CN113854548A - Lysozyme-containing microecological preparation and preparation method thereof - Google Patents
Lysozyme-containing microecological preparation and preparation method thereof Download PDFInfo
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- CN113854548A CN113854548A CN202110804615.XA CN202110804615A CN113854548A CN 113854548 A CN113854548 A CN 113854548A CN 202110804615 A CN202110804615 A CN 202110804615A CN 113854548 A CN113854548 A CN 113854548A
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- 102000016943 Muramidase Human genes 0.000 title claims abstract description 51
- 108010014251 Muramidase Proteins 0.000 title claims abstract description 51
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 title claims abstract description 51
- 229960000274 lysozyme Drugs 0.000 title claims abstract description 51
- 235000010335 lysozyme Nutrition 0.000 title claims abstract description 51
- 239000004325 lysozyme Substances 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 43
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 14
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 14
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 14
- 239000008103 glucose Substances 0.000 claims abstract description 14
- 241000192125 Firmicutes Species 0.000 claims abstract description 9
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 9
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims abstract description 9
- 235000003704 aspartic acid Nutrition 0.000 claims abstract description 9
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 235000013922 glutamic acid Nutrition 0.000 claims abstract description 9
- 239000004220 glutamic acid Substances 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims description 37
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 238000003756 stirring Methods 0.000 claims description 24
- 239000002244 precipitate Substances 0.000 claims description 21
- 239000012153 distilled water Substances 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 18
- 238000005406 washing Methods 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 13
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims description 12
- 102000002322 Egg Proteins Human genes 0.000 claims description 12
- 108010000912 Egg Proteins Proteins 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 12
- 229940044197 ammonium sulfate Drugs 0.000 claims description 12
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 12
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 12
- 235000014103 egg white Nutrition 0.000 claims description 12
- 210000000969 egg white Anatomy 0.000 claims description 12
- 239000011347 resin Substances 0.000 claims description 12
- 229920005989 resin Polymers 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 238000011049 filling Methods 0.000 claims description 6
- 230000001376 precipitating effect Effects 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 235000015278 beef Nutrition 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 238000007789 sealing Methods 0.000 claims description 5
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 3
- 230000002378 acidificating effect Effects 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 239000003729 cation exchange resin Substances 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 235000018102 proteins Nutrition 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 238000000967 suction filtration Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 5
- 239000000463 material Substances 0.000 abstract description 5
- 244000144972 livestock Species 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 3
- 230000036039 immunity Effects 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000012136 culture method Methods 0.000 abstract description 2
- 244000005700 microbiome Species 0.000 description 3
- 238000012258 culturing Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 206010010774 Constipation Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention relates to the technical field of microecological preparations, in particular to a microecological preparation containing lysozyme, which comprises 15-25% of lysozyme, 10-20% of gram-positive bacteria, 3-5% of glutamic acid (Glu35), 3-5% of aspartic acid (Asp52), 10-20% of bacillus subtilis and 25-41% of glucose solution. The materials and the culture method used in the invention are simple, can quickly adjust the micro-ecology of the intestinal tracts of human bodies or livestock, mainly balance the micro-ecology of the intestinal tracts, strengthen the physique and further achieve the effect of improving the immunity of the bodies, and the materials used in the culture are simple, have complex environment and technology for operation and preparation, and are suitable for mass production.
Description
Technical Field
The invention relates to the technical field of microecologics, in particular to a lysozyme-containing microecologics and a preparation method thereof.
Background
A microecological preparation is a living microbial preparation prepared from normal microorganisms or substances for promoting the growth of the microorganisms, all preparations capable of promoting the growth and the reproduction of normal microorganisms and inhibiting the growth and the reproduction of pathogenic bacteria are called 'microecological preparations', the microecological preparation can quickly establish the microecological balance of the intestinal tract due to the effect of regulating the intestinal tract, diarrhea and constipation can be prevented and treated no matter in infants, old people or new livestock, the existing microecological preparation containing lysozyme has the defects of complexity and preparation method, cannot quickly regulate the microecology of the intestinal tract of human bodies or livestock to balance the intestinal tract, cannot strengthen physique to improve the immunity effect of the bodies, the materials used for culturing are expensive to increase the culturing cost, the environment and the technology for operation and preparation are complicated, and the preparation is not suitable for mass production.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a lysozyme-containing microecological preparation and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a microecological preparation containing lysozyme comprises 15% -25% of lysozyme, 10% -20% of gram-positive bacteria, 3% -5% of glutamic acid (Glu35), 3% -5% of aspartic acid (Asp52), 10% -20% of bacillus subtilis and 25% -41% of glucose solution.
