basic ration is fed to a control group, wherein the basic ration formula comprises 10 parts of white fish meal, 30 parts of bean pulp, 18 parts of rapeseed meal, 12 parts of cotton pulp, 4.67 parts of flour, 5 parts of wheat bran, 5 parts of corn starch, 3.3 parts of fish oil, 3.2 parts of soybean oil, 0.39 part of vitamin premix, 5 parts of mineral premix, 0.11 part of choline chloride and 2.5 parts of carboxymethyl cellulose. The flavomycin group preferably comprises 0.5% flavomycin and the yeast cell wall group comprises 0.2% yeast cell wall in the basic ration, and three parallel groups are used for each experiment. Feeding test is started, 3 times per day, feeding amount is 1.6% of the weight of the fish body of each test group, feeding amount is adjusted according to feeding condition of the fish, and test period is 4 weeks. The results of the growth-promoting test of the antibacterial agent for fishing on carassius auratus gibelio are shown in Table 2.
TABLE 2 Effect of Yeast cell wall on Carassius gibelio growth
Note that: the results of the growth-promoting experiments in Table 2 are the average of 150 carassius auratus gibelio groups
Specific growth rate = 100 x (lnW Powder (D) -lnW Initially, the method comprises )/t
Wherein W is Powder (D) Is the weight, W of the carassius auratus gibelio at the end Initially, the method comprises The weight of the carassius auratus gibelio at the beginning is given, and t is the number of days of cultivation.
As can be seen from Table 2, the specific growth rate of the test group to which the yeast cell wall was added was superior to that of the control group, and had a remarkable growth-promoting effect.
The results of the test of the effect of the yeast cell wall on the serum immune index of carassius auratus gibelio are shown in Table 3.
TABLE 3 influence of Yeast cell wall on serum Immunity index of Carassius gibelio
Note that: the same column data shoulder marks with different lower case letters represent significant differences (P < 0.05)
As can be seen from table 3, the data processing and analysis used SPSS15.0 software, t-test analysis, the lysozyme activity of the allogynous crucian carp fed to the yeast cell wall was significantly higher than that of the control group and the flavomycin group (P < 0.05); however, the lysozyme activity of the flavomycin group was not significantly different from that of the control group (P > 0.05). From the measurement results of the phagocytic activity of the leucocytes, the phagocytic activity of the yeast cell wall group is significantly higher than that of the control group, while the phagocytic activity of the leucocytes of the flavomycin group is lower than that of the control group, but there is no significant difference (P > 0.05).
The death of the control group and the yeast cell wall group after challenge with Aeromonas hydrophila are shown in Table 4.
TABLE 4 protection of the yeast cell walls against Carassius gibelio after challenge with Aeromonas hydrophila
Note that: protection rate = 100 (1-mortality/control mortality)
As can be seen from Table 4, the mortality of fish bodies of two test groups added with yeast cell walls and flavomycin in the feed was significantly lower than that of the control group, wherein the protection rate of flavomycin was higher, and the protection rate of the yeast cell walls after challenge was close to that of flavomycin.
Example two
Preparation of Yeast cell wall
The yeast cell wall is prepared by the following method:
(1) Saccharomyces cerevisiae FX-2 (Saccharomyces cerevisiae) accession number was used as the Saccharomyces cerevisiae strain: the CCTCC NO is M2016418, the carbon source is molasses, the nitrogen source is urea, the pH value is 6.0, the culture temperature is 28 ℃, and the fermentation period is 24 hours, so as to obtain the yeast raw material.
(2) Autolysis treatment is carried out on the yeast raw material, sodium chloride is added to ensure that the mass concentration of the sodium chloride is 4.5%, the pH value is 6.5, the temperature is 75 ℃, after autolysis is carried out for 30 hours, the yeast cell wall precipitate is obtained after centrifugal separation.
(3) Enzymolysis:
a. diluting the precipitate with water to 20%, adding alkaline protease (calculated as yeast cell wall dry matter, the same shall apply hereinafter) with a mass concentration of 5.5%, controlling the temperature at 40deg.C, pH value at 6.0, and hydrolyzing for 10 hr;
b. Continuously adding 5.0 per mill of mannase with mass concentration, controlling the temperature to 60 ℃, controlling the pH value to 3.0, and controlling the hydrolysis time to 10 hours;
c. continuously adding beta-glucanase with the mass concentration of 0.8 per mill, controlling the temperature to 60 ℃, adjusting the pH value to 5.0, and controlling the hydrolysis time to 8 hours;
d. adjusting the temperature to 50 ℃, adding cellulase with the mass concentration of 0.8 per mill, adjusting the pH value to 5.5, and controlling the hydrolysis time to 10 hours;
(4) After the reaction is finished, the temperature is raised to 90 ℃ and kept for 1h, and the yeast cell wall is obtained after drying.
