WO2014194707A1 - 结石酶及其制备方法 - Google Patents
结石酶及其制备方法 Download PDFInfo
- Publication number
- WO2014194707A1 WO2014194707A1 PCT/CN2014/073456 CN2014073456W WO2014194707A1 WO 2014194707 A1 WO2014194707 A1 WO 2014194707A1 CN 2014073456 W CN2014073456 W CN 2014073456W WO 2014194707 A1 WO2014194707 A1 WO 2014194707A1
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- Prior art keywords
- enzyme
- parts
- culture
- fermentation
- powder
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 96
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 96
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 52
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000002363 auxin Substances 0.000 claims abstract description 26
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000000411 inducer Substances 0.000 claims abstract description 26
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- 235000015097 nutrients Nutrition 0.000 claims description 25
- 235000009496 Juglans regia Nutrition 0.000 claims description 22
- 235000020234 walnut Nutrition 0.000 claims description 22
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 18
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- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 10
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 8
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 8
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- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 6
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 6
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- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
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- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 4
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- 235000009337 Spinacia oleracea Nutrition 0.000 description 1
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- 235000013339 cereals Nutrition 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention relates to the technical field of enzyme or composite enzyme products, in particular to a stone enzyme for analysing human body stones and a preparation method thereof.
- the stone enzyme is an enzyme capable of degrading stones, which can be well applied to the decomposition of human stones; it is an enzyme obtained by microbial fermentation, extraction, concentration, and refining by using a solid-state fermentation method for raw and auxiliary materials.
- a complex enzyme product is an enzyme capable of degrading stones, which can be well applied to the decomposition of human stones; it is an enzyme obtained by microbial fermentation, extraction, concentration, and refining by using a solid-state fermentation method for raw and auxiliary materials.
- Reform and opening up have enabled our people to gradually move from poverty to prosperity. After entering a well-off society, people have eaten more and eat well, but there are more people who have stones. The reason is that substances that are eaten more are not absorbed into beneficial substances, but are transformed or accumulated as harmful substances. For example, eating high-fat foods, high-calcium foods (such as milk books, cheese, calcium-added foods), high-oxalic foods (such as spinach, tomatoes, potatoes, beets, etc.), sorghum foods, drugs, etc. People and people with diabetes are prone to stones (such as stomach stones, gallstones, kidney stones, urinary stones, etc.); stones can be tolerated at a very young age. When growing to a certain extent, they must use Western medicine. Means to remove stones and even remove the internal organs of the human body, pain and damage to the body are inevitable.
- high-calcium foods such as milk books, cheese, calcium-added foods
- high-oxalic foods such as spinach, tomatoes, potatoes, beets
- the object of the present invention is to provide a stone enzyme and a preparation method thereof according to the deficiencies of the prior art, and the stone enzyme can enter the human body in a completely painless and gentle manner to decompose the structure of the stone, which is milder than the traditional Chinese medicine. More specific, completely remove the stones in more than 7 days by means of lysis; the stone enzyme can be made into food, drink, health care products, medicines or injections, for people to use any way or at the same time to use various methods to resolve stones; Clinical practice has proved that the use of stone enzymes for the resolution of stones in the human body has a significant effect; the preparation method is simple and the production cost is low.
- the carbon source refers to D-glucose, D-fructose, D-mannose, D-sorbitol, sucrose, starch hydrolyzed sugar, starch, flour, corn flour, cellulosic powder, straw, chaff, sunflower dish, Wheat bran, malt, wort, papaya, pineapple, persimmon, banana, orange, orange, apple, fruit peel, fruit slag, beet, beet pulp, celery, vegetables, vegetable dregs, pearl powder, walnut powder, walnut residue, nuts More than one kind of material in slag, plant powder and plant residue.
- the nitrogen source is yeast extract, dry yeast, fish meal, peptone, beef extract, protein, snail, bean cake powder, peanut cake powder, fish skin, crab shell, shrimp shell, chitin, urea, amine, amide, hydrazine
- the nutrient salt is a mixed salt solution containing at least one of phosphorus, sulfur, magnesium, potassium, sodium, iron, calcium, zinc, cobalt, manganese, and selenium.
- the auxin is one or more substances selected from the group consisting of amino acids, purines, pyrimidines, vitamins, corn syrup, and bio-metabolizing agents.
- the enzyme-producing inducer is a material of artificial bovine gallstone or human stone crushed material.
- the stone enzyme is used as an enzyme for degrading stones, and is specifically used as a food, a drink, a health care product, a medicine or an injection in the decomposition of human stones; the solid form is white, light brown powder or granular, and the liquid is brown or transparent.
- the temperature is 25 ⁇ 50°C, the temperature is 15 ⁇ 50°C, and the enzyme concentration is 0. 5 ⁇ 5°C, the enzyme concentration is 0. l ⁇ 500g/L; the optimum F3 ⁇ 43. 5 ⁇ 6. 5, temperature 25 ⁇ 50°C, enzyme concentration 5 ⁇ 30g/L.
- the preparation method of the stone enzyme includes the following steps:
- the microbial strain is cultured in a slant surface of the microbial strain, and the microbial strain selected in the slant culture of the microbial strain is one or more strains of Aspergillus niger, Penicillium oxalicum, Yeast, Trichoderma, and Aspergillus oryzae;
- Microbial culture test tube slant culture The culture medium of the microbial strain is divided into 15 ⁇ 25 ml test tubes, and after sterilization, it is taken out to room temperature, and inoculated in a sterile room, the temperature is kept at a constant temperature incubator at 20-33 ° C. The culture is carried out for 24 to 72 hours to obtain a microbial strain cultured in a test tube;
- Expansion of microbial strains of triangular flasks The culture medium of the microbial strains is divided into 500 ml to 1000 ml flasks, and after sterilization, it is taken out to room temperature, and inoculated in a sterile room and cultured at a constant temperature of 20-33 ° C. ⁇ 72 hours, get a triangular bottle to expand Cultured microbial strains;
- the raw and auxiliary materials are selected, the raw and auxiliary materials to be pulverized are pulverized, and the raw and auxiliary materials are prepared according to the following parts by weight: Carbon source 20 ⁇ 30 parts
- the carbon source refers to D-glucose, D-fructose, D-mannose, D-sorbitol, sucrose, starch hydrolyzed sugar, starch, flour, corn flour, cellulosic powder, straw, chaff, sunflower dish , wheat bran, malt, wort, papaya, pineapple, persimmon, banana, orange, orange, apple, fruit peel, fruit slag, beet, beet pulp, celery, vegetables, vegetable residue, pearl powder, walnut powder, walnut residue, More than one material in nut residue, vegetable powder, and vegetable residue.
- the nitrogen source is yeast extract, dry yeast, fish meal, peptone, beef extract, protein, snail, bean cake powder, peanut cake powder, fish skin, crab shell, shrimp shell, chitin, urea, amine, amide, guanidine
- One or more materials such as purine base, pyrimidine base, ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium chloride, ammonium carbonate, ammonia, ammonia, and liquid ammonia.
- the nutrient salt is a mixed salt solution containing more than one of phosphorus, sulfur, magnesium, potassium, sodium, iron, calcium, zinc, cobalt, manganese, and selenium.
- the auxin is one or more of amino acids, purines, pyrimidines, vitamins, corn syrup, and biological metabolizing agents.
- the enzyme-producing inducer is a material of artificial bovine gallstone or human stone crushed material.