A method for preparing lysozyme-containing microecological preparation, wherein the lysozyme has positive molecular charge in a wide pH value range below the isoelectric point, can be adsorbed on weakly acidic cation exchange resin, and is eluted and salted out to obtain lysozyme precipitate, and then refined to obtain the finished product, and the method comprises the following specific operation steps:
step one, washing with egg white adsorption ↓724resin, eluting with ↓buffersolution ↓ammoniumsulfate solution, dialyzing, removing alkaline protein ↓naohfreeze-dried lysozyme freeze-dried powder, precipitating and drying lysozyme, adding 540kg fresh egg white into treated 80kg724 resin, stirring and adsorbing for 6h, and standing for 12h at 0-5 ℃;
step two, standing, pouring out an upper layer of egg white resin, centrifuging and spin-drying, washing adhered egg white with distilled water repeatedly, then filling the resin into a column, washing the resin with about 150L of 15mol/L, pH-6.5 phosphoric acid buffer solution, eluting with 600L of 10% ammonium sulfate solution, collecting eluent, adding ammonium sulfate into the eluent to ensure that the content of ammonium sulfate is 40%, generating white precipitate, standing in a cold place for 12h, performing precipitation, suction-filtration and drying on supernatant liquor of siphoning, dissolving the precipitate with 1 time of distilled water to form a thin paste, and filling into a dialysis bag to dialyze distilled water for about 24h at the temperature of 3-5 ℃;
step three, changing water for 2-3 times within 24h after dialysis with distilled water, centrifuging to remove precipitates, washing the precipitates with a small amount of water once, combining washing liquor and centrifugate, slowly adding 1mol/L sodium hydroxide solution into dialyzed clear liquid, continuously stirring to increase the pH value to 8.0-9.0, centrifuging to remove the precipitates if white precipitates exist, then adjusting the pH value to 5.0 with 3mol/L hydrochloric acid, and freeze-drying to obtain white flaky lysozyme; and
or adjusting pH of the centrifugate to 3.5 with 3mol/L hydrochloric acid, slowly adding 5% solid sodium chloride under stirring, standing at about 5 deg.C for 48 hr, centrifuging, precipitating, washing with distilled water, and drying to obtain lysozyme.
Preferably, the culture medium of bacillus subtilis: 20L-40L of distilled water, 40kg-80kg of glucose, 30kg-60kg of peptone, 10kg-20kg of sodium chloride, 1kg-2kg of beef extract and 40kg-80kg of agar, wherein the pH value is 7 at the temperature of 30-37 ℃.
A preparation method of a lysozyme-containing microecological preparation comprises the following specific steps:
the method comprises the following steps: pouring 25% -41% of glucose solution into a reaction tank, keeping the temperature in the reaction tank to 30-37 ℃, then adding 15% -25% of lysozyme and 10% -20% of gram-positive bacteria, stirring for 1h, then sealing the reaction tank to prevent other bacteria from being added, and then keeping the temperature at 30-37 ℃ and standing for 12 h;
step two: opening a reaction tank which is kept stand for 12 hours, adding 3-5% of glutamic acid (Glu35) and 3-5% of aspartic acid (Asp52) into the reaction tank, stirring the mixture for 1 hour, covering the reaction tank, keeping the temperature between 30 ℃ and 37 ℃ and keeping stand for 6 hours;
step three: opening a reaction tank which is kept stand for 6h, adding 10-20% of bacillus subtilis, stirring for 1h, covering the reaction tank, keeping the temperature of 30-37 ℃, and keeping stand for 24h to obtain the lysozyme-containing microecological preparation.