(II) in vitro bacteriostasis test of Yeast cell wall
The determination of the bacteriostatic effect on the yeast cell wall was carried out in the same manner as in example one.
The measurement results are shown in Table 5. As can be seen from Table 5, the prepared yeast cell wall has obvious antibacterial effects on Pseudomonas fluorescens, aeromonas comfort, aeromonas hydrophila, aeromonas verrucosa, aeromonas froglena and Streptococcus agalactiae, and the in vitro antibacterial effects of the yeast cell wall are similar to those of flavomycin.
TABLE 5 in vitro bacteriostatic effects of fishing antibacterial agents on common pathogenic bacteria of aquatic products
(III) application effect of yeast cell wall on carassius auratus gibelio
The effect test of the yeast cell wall on the allogynous crucian carp was performed in the same manner as in example one. Unlike example one, the yeast cell wall group added 0.01% yeast cell wall in the basal ration.
TABLE 6 Effect of Yeast cell wall on Carassius gibelio growth
As can be seen from Table 6, the specific growth rate of the test group to which the yeast cell wall was added was superior to that of the control group, and had a remarkable growth-promoting effect.
TABLE 7 influence of Yeast cell wall on serum Immunity index of Carassius gibelio
Note that: the same column data shoulder marks with different lower case letters represent significant differences (P < 0.05)
As can be seen from table 7, the lysozyme activity of the carassius auratus gibelio fed the yeast cell wall was significantly higher than that of the control group and the flavomycin group (P < 0.05); however, the lysozyme activity of the flavomycin group was not significantly different from that of the control group (P > 0.05). From the measurement results of the phagocytic activity of the leucocytes, the phagocytic activity of the yeast cell wall group is significantly higher than that of the control group, while the phagocytic activity of the leucocytes of the flavomycin group is lower than that of the control group, but there is no significant difference (P > 0.05).
TABLE 8 protection of Carassius gibelio by Yeast cell wall after challenge with Aeromonas hydrophila
As can be seen from Table 8, the mortality of fish bodies of two test groups added with yeast cell walls and flavomycin in the feed was significantly lower than that of the control group, wherein the protection rate of flavomycin was higher, and the protection rate of the yeast cell walls after challenge was close to that of flavomycin.
Example III
Preparation of Yeast cell wall
The yeast cell wall is prepared by the following method:
(1) Saccharomyces cerevisiae FX-2 (Saccharomyces cerevisiae) accession number was used as the Saccharomyces cerevisiae strain: the CCTCC NO is M2016418, the carbon source is molasses, the nitrogen source is urea, the pH value is 4.0, the culture temperature is 30 ℃, and the fermentation period is 20 hours, so as to obtain the yeast raw material.
(2) Autolysis treatment is carried out on the yeast raw material, sodium chloride is added to ensure that the mass concentration of the sodium chloride is 4%, the pH value is 6.0, the temperature is 60 ℃, after autolysis is carried out for 30 hours, the yeast cell wall precipitate is obtained after centrifugal separation.
(3) Enzymolysis:
a. diluting the precipitate with water to 10%, adding alkaline protease (calculated as yeast cell wall dry matter, the same shall apply below) with mass concentration of 4%, controlling temperature at 60deg.C, pH value at 7.0, and hydrolyzing for 10 hr;
b. adding mannase with mass concentration of 2.0%o, controlling the temperature to 60 ℃, controlling the pH value to 8.0, and controlling the hydrolysis time to 10 hours;
c. continuously adding beta-glucanase with the mass concentration of 0.2 per mill, controlling the temperature to 55 ℃, adjusting the pH to 5.0, and controlling the hydrolysis time to 5 hours;
d. adjusting the temperature to 65 ℃, adding cellulase with the mass concentration of 0.3 per mill, adjusting the pH value to 5.5, and controlling the hydrolysis time to 8 hours;
(4) After the reaction is finished, the temperature is raised to 90 ℃ and kept for 1h, and the yeast cell wall is obtained after drying.