- the carbon source, the nitrogen source and the water are put into the fermentation incubator according to the parts by weight and the fixed volume of the fermentation incubator, and the raw materials that have been put into the fermentation incubator are mixed, stirred and hooked, and disinfected for use. ;
- the auxiliary material nutrient salt and auxin are put into the spare container according to the parts by weight, stirred and hooked, and disinfected for use;
- the enzyme-producing inducer is put into the spare container according to the weight fraction thereof, stirred and hooked, and disinfected for use. ;
- step (2) Mixing the auxiliary material and the enzyme-producing inducer of step (2) with the raw materials that have been put into the fermentation incubator, and stirring and hooking, so that the fermentation culture device has a fixed volume of 30% ⁇ 70% ;
- the expanded culture microbial strain obtained in the step (1) is inoculated into the fermentation incubator, and the inoculum amount is 5 to 30% ; the air is processed into the fermentation incubator by the air compressor and the purification system, and the pure air ventilation amount is 0. 1 ⁇ 1. OL/L'min;
- the culture time is 48 to 96 hours; the temperature is 20 to 33 ° C; the enthalpy value is 3. 5 ⁇ 7. 1; the culture time is 48 to 96 hours;
- culture leaching the culture in the step (3) after the termination of the culture is leached, adding water 1 to 5 times, leaching time 1 to 24 hours, plate frame pressure filtration or centrifugal filtration to obtain liquid and Residue
- concentration the liquid separated in the step (4) is subjected to ultrafiltration or vacuum concentration to obtain a concentrated liquid, and the concentrated liquid is packaged to obtain a crude liquid;
- the concentrated liquid obtained in the step (5) is subjected to filtration and purification to obtain a purified liquid, and the purified liquid is packaged to obtain a liquid product;
- the purified liquid obtained in the step (7) is sedimented, centrifuged, and lyophilized to obtain a lyophilized product as a solid product.
- composition and weight composition of the culture medium of the microbial strain described in the step (1) are as follows:
- the protein-rich residue separated in the step (4) is used for the second fermentation or production of a protein term or production of an organic fertilizer.
- the beneficial effects of the invention are as follows: 1.
- the stone enzyme can enter the human body in a completely painless and gentle manner to dissolve the structure of the broken stone, which is gentler and more specific than the traditional Chinese medicine, and is used for conditioning, inhibition and lysis. More than 7 days to completely remove the stones, remove the stones more completely and thoroughly.
- the stone enzyme can be made into a food, a drink, a health care product, a medicine or an injection, and can be used to inhibit and dissolve stones in any way or in various ways. 3. It has been proved by clinical practice that due to the specificity of the stone enzyme, there is no need to pass the gravel.
- the stone enzyme is used for different sizes of stones in different parts of the stone body, and has obvious curative effect, convenient to take, and no toxic and side effects.
- Embodiment 1 The stone enzyme according to the present embodiment is obtained by microbial fermentation culture, leaching, concentration and refining by the following raw parts by weight:
- the carbon source refers to a material of D-glucose, flour, chaff, wort, persimmon, beet, and walnut residue.
- the nitrogen source is a material of dry yeast, fish meal, bean cake powder, chitin, ammonium phosphate, ammonium nitrate.
- the nutrient salt is a mixed salt solution containing magnesium, potassium, calcium, zinc and selenium.
- auxin is a material of corn syrup.
- the enzyme-producing inducer is a human stone crushed material.
- the stone enzyme is used as an enzyme for degrading stones, and is specifically used as a food, a drink, a health care product, a medicine or an injection in the decomposition of human stones; the solid form is white, light brown powder or granular, and the liquid is brown or transparent. 5 ⁇
- the temperature is 15 ⁇ 85 ° C, the enzyme concentration is 0. l ⁇ 500g / L; optimum ⁇ 4. 5, temperature 40 ° C, enzyme concentration 15g / L.
- the preparation method of the stone enzyme includes the following steps:
- Microbial strain slant culture the microbial strain selected in the slant culture of microbial strains is Penicillium oxalicum
- Microbial strain test tube slant culture The culture medium of the microbial strain is divided into 20 ml test tubes. After sterilization, it was taken out to room temperature, and inoculated in a sterile room, and then cultured at a constant temperature incubator at 30 ° C for 48 hours to obtain a microbial strain cultured in a test tube slant;
- Expanded culture of microbial strains of triangular flasks Dispense the culture medium of microbial strains in a 1000 ml flask, remove them to room temperature after sterilization, and inoculate them in a sterile chamber for 48 hours at 30 ° C to obtain a triangle. The bottle expands the cultured microbial strain;
- the raw and auxiliary materials are selected, the raw and auxiliary materials to be pulverized are pulverized, and the raw and auxiliary materials are prepared according to the following parts by weight: 30 carbon sources
- the carbon source refers to a material of D-glucose, flour, chaff, wort, persimmon, beet, and walnut residue.
- the nitrogen source is a material of dry yeast, fish meal, bean cake powder, chitin, ammonium phosphate.
- the nutrient salt is a mixed salt solution containing magnesium, potassium, calcium, zinc and selenium.
- auxin is a material of corn syrup.
- the enzyme-producing inducer is a human stone crushed material.
- the carbon source, the nitrogen source and the water are put into the fermentation incubator according to the parts by weight and the fixed volume of the fermentation incubator, and the raw materials that have been put into the fermentation incubator are mixed, stirred and hooked, and disinfected for use. ;
- the auxiliary material nutrient salt and auxin are put into the spare container according to the parts by weight, stirred and hooked, and disinfected for use;
- the enzyme-producing inducer is put into the spare container according to the weight fraction thereof, stirred and hooked, and disinfected for use. ;
- the expanded culture microbial strain obtained in the step (1) is inoculated into the fermentation incubator, and the inoculum amount is 15%;
- the air is ventilated by the air compressor and the purification system, and the air is ventilated to a volume of 0. 5 / L * min;
- the fermentation culture apparatus is subjected to microbial fermentation culture using the following culture conditions: a temperature of 30 ° C ; a 13 ⁇ 4 value of 5.5; a culture time of 72 hours;
- culture leaching the culture in the step (3) after the termination of the culture is leached, water is added 3 times, the leaching time is 12 hours, and the plate and frame are subjected to pressure filtration or centrifugal filtration to obtain a liquid and a residue;
- the concentrated liquid obtained in the step (5) is filtered and purified to obtain a purified liquid, and the purified liquid is packaged to obtain a liquid product;
- the purified liquid obtained in the step (7) is sedimented, centrifuged, and lyophilized to obtain a lyophilized product. It is a solid boutique.
- composition and weight composition of the culture medium of the microbial strain described in the step (1) are as follows:
- the protein-rich residue separated in the step (4) is used for the second fermentation or production of a protein term or production of an organic fertilizer.
- the second embodiment is the stone enzyme according to the present embodiment, which is obtained by microbial fermentation culture, leaching, concentration and refining by the following raw parts by weight:
- the carbon source refers to a material of wheat bran, sucrose, orange, orange, apple, and banana.
- the nitrogen source is a material of beef extract, dry yeast, peanut cake powder, shrimp shell, ammonium sulfate.
- the nutrient salt is a mixed salt solution containing phosphorus, sulfur, sodium, iron, calcium, zinc, cobalt and manganese ions.
- auxin is a vitamin material.
- the enzyme-producing substance is a material of artificial bovine gallstones and human stone crushing materials.
- the stone enzyme is used as an enzyme for degrading stones, and is specifically used as a food, a drink, a health care product, a medicine or an injection in the decomposition of human stones; the solid form is white, light brown powder or granular, and the liquid is brown or transparent.
- the temperature is 50.
- the temperature is 50, and the enzyme concentration is 5g/L.