The invention provides a lysozyme-containing microecological preparation and a preparation method thereof, and the beneficial effects are that:
the materials and the culture method used in the invention are simple, can quickly adjust the micro-ecology of the intestinal tracts of human bodies or livestock, mainly balance the micro-ecology of the intestinal tracts, strengthen the physique and further achieve the effect of improving the immunity of the bodies, and the materials used in the culture are simple, have complex environment and technology for operation and preparation, and are suitable for mass production.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "front", "rear", "both ends", "one end", "the other end", etc., indicate orientations or positional relationships based on the illustrated orientations or positional relationships, and are only for convenience of description and simplicity of description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it is to be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "disposed," "connected," and the like are to be construed broadly, such as "connected," which may be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
A microecological preparation containing lysozyme comprises 15% -25% of lysozyme, 10% -20% of gram-positive bacteria, 3% -5% of glutamic acid (Glu35), 3% -5% of aspartic acid (Asp52), 10% -20% of bacillus subtilis and 25% -41% of glucose solution.
A method for preparing lysozyme-containing microecological preparation, wherein the lysozyme has positive molecular charge in a wide pH value range below the isoelectric point, can be adsorbed on weakly acidic cation exchange resin, and is eluted and salted out to obtain lysozyme precipitate, and then refined to obtain the finished product, and the method comprises the following specific operation steps:
step one, washing with egg white adsorption ↓724resin, eluting with ↓buffersolution ↓ammoniumsulfate solution, dialyzing, removing alkaline protein ↓naohfreeze-dried lysozyme freeze-dried powder, precipitating and drying lysozyme, adding 540kg fresh egg white into treated 80kg724 resin, stirring and adsorbing for 6h, and standing for 12h at 0-5 ℃;
step two, standing, pouring out an upper layer of egg white resin, centrifuging and spin-drying, washing adhered egg white with distilled water repeatedly, then filling the resin into a column, washing the resin with about 150L of 15mol/L, pH-6.5 phosphoric acid buffer solution, eluting with 600L of 10% ammonium sulfate solution, collecting eluent, adding ammonium sulfate into the eluent to ensure that the content of ammonium sulfate is 40%, generating white precipitate, standing in a cold place for 12h, performing precipitation, suction-filtration and drying on supernatant liquor of siphoning, dissolving the precipitate with 1 time of distilled water to form a thin paste, and filling into a dialysis bag to dialyze distilled water for about 24h at the temperature of 3-5 ℃;
step three, changing water for 2-3 times within 24h after dialysis with distilled water, centrifuging to remove precipitates, washing the precipitates with a small amount of water once, combining washing liquor and centrifugate, slowly adding 1mol/L sodium hydroxide solution into dialyzed clear liquid, continuously stirring to increase the pH value to 8.0-9.0, centrifuging to remove the precipitates if white precipitates exist, then adjusting the pH value to 5.0 with 3mol/L hydrochloric acid, and freeze-drying to obtain white flaky lysozyme; and
or adjusting pH of the centrifugate to 3.5 with 3mol/L hydrochloric acid, slowly adding 5% solid sodium chloride under stirring, standing at about 5 deg.C for 48 hr, centrifuging, precipitating, washing with distilled water, and drying to obtain lysozyme.
The first embodiment is as follows: culture medium of bacillus subtilis: 20L of distilled water, 40kg of glucose, 30kg of peptone, 10kg of sodium chloride, 1kg of beef extract and 40kg of agar, wherein the pH value is 7 at the temperature of 30-37 ℃.
A preparation method of a lysozyme-containing microecological preparation comprises the following specific steps:
the method comprises the following steps: pouring 41 percent of glucose solution into a reaction tank, keeping the temperature in the reaction tank to be 30-37 ℃, then adding 15 percent of lysozyme and 10 percent of gram-positive bacteria, stirring for 1h, then sealing the reaction tank to prevent other bacteria from being added, and then keeping the temperature to be 30-37 ℃ and standing for 12 h;
step two: opening a reaction tank which is kept stand for 12h, adding 3 percent of glutamic acid (Glu35) and 3 percent of aspartic acid (Asp52), stirring for 1h, covering the reaction tank, keeping the temperature at 30-37 ℃, and keeping stand for 6 h;
step three: and opening the reaction tank for standing for 6h, adding 10 percent of bacillus subtilis, stirring for 1h, covering the reaction tank, and then keeping the temperature of 30-37 ℃ for standing for 24h to obtain the lysozyme-containing microecological preparation.