(II) in vitro bacteriostasis test of Yeast cell wall
The determination of the bacteriostatic effect on the yeast cell wall was carried out in the same manner as in example one.
The measurement results are shown in Table 9. As can be seen from Table 9, the prepared yeast cell wall has obvious antibacterial effects on Pseudomonas fluorescens, aeromonas comfort, aeromonas hydrophila, aeromonas verrucosa, aeromonas froglena and Streptococcus agalactiae, and the in vitro antibacterial effects of the yeast cell wall are similar to those of flavomycin.
Table 9 in vitro antibacterial effect of antibacterial agent for fishing on aquatic common pathogenic bacteria
(III) application effect of yeast cell wall on carassius auratus gibelio
The effect test of the yeast cell wall on the allogynous crucian carp was performed in the same manner as in example one. Unlike example one, the yeast cell wall group added 0.5% yeast cell wall in the basal ration.
TABLE 10 Effect of Yeast cell wall on Carassius gibelio growth
As can be seen from Table 10, the specific growth rate of the test group to which the yeast cell wall was added was superior to that of the control group, and had a remarkable growth-promoting effect.
TABLE 11 influence of Yeast cell wall on serum Immunity index of Carassius gibelio
And (3) injection: the same column data shoulder marks with different lower case letters represent significant differences (P < 0.05)
As can be seen from table 11, the lysozyme activity of the carassius auratus gibelio fed the yeast cell wall was significantly higher than that of the control group and the flavomycin group (P < 0.05); however, the lysozyme activity of the flavomycin group was not significantly different from that of the control group (P > 0.05). From the measurement results of the phagocytic activity of the leucocytes, the phagocytic activity of the yeast cell wall group is significantly higher than that of the control group, while the phagocytic activity of the leucocytes of the flavomycin group is lower than that of the control group, but there is no significant difference (P > 0.05).
TABLE 12 protection of Carassius gibelio by Yeast cell wall after challenge with Aeromonas hydrophila
As can be seen from Table 12, the mortality of fish bodies of two test groups added with yeast cell walls and flavomycin in the feed was significantly lower than that of the control group, wherein the protection rate of flavomycin was higher, and the protection rate of the yeast cell walls after challenge was close to that of flavomycin.
Example IV
Preparation of Yeast cell wall
The yeast cell wall is prepared by the following method:
(1) Saccharomyces cerevisiae FX-2 (Saccharomyces cerevisiae) accession number was used as the Saccharomyces cerevisiae strain: the CCTCC NO is M2016418, the carbon source is molasses, the nitrogen source is urea, the pH value is 5.0, the culture temperature is 30 ℃, and the fermentation period is 20 hours, so as to obtain the yeast raw material.
(2) Autolysis treatment is carried out on the yeast raw material, sodium chloride is added to ensure that the mass concentration of the sodium chloride is 3 percent, the pH value is 6.5, the temperature is 60 ℃, after autolysis is carried out for 25 hours, the yeast cell wall precipitate is obtained after centrifugal separation.
(3) Enzymolysis:
a. diluting the precipitate with water to 15%, adding alkaline protease (based on yeast cell wall dry matter, the same shall apply below) at 55deg.C, pH value of 8.0, and hydrolyzing for 7.5 hr;
b. continuing to add mannase with mass concentration of 3.0 per mill, controlling the temperature to 50 ℃, controlling the pH value to 7.0, and controlling the hydrolysis time to 12 hours;
c. continuously adding beta-glucanase with the mass concentration of 0.5 per mill, controlling the temperature to 60 ℃, adjusting the pH value to 5.5, and controlling the hydrolysis time to 7 hours;
d. adjusting the temperature to 55 ℃, adding cellulase with the mass concentration of 0.5 per mill, adjusting the pH value to 5.0, and controlling the hydrolysis time to 9 hours;
(4) After the reaction is finished, the temperature is raised to 90 ℃ and kept for 1h, and the yeast cell wall is obtained after drying.
(II) in vitro bacteriostasis test of Yeast cell wall
The determination of the bacteriostatic effect on the yeast cell wall was carried out in the same manner as in example one.