- the temperature is 15 ⁇ 85 °C, the enzyme concentration is 0. l ⁇ 500g/L;
- the preparation method of the stone enzyme includes the following steps:
- microbial strain slant culture the microbial strain selected in the cultivation of microbial strain slant is Aspergillus
- Microbial strain test tube slant culture The culture medium of the microbial strain is divided into 25 ml test tubes, and after sterilization, it is taken out to room temperature, inoculated in a sterile room, and cultured at a constant temperature incubator at 33 ° C for 72 hours. Obtaining a microbial strain cultured in a test tube;
- Expanded culture of microbial strains The medium of microbial strains is divided into 1000ml flasks, sterilized and taken to room temperature. After inoculation in a sterile room, incubate at 33 °C for 72 hours to obtain a triangle. The bottle expands the cultured microbial strain;
- the raw and auxiliary materials are selected, the raw and auxiliary materials to be pulverized are pulverized, and the raw and auxiliary materials are prepared in the following parts by weight: Carbon source 25 parts
- the carbon source refers to a material of wheat bran, sucrose, orange, orange, apple, and banana.
- the nitrogen source is a material of beef extract, dry yeast, peanut cake powder, shrimp shell, ammonium sulfate.
- the nutrient salt is a mixed salt solution containing phosphorus, sulfur, sodium, iron, calcium, zinc, cobalt and manganese ions.
- auxin is a vitamin material.
- the enzyme-producing substance is a material of artificial bovine gallstones and human stone crushing materials.
- the carbon source, the nitrogen source and the water are put into the fermentation incubator according to the parts by weight and the fixed volume of the fermentation incubator, and the raw materials that have been put into the fermentation incubator are mixed, stirred and hooked, and disinfected for use. ;
- the auxiliary material nutrient salt and auxin are put into the spare container according to the parts by weight, stirred and hooked, and disinfected for use;
- the enzyme-producing inducer is put into the spare container according to the weight fraction thereof, stirred and hooked, and disinfected for use. ;
- the expanded culture microbial strain obtained in the step (1) is inoculated into the fermentation incubator, and the inoculum amount is 30%;
- the air is processed into the fermentation incubator after being processed by the air compressor and the purification system, and the pure air ventilation is 1. 0L/L* min;
- the fermentation culture apparatus is subjected to microbial fermentation culture using the following culture conditions: a temperature of 33 ° C ; a 13 ⁇ 4 value of 6.5; a culture time of 96 hours; (4), culture leaching: the culture in the step (3) after the termination of the culture is leached, water is added 5 times, the leaching time is 24 hours, plate and frame filtration or centrifugal filtration to obtain liquid and residue;
- concentration the liquid separated in the step (4) is subjected to ultrafiltration or vacuum concentration to obtain a concentrated liquid, and the concentrated liquid is packaged to obtain a crude liquid;
- the concentrated liquid obtained in the step (5) is subjected to filtration and purification to obtain a purified liquid, and the purified liquid is packaged to obtain a liquid product;
- the purified liquid obtained in the step (7) is sedimented, centrifuged, and lyophilized to obtain a lyophilized product as a solid product.
- composition and weight composition of the culture medium of the microbial strain described in the step (1) are as follows:
- the protein-rich residue separated in the step (4) is used for the second fermentation or production of a protein term or production of an organic fertilizer.
- Embodiment 3 The stone enzyme according to the embodiment is obtained by microbial fermentation culture, leaching, concentration and refining by the following raw parts by weight of raw materials and auxiliary materials:
- carbon source refers to D-fructose, D-sorbitol, starch hydrolyzed sugar, starch, corn flour, straw, sunflower dish, malt, papaya, pineapple, fruit peel, fruit residue, beet pulp, celery, pearl powder, Walnut powder material.
- the nitrogen source is yeast extract, fish meal, peptone, protein, snail, fish skin, crab shell, chitin, urea, A material of amine, amide, purine base, pyrimidine base, ammonium phosphate, ammonium chloride, ammonium carbonate, liquid ammonia.
- the nutrient salt is a mixed salt solution containing phosphorus, sulfur, magnesium, potassium, sodium, iron, calcium, zinc, cobalt, manganese, and selenium ions.
- the auxin is a material of an amino acid, a purine or a pyrimidine.
- the enzyme-producing inducer is a material of artificial bovine gallstones.
- the stone enzyme is used as an enzyme for degrading stones, and is specifically used as a food, a drink, a health care product, a medicine or an injection in the decomposition of human stones; the solid form is white, light brown powder or granular, and the liquid is brown or transparent. ; using conditions PH2 5 ⁇ 7 5, a temperature of 15 ⁇ 85 ° C, an enzyme concentration of 0. l ⁇ 500g / L;... the optimum ⁇ 6 5, a temperature of 25 ° C, an enzyme concentration of 30g / L o
- the preparation method of the stone enzyme includes the following steps:
- Microbial strain slant culture the microbial strain selected in the cultivating of microbial strains is yeast;
- microbial strain test tube slant culture the culture medium of the microbial strain is divided into 15 ml test tubes, After sterilization, it was taken out to room temperature, and inoculated in a sterile room, and then cultured at a constant temperature incubator at 20 ° C for 24 hours to obtain a microbial strain cultured in a test tube slant;
- Expansion of microbial strains of triangular flasks The culture medium of the microbial strains is divided into 500 ml triangular flasks, and after sterilization, it is taken out to room temperature, inoculated in a sterile room, and cultured at 20 ° C for 24 hours at constant temperature to obtain a triangle.
- the bottle expands the cultured microbial strain;
- the raw and auxiliary materials are selected, the raw and auxiliary materials to be pulverized are pulverized, and the raw and auxiliary materials are prepared in the following parts by weight: Carbon source 20 parts
- carbon source refers to D-fructose, D-sorbitol, starch hydrolyzed sugar, starch, corn flour, straw, sunflower dish, malt, papaya, pineapple, fruit peel, fruit residue, beet pulp, celery, pearl powder, Walnut powder material.
- the nitrogen source is yeast extract, fish meal, peptone, protein, snail, fish skin, crab shell, chitin, urea, amine, amide, purine base, pyrimidine base, ammonium phosphate, ammonium chloride, ammonium carbonate, liquid Ammonia material.
- the nutrient salt is a mixed salt solution containing phosphorus, sulfur, magnesium, potassium, sodium, iron, calcium, zinc, cobalt, manganese, and selenium ions.
- the auxin is a material of an amino acid, a purine or a pyrimidine.
- the enzyme-producing inducer is a material of artificial bovine gallstones.
- the carbon source, the nitrogen source and the water are put into the fermentation incubator according to the parts by weight and the fixed volume of the fermentation incubator, and the raw materials that have been put into the fermentation incubator are mixed, stirred and hooked, and disinfected for use. ;
- the auxiliary material nutrient salt and auxin are put into the spare container according to the parts by weight, stirred and hooked, and disinfected for use;
- the enzyme-producing inducer is put into the spare container according to the weight fraction thereof, stirred and hooked, and disinfected for use. ;
- the expanded culture microbial strain obtained in the step (1) is inoculated into the fermentation incubator, and the inoculum amount is 5%;
- the air is ventilated through the air compressor and the purification system, and the air is ventilated to a volume of 0.1 L / L * min;
- the fermentation culture apparatus is subjected to microbial fermentation culture under the following culture conditions: a temperature of 20 ° C ; a ra value of 7.1; a culture time of 24 hours;
- culture leaching the culture in the step (3) after the termination of the culture is leached, water is added 1 time, the leaching time is 1 hour, and the plate is subjected to pressure filtration or centrifugal filtration to obtain a liquid and a residue;
- the concentrated liquid obtained in the step (5) is filtered and purified to obtain a purified liquid, and the purified liquid is packaged to obtain a liquid product;
- the purified liquid obtained in the step (7) is sedimented, centrifuged, and lyophilized to obtain a lyophilized product as a solid product.
- composition and weight composition of the culture medium of the microbial strain described in the step (1) are as follows:
- step (4) the protein-rich residue separated in step (4) is used for the second fermentation or production of protein materials. Or produce organic fertilizer.