Example two: culture medium of bacillus subtilis: 30L of distilled water, 60kg of glucose, 45kg of peptone, 15kg of sodium chloride, 1.5kg of beef extract and 60kg of agar, wherein the pH value is 7 at the temperature of 30-37 ℃.
A preparation method of a lysozyme-containing microecological preparation comprises the following specific steps:
the method comprises the following steps: pouring 32% of glucose solution into a reaction tank, keeping the temperature in the reaction tank to be 30-37 ℃, then adding 20% of lysozyme and 15% of gram-positive bacteria, stirring for 1h, then sealing the reaction tank to prevent other bacteria from being added, and then keeping the temperature to be 30-37 ℃ and standing for 12 h;
step two: opening a reaction tank which is kept stand for 12h, adding 4 percent of glutamic acid (Glu35) and 4 percent of aspartic acid (Asp52), stirring for 1h, covering the reaction tank, keeping the temperature at 30-37 ℃, and keeping stand for 6 h;
step three: and opening the reaction tank for standing for 6h, adding 15 percent of bacillus subtilis, stirring for 1h, covering the reaction tank, and then keeping the temperature of 30-37 ℃ for standing for 24h to obtain the lysozyme-containing microecological preparation.
Example two: culture medium of bacillus subtilis: 40L of distilled water, 80kg of glucose, 60kg of peptone, 20kg of sodium chloride, 2kg of beef extract and 80kg of agar, wherein the pH value is 7 at the temperature of 30-37 ℃.
A preparation method of a lysozyme-containing microecological preparation comprises the following specific steps:
the method comprises the following steps: pouring 25% of glucose solution into a reaction tank, keeping the temperature in the reaction tank to be 30-37 ℃, then adding 25% of lysozyme and 20% of gram-positive bacteria, stirring for 1h, then sealing the reaction tank to prevent other bacteria from being added, and then keeping the temperature to be 30-37 ℃ and standing for 12 h;
step two: opening a reaction tank which is kept stand for 12h, adding 5 percent of glutamic acid (Glu35) and 5 percent of aspartic acid (Asp52), stirring for 1h, covering the reaction tank, keeping the temperature at 30-37 ℃, and keeping stand for 6 h;
step three: opening a reaction tank which is kept stand for 6h, adding bacillus subtilis with the concentration of 20 percent, stirring for 1h, covering the reaction tank, keeping the temperature of 30-37 ℃, and keeping stand for 24h to obtain the lysozyme-containing microecological preparation.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (4)
1. A lysozyme-containing microecological preparation is characterized in that: comprises 15-25% of lysozyme, 10-20% of gram-positive bacteria, 3-5% of glutamic acid (Glu35), 3-5% of aspartic acid (Asp52), 10-20% of bacillus subtilis and 25-41% of glucose solution.