The measurement results are shown in Table 13. As can be seen from Table 13, the prepared yeast cell wall has obvious antibacterial effects on Pseudomonas fluorescens, aeromonas comfort, aeromonas hydrophila, aeromonas verrucosa, aeromonas froglena and Streptococcus agalactiae, and the in vitro antibacterial effects of the yeast cell wall are similar to those of flavomycin.
Table 13 in vitro antibacterial effect of fishing antibacterial agent on aquatic common pathogenic bacteria
(III) application effect of yeast cell wall on carassius auratus gibelio
The effect test of the yeast cell wall on the allogynous crucian carp was performed in the same manner as in example one. Unlike example one, the yeast cell wall group added 0.01% yeast cell wall in the basal ration.
TABLE 14 Effect of Yeast cell wall on Carassius gibelio growth
As can be seen from Table 14, the specific growth rate of the test group to which the yeast cell wall was added was superior to that of the control group, and had a remarkable growth-promoting effect.
TABLE 15 influence of Yeast cell wall on serum Immunity index of Carassius gibelio
Note that: the same column data shoulder marks with different lower case letters represent significant differences (P < 0.05)
As can be seen from table 15, the lysozyme activity of the carassius auratus gibelio fed the yeast cell wall was significantly higher than that of the control group and the flavomycin group (P < 0.05); however, the lysozyme activity of the flavomycin group was not significantly different from that of the control group (P > 0.05). From the measurement results of the phagocytic activity of the leucocytes, the phagocytic activity of the yeast cell wall group is significantly higher than that of the control group, while the phagocytic activity of the leucocytes of the flavomycin group is lower than that of the control group, but there is no significant difference (P > 0.05).
TABLE 16 protection of Carassius gibelio after challenge with Aeromonas hydrophila by Yeast cell wall
As can be seen from Table 16, the mortality of fish bodies of two test groups added with yeast cell walls and flavomycin in the feed was significantly lower than that of the control group, wherein the protection rate of flavomycin was higher and the yeast cell walls had a certain protection rate.
Example five
Preparation of Yeast cell wall
Yeast cell walls were prepared in the same manner as in example four.
(II) in vitro bacteriostasis test of Yeast cell wall
The determination of the bacteriostatic effect on the yeast cell wall was carried out in the same manner as in example four.
(III) application effect of yeast cell wall on carassius auratus gibelio
The application effect test on carassius auratus gibelio was performed on the yeast cell wall in the same manner as in example four. The difference from example four is that the yeast cell wall group adds 0.2% yeast cell wall in the basal ration.
TABLE 18 Effect of Yeast cell wall on Carassius gibelio growth
As can be seen from Table 18, the specific growth rate of the test group to which the yeast cell wall was added was superior to that of the control group, and had a remarkable growth-promoting effect.
TABLE 19 influence of yeast cell walls on serum immune index of Carassius gibelio
Note that: the same column data shoulder marks with different lower case letters represent significant differences (P < 0.05)
As can be seen from table 19, the lysozyme activity of the carassius auratus gibelio fed the yeast cell wall was significantly higher than that of the control group and the flavomycin group (P < 0.05); however, the lysozyme activity of the flavomycin group was not significantly different from that of the control group (P > 0.05). From the measurement results of the phagocytic activity of the leucocytes, the phagocytic activity of the yeast cell wall group is significantly higher than that of the control group, while the phagocytic activity of the leucocytes of the flavomycin group is lower than that of the control group, but there is no significant difference (P > 0.05).
TABLE 20 protection of Carassius gibelio after challenge with Aeromonas hydrophila by Yeast cell wall
As can be seen from Table 20, the mortality of fish bodies of two test groups added with yeast cell walls and flavomycin in the feed was significantly lower than that of the control group, wherein the protection rate of flavomycin was higher, and the protection rate of the yeast cell walls after challenge was close to that of flavomycin.
Comparative example one
Preparation of Yeast cell wall
Yeast cell walls were prepared in the same manner as in example five. In contrast to example five, the enzymatic hydrolysis process of comparative example one does not comprise alkaline protease.
(II) in vitro bacteriostasis test of Yeast cell wall
The determination of the bacteriostatic effect on the yeast cell wall was carried out in the same manner as in example five.
The measurement results are shown in Table 21. As can be seen from Table 21, the prepared yeast cell wall has no obvious antibacterial effect on Pseudomonas fluorescens, aeromonas comfort, aeromonas hydrophila, aeromonas veronii, aeromonas frog killing and Streptococcus agalactiae.