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Abstract
提供了一种结石酶及其制备方法,该酶能够降解结石,其制备方法包括由下列重量份数的原料利用固态发酵法进行微生物发酵培养,经浸提、浓缩、精制获得该酶:碳源20-30份、氮源1-10份、水40-94份、营养盐1-10份、生长素1-5份、产酶诱导物1-10份,所述产酶诱导物是人工牛胆结石或人体结石粉碎物。
Description
结石酶及其制备方法
技术领域
本发明涉及酶或复合型酶产品技术领域, 特别是涉及一种用于化解人体结石的结石酶及 其制备方法。
背景技术
结石酶是一种能降解结石的酶,说能很好的运用到化解人体结石; 它是由有关原辅材料利 用固态发酵法进行微生物发酵培养、 经浸提、 浓缩、 精制而获得的酶或复合型酶产品。
改革开放使我国人民逐步从贫穷迈向富裕, 进入小康后, 人们吃得多了, 吃得好了, 但 是, 得结石的人也多了。 原因是吃多了的物质没有被吸收转化为有益物质, 而是转变或积累 为有害物质。 例如吃高脂肪食物、 高钙食物 (如牛奶书、 奶酪、 加钙食品)、 高草酸食物 (如 菠菜、 番茄、 土豆、 甜菜等)、 高嘌吟食物、 药物等而又少活动的亚健康人群以及糖尿病患 者等, 均容易患上结石 (如胃结石、 胆结石、 肾结石、 尿结石等); 结石在很小的时候还能 忍受, 当长大到一定程度时, 就要动用西医的手段来去除结石甚至去除人体的内脏, 疼痛和 对身体的损伤实在难免。
临床实践证明, 单纯使用西药治疗结石病其效果不理想, 且长期服用西药, 其副作用大, 病人难以耐受。 当结石病严重时, 需要采取手术才能解决问题, 给人们造成极大的痛苦。 近 些年人们研究使用中药治疗结石病, 但有缺陷, 由于中药的专一性不强并主要是以凉性药为 主, 其副作用难以用于平时对结石病的预防, 遇到大的结石还是要先碎石再使用中药治疗去 除结石, 治疗时间长, 疗效不显著。
因此, 人们迫切希望一种疗效显著、 服用方便、 无毒副作用的针对人体任何部位无论大 小结石都能治疗结石和平时预防结石的结石酶问世。 所述结石酶经临床实践证明, 将结石酶 用于化解人体内的结石有着显著的疗效。
发明内容
本发明的目的在于针对现有技术的不足, 提供一种结石酶及其制备方法, 该结石酶可以 完全无疼痛的、 温和的方式进入人体以化解方式破坏结石的结构, 比中药使用起来更温和更 专一, 以化解的方式 7天以上完全彻底的去除结石; 该结石酶可做成食品、 饮品、 保健品、 药品或针剂, 供人们采用任一方式或同时采用多种方式化解结石; 经临床实践证明, 将结石 酶用于化解人体内的结石有着显著的效果; 该制备方法简单, 生产成本低。
为实现上述目的, 本发明采用以下技术方案:
结石酶,它是由下列重量份数的原辅材料利用固态发酵法进行微生物发酵培养、经浸提、 浓缩、 精制而获得的:
碳源 20〜30份
氮源 1〜10份
水 40〜94份
营养盐 1〜10份
生长素 1〜5份
产酶诱导物 1〜10份。
其中所述碳源是指 D-葡萄糖、 D-果糖、 D-甘露糖、 D-山梨醇、蔗糖、淀粉水解糖、淀粉、 面粉、 玉米粉、 纤维质粉、 秸秆、 谷壳、 葵花盘、 麦麸、 麦芽、 麦芽汁、 木瓜、 菠萝、 柿子、 香蕉、 橘子、 橙子、 苹果、 水果皮、 水果渣、 甜菜、 甜菜渣、 芹菜、 蔬菜、 蔬菜渣、 珍珠粉、 核桃粉、 核桃渣、 坚果渣、 植物质粉、 植物渣中一种以上的物料。
其中所述氮源是酵母膏、 干酵母、 鱼粉、 蛋白胨、 牛肉膏、 蛋白质、 蜗牛、 豆饼粉、 花 生饼粉、 鱼皮、 蟹壳、 虾壳、 甲壳素、 尿素、 胺、 酰胺、 嘌吟碱、 嘧啶碱、 硫酸铵、 磷酸铵、 硝酸铵、 氯化铵、 碳酸铵、 氨水、 氨气、 液氨中一种以上的物料。
其中所述营养盐是含磷、 硫、 镁、 钾、 钠、 铁、 钙、 锌、 钴、 锰、 硒中一种以上离子的 混合盐液。
其中所述生长素是氨基酸、 嘌吟、 嘧啶、 维生素、 玉米浆、 生物代谢剂中一种以上的物 料。
其中所述产酶诱导物是人工牛胆结石或人体结石粉碎物的物料。
所述结石酶作为降解结石的酶, 具体作为食品、 饮品、 保健品、 药品或针剂在化解人体 结石中的应用; 其形态固体为白色、 浅褐色粉状或颗粒状, 液体为褐色或透明状; 其使用条 件为 ΓΉ2. 5〜7. 5, 温度 15〜85°C, 酶浓度 0. l〜500g/L; 最适 F¾3. 5〜6. 5, 温度 25〜50°C, 酶浓度 5〜30g/L。 结石酶的制备方法, 包括如下步骤:
( 1 ) 、 微生物菌种斜面培养, 在微生物菌种斜面培养中选用的的微生物菌种是黑曲霉 菌、 草酸青霉菌、 酵母菌、 木霉菌、 米曲霉菌中一种以上的菌种;
微生物菌种试管斜面培养: 将微生物菌种的培养基分装于 15〜25 毫升的试管中, 经灭 菌后取出降至室温, 在无菌室接种后于恒温培养箱 20-33°C恒温培养 24〜72小时, 获得试管 斜面培养的微生物菌种;
微生物菌种三角瓶扩大培养:将微生物菌种的培养基分装于 500ml〜1000ml的三角瓶中, 经灭菌后取出降至室温, 在无菌室接种后于 20-33°C恒温培养 24〜72小时, 获得三角瓶扩大
培养的微生物菌种;
( 2 )、 原辅材料前处理:
将原辅材料选材,对需要粉碎的原辅材料进行粉碎,再将原辅材料按以下重量份数备料: 碳源 20〜30份
氮源 1〜10份
水 40〜94份
营养盐 1〜10份
生长素 1〜5份
产酶诱导物 1〜10份。
其中: 所述碳源是指 D-葡萄糖、 D-果糖、 D-甘露糖、 D-山梨醇、 蔗糖、 淀粉水解糖、 淀 粉、 面粉、 玉米粉、 纤维质粉、 秸秆、 谷壳、 葵花盘、 麦麸、 麦芽、 麦芽汁、 木瓜、 菠萝、 柿子、 香蕉、 橘子、 橙子、 苹果、 水果皮、 水果渣、 甜菜、 甜菜渣、 芹菜、 蔬菜、 蔬菜渣、 珍珠粉、 核桃粉、 核桃渣、 坚果渣、 植物质粉、 植物渣中一种以上的物料。
其中: 所述氮源是酵母膏、 干酵母、 鱼粉、 蛋白胨、 牛肉膏、 蛋白质、 蜗牛、 豆饼粉、 花生饼粉、 鱼皮、 蟹壳、 虾壳、 甲壳素、 尿素、 胺、 酰胺、 嘌吟碱、 嘧啶碱、 硫酸铵、 磷酸 铵、 硝酸铵、 氯化铵、 碳酸铵、 氨水、 氨气、 液氨中一种以上的物料。
其中: 所述营养盐是含磷、 硫、 镁、 钾、 钠、 铁、 钙、 锌、 钴、 锰、 硒中一种以上离子 的混合盐液。
其中: 所述生长素是氨基酸、 嘌吟、 嘧啶、 维生素、 玉米浆、 生物代谢剂中一种以上的 物料。
其中: 所述产酶诱导物是人工牛胆结石或人体结石粉碎物的物料。