2. The method for preparing a microecological preparation comprising lysozyme of claim 1, wherein the lysozyme has a positive molecular charge in a wide pH range below the isoelectric point, and can be adsorbed on a weakly acidic cation exchange resin, eluted and salted out to obtain a precipitate of lysozyme, and then refined to obtain a finished product, wherein the specific steps of the method are as follows:
step one, washing with egg white adsorption ↓724resin, eluting with ↓buffersolution ↓ammoniumsulfate solution, dialyzing, removing alkaline protein ↓naohfreeze-dried lysozyme freeze-dried powder, precipitating and drying lysozyme, adding 540kg fresh egg white into treated 80kg724 resin, stirring and adsorbing for 6h, and standing for 12h at 0-5 ℃;
step two, standing, pouring out an upper layer of egg white resin, centrifuging and spin-drying, washing adhered egg white with distilled water repeatedly, then filling the resin into a column, washing the resin with about 150L of 15mol/L, pH-6.5 phosphoric acid buffer solution, eluting with 600L of 10% ammonium sulfate solution, collecting eluent, adding ammonium sulfate into the eluent to ensure that the content of ammonium sulfate is 40%, generating white precipitate, standing in a cold place for 12h, performing precipitation, suction-filtration and drying on supernatant liquor of siphoning, dissolving the precipitate with 1 time of distilled water to form a thin paste, and filling into a dialysis bag to dialyze distilled water for about 24h at the temperature of 3-5 ℃;
step three, changing water for 2-3 times within 24h after dialysis with distilled water, centrifuging to remove precipitates, washing the precipitates with a small amount of water once, combining washing liquor and centrifugate, slowly adding 1mol/L sodium hydroxide solution into dialyzed clear liquid, continuously stirring to increase the pH value to 8.0-9.0, centrifuging to remove the precipitates if white precipitates exist, then adjusting the pH value to 5.0 with 3mol/L hydrochloric acid, and freeze-drying to obtain white flaky lysozyme; and
or adjusting pH of the centrifugate to 3.5 with 3mol/L hydrochloric acid, slowly adding 5% solid sodium chloride under stirring, standing at about 5 deg.C for 48 hr, centrifuging, precipitating, washing with distilled water, and drying to obtain lysozyme.
3. The method for preparing a lysozyme-containing microecological preparation according to any one of claims 1 to 2, wherein the culture medium of Bacillus subtilis: 20L-40L of distilled water, 40kg-80kg of glucose, 30kg-60kg of peptone, 10kg-20kg of sodium chloride, 1kg-2kg of beef extract and 40kg-80kg of agar, wherein the pH value is 7 at the temperature of 30-37 ℃.
4. The method for preparing a microecological preparation comprising lysozyme, according to claim 1, comprising the following steps:
the method comprises the following steps: pouring 25% -41% of glucose solution into a reaction tank, keeping the temperature in the reaction tank to 30-37 ℃, then adding 15% -25% of lysozyme and 10% -20% of gram-positive bacteria, stirring for 1h, then sealing the reaction tank to prevent other bacteria from being added, and then keeping the temperature at 30-37 ℃ and standing for 12 h;
step two: opening a reaction tank which is kept stand for 12 hours, adding 3-5% of glutamic acid (Glu35) and 3-5% of aspartic acid (Asp52) into the reaction tank, stirring the mixture for 1 hour, covering the reaction tank, keeping the temperature between 30 ℃ and 37 ℃ and keeping stand for 6 hours;
step three: opening a reaction tank which is kept stand for 6h, adding 10-20% of bacillus subtilis, stirring for 1h, covering the reaction tank, keeping the temperature of 30-37 ℃, and keeping stand for 24h to obtain the lysozyme-containing microecological preparation.
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| CN104800095A (en) * | 2015-04-22 | 2015-07-29 | 江苏雪豹日化有限公司 | Biological lysozyme compound preparation and preparation method thereof |
| CN110438107A (en) * | 2019-08-21 | 2019-11-12 | 徐州生物工程职业技术学院 | A kind of high-efficiency method for producing of lysozyme |
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- 2021-07-16 CN CN202110804615.XA patent/CN113854548A/en active Pending
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| CN1475567A (en) * | 2002-08-13 | 2004-02-18 | 阎洪世 | Preparation method of lysozyme |
| CN101530161A (en) * | 2008-03-10 | 2009-09-16 | 北京大北农科技集团股份有限公司 | Novel feed additive and preparation method thereof |
| CN102409015A (en) * | 2011-12-06 | 2012-04-11 | 北京大北农科技集团股份有限公司 | Composite micro-ecological preparation as well as premixed material and application of preparation in feed additive |
| CN104800095A (en) * | 2015-04-22 | 2015-07-29 | 江苏雪豹日化有限公司 | Biological lysozyme compound preparation and preparation method thereof |
| CN110438107A (en) * | 2019-08-21 | 2019-11-12 | 徐州生物工程职业技术学院 | A kind of high-efficiency method for producing of lysozyme |
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