Table 21 in vitro antibacterial effect of fishing antibacterial agent on aquatic product common pathogenic bacteria
(III) application effect of yeast cell wall on carassius auratus gibelio
The effect test of the yeast cell wall on the allogynous crucian carp was performed in the same manner as in example five.
TABLE 22 Effect of Yeast cell walls on Carassius gibelio growth
As can be seen from Table 22, the test group to which the yeast cell wall was added had a lower specific growth rate than the control group, and had no growth-promoting effect.
TABLE 23 influence of yeast cell walls on serum Immunity index of Carassius gibelio
Note that: the same column data shoulder marks with different lower case letters represent significant differences (P < 0.05)
As can be seen from Table 23, the lysozyme activity of the carassius auratus gibelio fed the yeast cell wall was not significantly different from that of the control group and the flavomycin group (P > 0.05). From the measurement results of the phagocytic activity of the leucocytes, the phagocytic activity of the yeast cell wall group was not significantly different from that of the control group, while the phagocytic activity of the flavomycin group was lower than that of the control group, but not significantly different (P > 0.05).
Table 24 protective action of Yeast cell wall on Carassius gibelio after challenge with Aeromonas hydrophila
As can be seen from Table 24, the mortality of fish in the test group with the addition of the flavomycin in the feed was significantly lower than that in the control group, while the protection rate of the yeast cell wall after challenge was zero. The yeast cell walls prepared according to such methods are shown to not greatly aid in aquatic animal production and immunization.
Comparative example two
Preparation of Yeast cell wall
Yeast cell walls were prepared in the same manner as in example five. In contrast to example five, the comparative example one did not include cellulase during the enzymatic hydrolysis.
(II) in vitro bacteriostasis test of Yeast cell wall
The determination of the bacteriostatic effect on the yeast cell wall was carried out in the same manner as in example five.
The measurement results are shown in Table 25. As can be seen from Table 25, the prepared yeast cell wall has no obvious antibacterial effect on Pseudomonas fluorescens, aeromonas comfort, aeromonas hydrophila, aeromonas veronii, aeromonas frog killing and Streptococcus agalactiae.
Table 25 in vitro antibacterial effect of fishing antibacterial agent on aquatic product common pathogenic bacteria
(III) application effect of yeast cell wall on carassius auratus gibelio
The effect test of the yeast cell wall on the allogynous crucian carp was performed in the same manner as in example five.
TABLE 26 Effect of Yeast cell wall on Carassius gibelio growth
As can be seen from Table 26, the specific growth rate of the test group to which the yeast cell wall was added was lower than that of the control group, and there was no growth-promoting effect.
TABLE 27 influence of yeast cell walls on serum Immunity index of Carassius gibelio
Note that: the same column data shoulder marks with different lower case letters represent significant differences (P < 0.05)
As can be seen from Table 27, the lysozyme activity of the carassius auratus gibelio fed the yeast cell wall was not significantly different from that of the control group and the flavomycin group (P > 0.05). From the measurement results of the phagocytic activity of the leucocytes, the phagocytic activity of the yeast cell wall group was not significantly different from that of the control group, while the phagocytic activity of the flavomycin group was lower than that of the control group, but not significantly different (P > 0.05). .
Table 28 protective action of Yeast cell wall on Carassius gibelio after challenge with Aeromonas hydrophila
As can be seen from Table 28, the mortality of fish in the test group with the addition of fulvin was significantly lower than that in the control group, while the protection rate of the yeast cell wall after challenge was zero. The yeast cell walls prepared according to such methods are shown to not greatly aid in aquatic animal production and immunization.
Comparative example three
Preparation of Yeast cell wall
Yeast cell walls were prepared in the same manner as in example five. The difference from example five is that the addition amounts of alkaline protease and cellulase of comparative example one were different, in which the addition amount of alkaline protease was 7% and the addition amount of cellulase was 0.1%.
(II) in vitro bacteriostasis test of Yeast cell wall
The determination of the bacteriostatic effect on the yeast cell wall was carried out in the same manner as in example five.
The measurement results are shown in Table 29. As can be seen from Table 29, the prepared yeast cell wall has no obvious antibacterial effect on Pseudomonas fluorescens, aeromonas comfort, aeromonas hydrophila, aeromonas veronii, aeromonas frog killing and Streptococcus agalactiae.