将碳源、氮源和水按照其重量份数及发酵培养器的投料定容的 80%投入到发酵培养器内, 将已投入到发酵培养器内的原材料混合, 搅拌均勾, 消毒待用;
将辅助材料营养盐、 生长素按照其重量份数投入到备用容器内, 搅拌均勾, 消毒待用; 将产酶诱导物按照其重量份数投入到备用容器内, 搅拌均勾, 消毒待用;
( 3 )、 发酵培养器培养:
将步骤 (2 ) 的辅助材料和产酶诱导物与已投入到发酵培养器内的原材料进行混合, 搅 拌均勾, 使发酵培养器的投料定容为 30%〜70%;
将步骤 (1 ) 获得的扩大培养的微生物菌种接种到发酵培养器内, 接种量为 5〜30%; 将空气经空气压缩机和净化系统处理后输入发酵培养器内, 纯净空气通风量为 0. 1〜 1. OL/L'min;
在发酵培养器内采用如下培养条件进行微生物发酵培养:温度为 20〜33°C ; ΓΉ值为 3. 5〜 7. 1; 培养时间为 48〜96小时;
( 4)、 培养物浸提: 将步骤 (3 ) 中的终止培养后的培养物进行浸提, 加水 1〜5倍, 浸 提时间 1〜24小时, 板框压滤或离心过滤获得液体和残渣;
( 5 )、 浓缩: 将步骤 (4 ) 中分离出的液体进行超滤或真空浓缩获得浓缩后的液体, 将 浓缩后的液体包装, 获得液体粗品;
( 6 )、 干燥: 将步骤 (5 ) 中获得的浓缩后的液体进行喷雾干燥, 获得固体粗品;
( 7 )、 精制: 将步骤 (5 ) 中获得的浓缩后的液体进行过滤精制, 获得精制后的液体, 将精制后的液体包装, 获得液体精品;
( 8 )、 冻干: 将步骤 (7 ) 中获得的精制后的液体进行沉降、 离心、 冻干, 获得冻干品 为固体精品。
其中, 步骤 (1 ) 中所述的微生物菌种的培养基每升成份、 重量组成如下:
试管斜面培养基配方
去皮马铃薯 10000〜30000 mg, 葡萄糖 100〜1000 mg, 柿饼细粒 100〜3000mg, 核桃粉 100〜1000 mg, 人体结石的粉碎物 100〜1000 mg, 琼脂 10000〜20000 mg, 用水定容至 1升 三角瓶扩大培养基配方
NaN03 1000〜 10000 mg, KC1 100〜1000 mg, K2HP04 500〜2000 mg, KH2P04 1000〜5000 mg, (NH4) 2S04 1000〜5000 mg , MgS04 · 7H20 100〜2000 mg, CaCl2 100〜2000 mg, FeS04 1〜 10 mg, MnS04 · 0 l〜8mg, ZnS04 · 0 l〜5mg, C。C12 0. l〜lmg, N¾Se030. 1〜0. 3mg, 尿素 100〜3000mg, 甲壳素 100〜3000mg, 香蕉粉 100〜1000 mg, 核桃粉 100〜1000 mg, 人体结 石的粉碎物 100〜1000 mg, 用水定容至 1升容量。
进一步的, 将步骤 (4 ) 中分离出的富含蛋白质的残渣用于第二次发酵或生产蛋白词料 或生产有机肥。
本发明的有益效果是: 1、 该结石酶可以完全无疼痛的、 温和的方式进入人体以化解方 式破环结石的结构, 比中药使用起来更温和更专一, 以调理、 抑制、 化解的方式 7天以上完 全彻底的去除结石, 去除结石更完全更彻底。 2、 该结石酶可做成食品、 饮品、 保健品、 药 品或针剂, 供人们采用任一方式或同时采用多种方式抑制和化解结石。 3、 经临床实践证明, 由于结石酶的专一性, 故不需经过碎石, 结石酶用于结石人体内不同部位的不同大小的结石 均有显著的疗效, 服用方便, 且无毒副作用。 4、 服用该结石酶可以在平时预防结石病。 5、 该结石酶的制备方法简单, 生产成本低。 6、 该结石酶在生产过程中不会出现废水、 废气和
废渣, 符合环保要求。 7、 所述结石酶自 2002年研究以来经实践证明, 将结石酶用于化解人 体内的结石有着以上显著的效果。
具体实施方式
下面用三个具体实施例对本发明作进一步详细的说明, 并不是把本发明的实施范围局限 于此。
实施例一, 本实施例所述的结石酶, 它是由下列重量份数的原辅材料利用固态发酵法进 行微生物发酵培养、 经浸提、 浓缩、 精制而获得的:
碳源 30份
氮源 1份
水 94份
营养盐 10份
生长素 5份
产酶诱导物 10份。
其中所述碳源是指 D-葡萄糖、 面粉、 谷壳、 麦芽汁、 柿子、 甜菜、 核桃渣的物料。 其中所述氮源是干酵母、 鱼粉、 豆饼粉、 甲壳素、 磷酸铵、 硝酸铵的物料。
其中所述营养盐是含镁、 钾、 钙、 锌、 硒的混合盐液。
其中所述生长素是玉米浆的物料。
其中所述产酶诱导物是人体结石粉碎物。
所述结石酶作为降解结石的酶, 具体作为食品、 饮品、 保健品、 药品或针剂在化解人体 结石中的应用; 其形态固体为白色、 浅褐色粉状或颗粒状, 液体为褐色或透明状; 其使用条 件为 PH2. 5〜7. 5, 温度 15〜85°C, 酶浓度 0. l〜500g/L; 最适 ΓΉ4. 5, 温度 40°C, 酶浓度 15g/L。 结石酶的制备方法, 包括如下步骤:
( 1 )、 微生物菌种斜面培养, 在微生物菌种斜面培养中选用的的微生物菌种是草酸青霉菌; 微生物菌种试管斜面培养: 将微生物菌种的培养基分装于 20 毫升的试管中, 经灭菌后 取出降至室温, 在无菌室接种后于恒温培养箱 30°C恒温培养 48小时, 获得试管斜面培养的 微生物菌种;
微生物菌种三角瓶扩大培养: 将微生物菌种的培养基分装于 1000ml 的三角瓶中, 经灭 菌后取出降至室温, 在无菌室接种后于 30°C恒温培养 48小时, 获得三角瓶扩大培养的微生 物菌种;
( 2)、 原辅材料前处理:
将原辅材料选材,对需要粉碎的原辅材料进行粉碎,再将原辅材料按以下重量份数备料:
碳源 30份
氮源 1份
水 94份
营养盐 10份
生长素 5份
产酶诱导物 10份。
其中所述碳源是指 D-葡萄糖、 面粉、 谷壳、 麦芽汁、 柿子、 甜菜、 核桃渣的物料。 其中所述氮源是干酵母、 鱼粉、 豆饼粉、 甲壳素、 磷酸铵的物料。
其中所述营养盐是含镁、 钾、 钙、 锌、 硒的混合盐液。
其中所述生长素是玉米浆的物料。
其中所述产酶诱导物是人体结石粉碎物。
将碳源、氮源和水按照其重量份数及发酵培养器的投料定容的 80%投入到发酵培养器内, 将已投入到发酵培养器内的原材料混合, 搅拌均勾, 消毒待用;
将辅助材料营养盐、 生长素按照其重量份数投入到备用容器内, 搅拌均勾, 消毒待用; 将产酶诱导物按照其重量份数投入到备用容器内, 搅拌均勾, 消毒待用;
( 3)、 发酵培养器培养:
将步骤 (2) 的辅助材料和产酶诱导物与已投入到发酵培养器内的原材料进行混合, 搅 拌均勾, 使发酵培养器的投料定容为 50%;
将步骤 (1 ) 获得的扩大培养的微生物菌种接种到发酵培养器内, 接种量为 15%;
将空气经空气压缩机和净化系统处理后输入发酵培养器内, 纯净空气通风量为 0. 5/L* min;
在发酵培养器内采用如下培养条件进行微生物发酵培养: 温度为 30°C ; 1¾值为5. 5; 培 养时间为 72小时;
(4)、 培养物浸提: 将步骤 (3) 中的终止培养后的培养物进行浸提, 加水 3倍, 浸提 时间 12小时, 板框压滤或离心过滤获得液体和残渣;
( 5)、 浓缩: 将步骤 (4) 中分离出的液体进行超滤或真空浓缩获得浓缩后的液体, 将 浓缩后的液体包装, 获得液体粗品;
(6)、 干燥: 将步骤 (5) 中获得的浓缩后的液体进行喷雾干燥, 获得固体粗品;
( 7)、 精制: 将步骤 (5 ) 中获得的浓缩后的液体进行过滤精制, 获得精制后的液体, 将精制后的液体包装, 获得液体精品;
( 8)、 冻干: 将步骤 (7 ) 中获得的精制后的液体进行沉降、 离心、 冻干, 获得冻干品
为固体精品。
其中, 步骤 (1 ) 中所述的微生物菌种的培养基每升成份、 重量组成如下:
试管斜面培养基配方
去皮马铃薯 20000 mg, 葡萄糖 500 mg, 柿饼细粒 1500mg, 核桃粉 500mg, 人体结石的 粉碎物 500 mg, 琼脂 15000 mg, 用水定容至 1升容量。
三角瓶扩大培养基配方
NaN03 5000 mg, KC1 500 mg , K2HP04 1200 mg, KH2P04 2500 mg , ( NH4) 2S04 2500 mg , MgS04 · 7H20 1000 mg, CaCl2 1200 mg, FeS04 5 mg, MnS04 · H20 4mg, ZnS04 · H20 3mg, C。C12 0. 5mg, N¾Se030. 2mg, 尿素 2000mg, 甲壳素 1500mg, 香蕉粉 500 mg, 核桃粉 500 mg, 人体 结石的粉碎物 500 mg, 用水定容至 1升容量。
进一步的, 将步骤 (4 ) 中分离出的富含蛋白质的残渣用于第二次发酵或生产蛋白词料 或生产有机肥。
实施例二, 本实施例所述的结石酶, 它是由下列重量份数的原辅材料利用固态发酵法进 行微生物发酵培养、 经浸提、 浓缩、 精制而获得的:
碳源 25份
氮源 6份
水 68份
营养盐 5份
生长素 3份
产酶诱导物 6份。
其中所述碳源是指麦麸、 蔗糖、 橘子、 橙子、 苹果、 香蕉的物料。
其中所述氮源是牛肉膏、 干酵母、 花生饼粉、 虾壳、 硫酸铵的物料。
其中所述营养盐是含磷、 硫、 钠、 铁、 钙、 锌、 钴、 锰离子的混合盐液。
其中所述生长素是维生素的物料。
其中所述产酶诱导物是人工牛胆结石和人体结石粉碎物的物料。
所述结石酶作为降解结石的酶, 具体作为食品、 饮品、 保健品、 药品或针剂在化解人体 结石中的应用; 其形态固体为白色、 浅褐色粉状或颗粒状, 液体为褐色或透明状; 其使用条 件为 PH2. 5〜7. 5, 温度 15〜85 °C, 酶浓度 0. l〜500g/L; 最适 ΓΉ3. 5, 温度 50 , 酶浓度 5g/L。 结石酶的制备方法, 包括如下步骤:
( 2 ) 、 微生物菌种斜面培养, 在微生物菌种斜面培养中选用的的微生物菌种是黑曲霉 菌;
微生物菌种试管斜面培养: 将微生物菌种的培养基分装于 25 毫升的试管中, 经灭菌后 取出降至室温, 在无菌室接种后于恒温培养箱 33°C恒温培养 72小时, 获得试管斜面培养的 微生物菌种;
微生物菌种三角瓶扩大培养: 将微生物菌种的培养基分装于 1000ml 的三角瓶中, 经灭 菌后取出降至室温, 在无菌室接种后于 33°C恒温培养 72小时, 获得三角瓶扩大培养的微生 物菌种;
( 2)、 原辅材料前处理:
将原辅材料选材,对需要粉碎的原辅材料进行粉碎,再将原辅材料按以下重量份数备料: 碳源 25份
氮源 6份
水 68份
营养盐 5份
生长素 3份
产酶诱导物 6份。
其中所述碳源是指麦麸、 蔗糖、 橘子、 橙子、 苹果、 香蕉的物料。
其中所述氮源是牛肉膏、 干酵母、 花生饼粉、 虾壳、 硫酸铵的物料。
其中所述营养盐是含磷、 硫、 钠、 铁、 钙、 锌、 钴、 锰离子的混合盐液。
其中所述生长素是维生素的物料。
其中所述产酶诱导物是人工牛胆结石和人体结石粉碎物的物料。
将碳源、氮源和水按照其重量份数及发酵培养器的投料定容的 80%投入到发酵培养器内, 将已投入到发酵培养器内的原材料混合, 搅拌均勾, 消毒待用;
将辅助材料营养盐、 生长素按照其重量份数投入到备用容器内, 搅拌均勾, 消毒待用; 将产酶诱导物按照其重量份数投入到备用容器内, 搅拌均勾, 消毒待用;
( 3)、 发酵培养器培养:
将步骤 (2) 的辅助材料和产酶诱导物与已投入到发酵培养器内的原材料进行混合, 搅 拌均勾, 使发酵培养器的投料定容为 70%;
将步骤 (1 ) 获得的扩大培养的微生物菌种接种到发酵培养器内, 接种量为 30%;
将空气经空气压缩机和净化系统处理后输入发酵培养器内, 纯净空气通风量为 1. 0L/L* min;
在发酵培养器内采用如下培养条件进行微生物发酵培养: 温度为 33°C ; 1¾值为6. 5; 培 养时间为 96小时;
( 4)、 培养物浸提: 将步骤 (3 ) 中的终止培养后的培养物进行浸提, 加水 5倍, 浸提 时间 24小时, 板框压滤或离心过滤获得液体和残渣;
( 5 )、 浓缩: 将步骤 (4 ) 中分离出的液体进行超滤或真空浓缩获得浓缩后的液体, 将 浓缩后的液体包装, 获得液体粗品;
( 6 )、 干燥: 将步骤 (5 ) 中获得的浓缩后的液体进行喷雾干燥, 获得固体粗品;
( 7 )、 精制: 将步骤 (5 ) 中获得的浓缩后的液体进行过滤精制, 获得精制后的液体, 将精制后的液体包装, 获得液体精品;
( 8 )、 冻干: 将步骤 (7 ) 中获得的精制后的液体进行沉降、 离心、 冻干, 获得冻干品 为固体精品。
其中, 步骤 (1 ) 中所述的微生物菌种的培养基每升成份、 重量组成如下:
试管斜面培养基配方
去皮马铃薯 30000 mg, 葡萄糖 1000 mg, 柿饼细粒 3000mg, 核桃粉 1000 mg, 人体结石 的粉碎物 1000 mg, 琼脂 20000 mg, 用水定容至 1升容量。
三角瓶扩大培养基配方