In vitro antibacterial effect of surface 29 fishing antibacterial agent on aquatic product common pathogenic bacteria
(III) application effect of yeast cell wall on carassius auratus gibelio
The effect test of the yeast cell wall on the allogynous crucian carp was performed in the same manner as in example five.
TABLE 30 Effect of yeast cell walls on Carassius gibelio growth
As can be seen from Table 30, the test group to which the yeast cell wall was added had a lower specific growth rate than the control group, and had no growth-promoting effect.
TABLE 31 influence of yeast cell walls on serum immune index of Carassius gibelio
Note that: the same column data shoulder marks with different lower case letters represent significant differences (P < 0.05)
As can be seen from Table 31, the lysozyme activity of the carassius auratus gibelio fed the yeast cell wall was not significantly different from that of the control group and the flavomycin group (P > 0.05). From the measurement results of the phagocytic activity of the leucocytes, the phagocytic activity of the yeast cell wall group was not significantly different from that of the control group, while the phagocytic activity of the flavomycin group was lower than that of the control group, but not significantly different (P > 0.05). .
Table 32 protective action of Yeast cell wall on Carassius gibelio after challenge with Aeromonas hydrophila
As can be seen from Table 32, the mortality of fish in the test group with the addition of fulvin was significantly lower than that in the control group, while the protection rate of the yeast cell wall after challenge was very low. The yeast cell walls prepared according to such methods are shown to not greatly aid in aquatic animal production and immunization.
Comparative example four
Preparation of Yeast cell wall
Yeast cell walls were prepared in the same manner as in example five. The difference from example five is that the addition amounts of alkaline protease and cellulase of comparative example one are different, wherein the addition amount of alkaline protease is 1% and the addition amount of cellulase is 3%.
(II) in vitro bacteriostasis test of Yeast cell wall
The determination of the bacteriostatic effect on the yeast cell wall was carried out in the same manner as in example five.
The measurement results are shown in Table 33. As can be seen from Table 33, the prepared yeast cell wall has no obvious antibacterial effect on Pseudomonas fluorescens, aeromonas comfort, aeromonas hydrophila, aeromonas veronii, aeromonas frog killing and Streptococcus agalactiae.
Table 33 in vitro antibacterial effect of antibacterial agent for fishing on aquatic common pathogenic bacteria
(III) application effect of yeast cell wall on carassius auratus gibelio
The effect test of the yeast cell wall on the allogynous crucian carp was performed in the same manner as in example five.
TABLE 34 Effect of Yeast cell walls on Carassius gibelio growth
As can be seen from Table 34, the specific growth rate of the test group to which the yeast cell wall was added was the same as that of the control group, and there was no growth-promoting effect.
TABLE 35 influence of yeast cell walls on serum immune index of Carassius gibelio
Note that: the same column data shoulder marks with different lower case letters represent significant differences (P < 0.05)
As can be seen from Table 35, the lysozyme activity of the carassius auratus gibelio fed the yeast cell wall was not significantly different from that of the control group and the flavomycin group (P > 0.05). From the measurement results of the phagocytic activity of the white blood cells, the phagocytic activity of the white blood cells of the yeast cell wall group and the flavomycin group was lower than that of the control group, but there was no significant difference (P > 0.05).
Table 36 protection of Yeast cell wall against Carassius gibelio after challenge with Aeromonas hydrophila
As can be seen from Table 36, the mortality of fish in the test group with the addition of fulvin was significantly lower than that in the control group, while the protection rate of the yeast cell wall after challenge was substantially zero. The yeast cell walls prepared according to such methods are shown to not greatly aid in aquatic animal production and immunization.
In conclusion, the yeast cell wall has obvious antibacterial effects on pseudomonas fluorescens, aeromonas comfort, aeromonas hydrophila, aeromonas verrucosa, aeromonas froglena and streptococcus agalactiae; compared with the test group added with the flavomycin, the specific growth rates are not significantly different and are higher than those of the control group; lysozyme activity and phagocytic activity were higher than those of the flavomycin group and the control group; the death rate after the aeromonas hydrophila is subjected to power toxin is greatly reduced compared with that of a control group, and the protection rate after the aeromonas hydrophila is subjected to toxin attack is close to that of the flavomycin. Therefore, the yeast cell wall of the invention is applied to the field of fish antibacterial agents, can inhibit fish pathogenic bacteria, promote fish growth, reduce disease occurrence rate and have the function of replacing antibiotics.
The above description is not intended to limit the invention in any way, but is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (25)
1. The application of the yeast cell wall in preparing the fish antibacterial agent is characterized in that the yeast cell wall is obtained by performing autolysis wall breaking on yeast, and then performing enzymolysis on alkaline protease, mannanase, beta-glucanase and cellulase one by one;
Wherein, based on the dry matter mass of the yeast cell wall, the addition amounts of the alkaline protease, the mannanase, the beta-glucanase and the cellulase are respectively 2.0 to 5.5 per mill, 2.0 to 5.0 per mill, 0.2 to 0.8 per mill and 0.3 to 0.8 per mill;
wherein the enzymolysis temperature of the alkaline protease is 40-60 ℃, the pH value is 6.0-8.5, and the hydrolysis time is 7-10 hours;
the enzymolysis temperature of the mannase is 40-60 ℃, the pH is 6.0-8.0, and the hydrolysis time is 10-15 hours;
the enzymolysis temperature of the beta-glucanase is 55-60 ℃, the pH is 5.0-6.0, and the hydrolysis time is 5-8 hours;
the enzymolysis temperature of the cellulase is 50-65 ℃, the pH is 4.0-5.5, and the hydrolysis time is 8-10 hours.
2. The use according to claim 1, wherein the yeast is Saccharomyces cerevisiae FX-2 (Saccharomyces cerevisiae FX-2) with a preservation number of CCTCC NO: M2016418.
3. Use according to claim 1 or 2, wherein the preparation of the yeast cell wall comprises the steps of:
(1) Autolyzing the yeast-containing material to break wall and separating to obtain yeast cell wall precipitate;
(2) And (3) carrying out enzymolysis on the yeast cell wall precipitate one by using alkaline protease, mannanase, beta-glucanase and cellulase to obtain the yeast cell wall.
4. The use according to claim 3, wherein in step (1), the autolytic wall breaking is performed at a salt concentration of 2-4.5%, a pH of 5.0-6.5 and a temperature of 55-75 ℃.
5. The use according to claim 3, wherein the yeast-containing feedstock is obtained by fermentation treatment using saccharomyces cerevisiae, molasses as carbon source and urea as nitrogen source.
6. The use according to claim 5, wherein the fermentation treatment has a pH of 4.0-6.0, a fermentation time of 16-24 hours and a fermentation temperature of 28-30 ℃.
7. The use according to claim 4, wherein the yeast-containing material is obtained by fermentation treatment using Saccharomyces cerevisiae, molasses as carbon source and urea as nitrogen source.
8. The use according to claim 7, wherein the fermentation treatment has a pH of 4.0-6.0, a fermentation time of 16-24 hours and a fermentation temperature of 28-30 ℃.
9. The use according to claim 3, wherein in step (2) the yeast cell wall precipitate is diluted to a suspension having a concentration of 10-20% based on dry matter of the yeast cell wall precipitate before subjecting the yeast cell wall precipitate to enzymatic hydrolysis with alkaline protease, mannanase, beta-glucanase and cellulase.
10. The use according to claim 4, wherein in step (2) the yeast cell wall precipitate is diluted to a suspension having a concentration of 10-20% based on dry matter of the yeast cell wall precipitate before subjecting the yeast cell wall precipitate to enzymatic hydrolysis with alkaline protease, mannanase, beta-glucanase and cellulase.
11. The use according to claim 5, wherein in step (2) the yeast cell wall precipitate is diluted to a suspension having a concentration of 10-20% based on dry matter of the yeast cell wall precipitate before subjecting the yeast cell wall precipitate to enzymatic hydrolysis with alkaline protease, mannanase, beta-glucanase and cellulase.
12. The use according to claim 6, wherein in step (2) the yeast cell wall precipitate is diluted to a suspension having a concentration of 10-20% based on dry matter of the yeast cell wall precipitate before subjecting the yeast cell wall precipitate to enzymatic hydrolysis with alkaline protease, mannanase, beta-glucanase and cellulase.
13. The use according to claim 7, wherein in step (2) the yeast cell wall precipitate is diluted to a suspension having a concentration of 10-20% based on dry matter of the yeast cell wall precipitate before subjecting the yeast cell wall precipitate to enzymatic hydrolysis with alkaline protease, mannanase, beta-glucanase and cellulase.