NaN03 10000 mg, KC1 1000 mg, K2HP04 2000 mg, KH2P04 5000 mg, (NH4) 2S04 5000 mg , MgS04 · 7H20 2000 mg, CaCl2 2000 mg, FeS04 10 mg, MnS04 · H20 8mg, ZnS04 · H20 5mg, C。C12 lmg, N¾Se030. 3mg, 尿素 3000mg, 甲壳素 3000mg, 香蕉粉 1000 mg, 核桃粉 1000 mg, 人体 结石的粉碎物 1000 mg, 用水定容至 1升容量。
进一步的, 将步骤 (4 ) 中分离出的富含蛋白质的残渣用于第二次发酵或生产蛋白词料 或生产有机肥。
实施例三, 本实施例所述的结石酶, 它是由下列重量份数的原辅材料利用固态发酵法进 行微生物发酵培养、 经浸提、 浓缩、 精制而获得的:
碳源 20份
氮源 10份
水 40份
营养盐 1份
生长素 1份
产酶诱导物 1份。
其中所述碳源是指 D-果糖、 D-山梨醇、 淀粉水解糖、 淀粉、 玉米粉、 秸秆、 葵花盘、 麦 芽、 木瓜、 菠萝、 水果皮、 水果渣、 甜菜渣、 芹菜、 珍珠粉、 核桃粉的物料。
其中所述氮源是酵母膏、 鱼粉、 蛋白胨、 蛋白质、 蜗牛、 鱼皮、 蟹壳、 甲壳素、 尿素、
胺、 酰胺、 嘌吟碱、 嘧啶碱、 磷酸铵、 氯化铵、 碳酸铵、 液氨的物料。
其中所述营养盐是含磷、 硫、 镁、 钾、 钠、 铁、 钙、 锌、 钴、 锰、 硒离子的混合盐液。 其中所述生长素是氨基酸、 嘌吟、 嘧啶的物料。
其中所述产酶诱导物是人工牛胆结石的物料。
所述结石酶作为降解结石的酶, 具体作为食品、 饮品、 保健品、 药品或针剂在化解人体 结石中的应用; 其形态固体为白色、 浅褐色粉状或颗粒状, 液体为褐色或透明状; 其使用条 件为 PH2. 5〜7. 5, 温度 15〜85°C, 酶浓度 0. l〜500g/L; 最适 ΓΉ6. 5, 温度 25°C, 酶浓度 30g/Lo
结石酶的制备方法, 包括如下步骤:
( 3) 、 微生物菌种斜面培养, 在微生物菌种斜面培养中选用的的微生物菌种是酵母菌; 微生物菌种试管斜面培养: 将微生物菌种的培养基分装于 15 毫升的试管中, 经灭菌后 取出降至室温, 在无菌室接种后于恒温培养箱 20°C恒温培养 24小时, 获得试管斜面培养的 微生物菌种;
微生物菌种三角瓶扩大培养: 将微生物菌种的培养基分装于 500ml的三角瓶中, 经灭菌 后取出降至室温, 在无菌室接种后于 20°C恒温培养 24小时, 获得三角瓶扩大培养的微生物 菌种;
( 2)、 原辅材料前处理:
将原辅材料选材,对需要粉碎的原辅材料进行粉碎,再将原辅材料按以下重量份数备料: 碳源 20份
氮源 10份
水 40份
营养盐 1份
生长素 1份
产酶诱导物 1份。
其中所述碳源是指 D-果糖、 D-山梨醇、 淀粉水解糖、 淀粉、 玉米粉、 秸秆、 葵花盘、 麦 芽、 木瓜、 菠萝、 水果皮、 水果渣、 甜菜渣、 芹菜、 珍珠粉、 核桃粉的物料。
其中所述氮源是酵母膏、 鱼粉、 蛋白胨、 蛋白质、 蜗牛、 鱼皮、 蟹壳、 甲壳素、 尿素、 胺、 酰胺、 嘌吟碱、 嘧啶碱、 磷酸铵、 氯化铵、 碳酸铵、 液氨的物料。
其中所述营养盐是含磷、 硫、 镁、 钾、 钠、 铁、 钙、 锌、 钴、 锰、 硒离子的混合盐液。 其中所述生长素是氨基酸、 嘌吟、 嘧啶的物料。
其中所述产酶诱导物是人工牛胆结石的物料。
将碳源、氮源和水按照其重量份数及发酵培养器的投料定容的 80%投入到发酵培养器内, 将已投入到发酵培养器内的原材料混合, 搅拌均勾, 消毒待用;
将辅助材料营养盐、 生长素按照其重量份数投入到备用容器内, 搅拌均勾, 消毒待用; 将产酶诱导物按照其重量份数投入到备用容器内, 搅拌均勾, 消毒待用;
( 3)、 发酵培养器培养:
将步骤 (2) 的辅助材料和产酶诱导物与已投入到发酵培养器内的原材料进行混合, 搅 拌均勾, 使发酵培养器的投料定容为 30%;
将步骤 (1 ) 获得的扩大培养的微生物菌种接种到发酵培养器内, 接种量为 5%;
将空气经空气压缩机和净化系统处理后输入发酵培养器内, 纯净空气通风量为 0. 1L/L* min;
在发酵培养器内采用如下培养条件进行微生物发酵培养: 温度为 20°C ; ra值为 7. 1 ; 培 养时间为 24小时;
(4)、 培养物浸提: 将步骤 (3) 中的终止培养后的培养物进行浸提, 加水 1 倍, 浸提 时间 1小时, 板框压滤或离心过滤获得液体和残渣;
( 5)、 浓缩: 将步骤 (4) 中分离出的液体进行超滤或真空浓缩获得浓缩后的液体, 将 浓缩后的液体包装, 获得液体粗品;
(6)、 干燥: 将步骤 (5) 中获得的浓缩后的液体进行喷雾干燥, 获得固体粗品;
( 7)、 精制: 将步骤 (5 ) 中获得的浓缩后的液体进行过滤精制, 获得精制后的液体, 将精制后的液体包装, 获得液体精品;
( 8)、 冻干: 将步骤 (7 ) 中获得的精制后的液体进行沉降、 离心、 冻干, 获得冻干品 为固体精品。
其中, 步骤 (1 ) 中所述的微生物菌种的培养基每升成份、 重量组成如下:
试管斜面培养基配方
去皮马铃薯 10000mg, 葡萄糖 100mg, 柿饼细粒 100mg, 核桃粉 100mg, 人体结石的粉碎 物 100mg, 琼脂 10000mg, 用水定容至 1升容量。
三角瓶扩大培养基配方
NaN03 lOOOmg, KCl lOOmg, K2HP04 500mg, KH2P04 lOOOmg, (NH4) 2S04 lOOOmg , MgS04 · 7 0 lOOmg, CaCl2 lOOmg, FeS04 lmg, MnS04 · 0 lmg, ZnS04 · 0 lmg, C。C12 0. Img, N¾Se030. Img, 尿素 100mg, 甲壳素 100mg, 香蕉粉 100 mg, 核桃粉 100mg, 人体结石的粉碎物 100mg, 用 水定容至 1升容量。
进一步的, 将步骤 (4) 中分离出的富含蛋白质的残渣用于第二次发酵或生产蛋白词料
或生产有机肥。
以上发明实施例, 并非用来限制本发明, 凡其类似的或近似的采用上述技术方案获得的 结石酶产品, 均包含在本发明专利申请的保护范围之内。
Claims
1、 结石酶, 其特征在于: 它是由下列重量份数的原辅材料利用固态发酵法进行微生物 发酵培养、 经浸提、 浓缩、 精制而获得的:
碳源 20〜30份
氮源 1〜10份
水 40〜94份
营养盐 1〜10份
生长素 1〜 5份
产酶诱导物 1〜10份。