14. The use according to claim 8, wherein in step (2) the yeast cell wall precipitate is diluted to a suspension having a concentration of 10-20% based on dry matter of the yeast cell wall precipitate before subjecting the yeast cell wall precipitate to enzymatic hydrolysis with alkaline protease, mannanase, beta-glucanase and cellulase.
15. The application of the yeast cell wall in preparing the fish feed is characterized in that the yeast cell wall is obtained by performing autolysis wall breaking on yeast, and then performing enzymolysis on alkaline protease, mannanase, beta-glucanase and cellulase one by one;
wherein, based on the dry matter mass of the yeast cell wall, the addition amounts of the alkaline protease, the mannanase, the beta-glucanase and the cellulase are respectively 2.0 to 5.5 per mill, 2.0 to 5.0 per mill, 0.2 to 0.8 per mill and 0.3 to 0.8 per mill;
wherein the enzymolysis temperature of the alkaline protease is 40-60 ℃, the pH value is 6.0-8.5, and the hydrolysis time is 7-10 hours;
the enzymolysis temperature of the mannase is 40-60 ℃, the pH is 6.0-8.0, and the hydrolysis time is 10-15 hours;
the enzymolysis temperature of the beta-glucanase is 55-60 ℃, the pH is 5.0-6.0, and the hydrolysis time is 5-8 hours;
the enzymolysis temperature of the cellulase is 50-65 ℃, the pH is 4.0-5.5, and the hydrolysis time is 8-10 hours.
16. The use according to claim 15, wherein the yeast cell wall is added to a fish feed for feeding aquatic animals.
17. The use according to claim 16, wherein 0.01% -0.5% of yeast cell wall is added by weight of the fish feed.
18. The use according to claim 15, wherein the yeast cell wall is mixed with a carrier to form a premix for feeding the aquatic animal.
19. The use according to claim 18, wherein the carrier is one or more of corn flour, wheat flour, soybean flour, rice bran or zeolite powder.
20. The use according to claim 15, wherein the yeast is saccharomyces cerevisiae FX-2 (Saccharomyces cerevisiae FX-2) with a preservation number of cctccc No. M2016418.
21. The use according to claim 15, wherein the preparation of the yeast cell wall comprises the steps of:
(1) Autolyzing the yeast-containing material to break wall and separating to obtain yeast cell wall precipitate;
(2) And (3) carrying out enzymolysis on the yeast cell wall precipitate one by using alkaline protease, mannanase, beta-glucanase and cellulase to obtain the yeast cell wall.
22. The use according to claim 21, wherein in step (1), the autolytic wall breaking is performed at a salt concentration of 2-4.5%, a pH of 5.0-6.5 and a temperature of 55-75 ℃.
23. The use according to claim 21, wherein the yeast-containing feedstock is obtained by fermentation using saccharomyces cerevisiae, molasses as a carbon source and urea as a nitrogen source.
24. Use according to claim 23, wherein the fermentation treatment has a pH of 4.0-6.0, a fermentation time of 16-24 hours and a fermentation temperature of 28-30 ℃.
25. The use according to claim 21, wherein in step (2) the yeast cell wall precipitate is diluted to a suspension having a concentration of 10-20% based on dry matter of the yeast cell wall precipitate before subjecting the yeast cell wall precipitate to enzymatic hydrolysis with alkaline protease, mannanase, beta-glucanase and cellulase.
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| JPS504290A (en) * | 1973-05-11 | 1975-01-17 | ||
| CN103589641A (en) * | 2012-08-17 | 2014-02-19 | 广州市纽溢乐商贸有限公司 | Three-step enzymatic hydrolysis method used for producing forage saccharomyces cerevisiae cells with broken-down walls |
| CN109303195A (en) * | 2017-07-28 | 2019-02-05 | 安琪酵母股份有限公司 | A kind of yeast cell wall and its preparation method and application of alternative antibiotic |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPS504290A (en) * | 1973-05-11 | 1975-01-17 | ||
| CN103589641A (en) * | 2012-08-17 | 2014-02-19 | 广州市纽溢乐商贸有限公司 | Three-step enzymatic hydrolysis method used for producing forage saccharomyces cerevisiae cells with broken-down walls |
| CN109303195A (en) * | 2017-07-28 | 2019-02-05 | 安琪酵母股份有限公司 | A kind of yeast cell wall and its preparation method and application of alternative antibiotic |
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