2、 根据权利要求 1所述的结石酶, 其特征在于: 所述碳源是指 D-葡萄糖、 D-果糖、 D- 甘露糖、 D-山梨醇、 蔗糖、 淀粉水解糖、 淀粉、 面粉、 玉米粉、 纤维质粉、 秸秆、 谷壳、 葵 花盘、 麦麸、 麦芽、 麦芽汁、 木瓜、 菠萝、 柿子、 香蕉、 橘子、 橙子、 苹果、 水果皮、 水果 渣、 甜菜、 甜菜渣、 芹菜、 蔬菜、 蔬菜渣、 珍珠粉、 核桃粉、 核桃渣、 坚果渣、 植物质粉、 植物渣中一种以上的物料。
3、 根据权利要求 1 所述的结石酶, 其特征在于: 所述氮源是酵母膏、 干酵母、 鱼粉、 蛋白胨、 牛肉膏、 蛋白质、 蜗牛、 豆饼粉、 花生饼粉、 鱼皮、 蟹壳、 虾壳、 甲壳素、 尿素、 胺、 酰胺、 嘌吟碱、 嘧啶碱、 硫酸铵、 磷酸铵、 硝酸铵、 氯化铵、 碳酸铵、 氨水、 氨气、 液 氨中一种以上的物料。
4、 根据权利要求 1所述的结石酶, 其特征在于: 所述营养盐是含磷、 硫、 镁、 钾、 钠、 铁、 钙、 锌、 钴、 锰、 硒中一种以上离子的混合盐液。
5、 根据权利要求 1 所述的结石酶, 其特征在于: 所述生长素是氨基酸、 嘌吟、 嘧啶、 维生素、 玉米浆、 生物代谢剂中一种以上的物料。
6、 根据权利要求 1 所述的结石酶, 其特征在于: 所述产酶诱导物是人工牛胆结石或人 体结石粉碎物的物料。
7、 权利要求 1〜6任一项所述的结石酶, 其特征在于: 作为降解结石的酶, 具体作为食 品、 饮品、 保健品、 药品或针剂在化解人体结石中的应用; 其形态固体为白色、 浅褐色粉状 或颗粒状, 液体为褐色或透明状; 其使用条件为 ΕΉ2. 5〜7. 5, 温度 15〜85°C, 酶浓度 0. 1〜 500g/L; 最适 PH3. 5〜6. 5, 温度 25〜50°C, 酶浓度 5〜30g/L。
8、 权利要求 1〜6任一项所述的结石酶的制备方法, 其特征在于, 包括如下步骤: ( 1 )、微生物菌种斜面培养,在微生物菌种斜面培养中选用的的微生物菌种是黑曲霉菌、 草酸青霉菌、 酵母菌、 木霉菌、 米曲霉菌中一种以上的菌种;
微生物菌种试管斜面培养: 将微生物菌种的培养基分装于 15〜25 毫升的试管中, 经灭 菌后取出降至室温, 在无菌室接种后于恒温培养箱 20-33°C恒温培养 24〜72小时, 获得试管 斜面培养的微生物菌种;
微生物菌种三角瓶扩大培养:将微生物菌种的培养基分装于 500ml〜1000ml的三角瓶中, 经灭菌后取出降至室温, 在无菌室接种后于 20-33°C恒温培养 24〜72小时, 获得三角瓶扩大 培养的微生物菌种;
( 2)、 原辅材料前处理:
将原辅材料选材,对需要粉碎的原辅材料进行粉碎,再将原辅材料按以下重量份数备料: 碳源 20〜30份
氮源 1〜10份
水 40〜94份
营养盐 1〜10份
生长素 1〜5份
产酶诱导物 1〜10份。
其中: 所述碳源是指 D-葡萄糖、 D-果糖、 D-甘露糖、 D-山梨醇、 蔗糖、 淀粉水解糖、 淀 粉、 面粉、 玉米粉、 纤维质粉、 秸秆、 谷壳、 葵花盘、 麦麸、 麦芽、 麦芽汁、 木瓜、 菠萝、 柿子、 香蕉、 橘子、 橙子、 苹果、 水果皮、 水果渣、 甜菜、 甜菜渣、 芹菜、 蔬菜、 蔬菜渣、 珍珠粉、 核桃粉、 核桃渣、 坚果渣、 植物质粉、 植物渣中一种以上的物料。
其中: 所述氮源是酵母膏、 干酵母、 鱼粉、 蛋白胨、 牛肉膏、 蛋白质、 蜗牛、 豆饼粉、 花生饼粉、 鱼皮、 蟹壳、 虾壳、 甲壳素、 尿素、 胺、 酰胺、 嘌吟碱、 嘧啶碱、 硫酸铵、 磷酸 铵、 硝酸铵、 氯化铵、 碳酸铵、 氨水、 氨气、 液氨中一种以上的物料。
其中: 所述营养盐是含磷、 硫、 镁、 钾、 钠、 铁、 钙、 锌、 钴、 锰、 硒中一种以上离子 的混合盐液。
其中: 所述生长素是氨基酸、 嘌吟、 嘧啶、 维生素、 玉米浆、 生物代谢剂中一种以上的 物料。
其中: 所述产酶诱导物是人工牛胆结石或人体结石粉碎物的物料。
将碳源、氮源和水按照其重量份数及发酵培养器的投料定容的 80%投入到发酵培养器内, 将已投入到发酵培养器内的原材料混合, 搅拌均勾, 消毒待用;
将辅助材料营养盐、 生长素按照其重量份数投入到备用容器内, 搅拌均勾, 消毒待用; 将产酶诱导物按照其重量份数投入到备用容器内, 搅拌均勾, 消毒待用;
( 3)、 发酵培养器培养:
将步骤 (2 ) 的辅助材料和产酶诱导物与已投入到发酵培养器内的原材料进行混合, 搅 拌均勾, 使发酵培养器的投料定容为 30%〜70%;
将步骤 (1 ) 获得的扩大培养的微生物菌种接种到发酵培养器内, 接种量为 5〜30%; 将空气经空气压缩机和净化系统处理后输入发酵培养器内, 纯净空气通风量为 0. 1〜 1. OL/L'min;
在发酵培养器内采用如下培养条件进行微生物发酵培养:温度为 20〜33°C; ΓΉ值为 3. 5〜 7. 1; 培养时间为 48〜96小时;
( 4)、 培养物浸提: 将步骤 (3 ) 中的终止培养后的培养物进行浸提, 加水 1〜5倍, 浸 提时间 1〜24小时, 板框压滤或离心过滤获得液体和残渣;
( 5 )、 浓缩: 将步骤 (4 ) 中分离出的液体进行超滤或真空浓缩获得浓缩后的液体, 将 浓缩后的液体包装, 获得液体粗品;
( 6 )、 干燥: 将步骤 (5 ) 中获得的浓缩后的液体进行喷雾干燥, 获得固体粗品;
( 7 )、 精制: 将步骤 (5 ) 中获得的浓缩后的液体进行过滤精制, 获得精制后的液体, 将精制后的液体包装, 获得液体精品;
( 8 )、 冻干: 将步骤 (7 ) 中获得的精制后的液体进行析出、 沉降、 离心、 冻干, 获得 冻干品为固体精品。
9、 根据权利要求 8所述的结石酶的制备方法, 其特征在于: 步骤 (1 ) 中所述的微生物 菌种的培养基每升成份、 重量组成如下:
试管斜面培养基配方
去皮马铃薯 10000〜30000 mg, 葡萄糖 100〜1000 mg, 柿饼细粒 100〜3000mg, 核桃粉 100〜1000 mg, 人体结石的粉碎物 100〜1000 mg, 琼脂 10000〜20000 mg, 用水定容至 1升 三角瓶扩大培养基配方
NaN03 1000〜 10000 mg, KC1 100〜1000 mg, K2HP04 500〜2000 mg, KH2P04 1000〜5000 mg, (NH4) 2S04 1000〜5000 mg , MgS04 · 7H20 100〜2000 mg, CaCl2 100〜2000 mg, FeS04 1〜 10 mg, MnS04 · 0 l〜8mg, ZnS04 · 0 l〜5mg, C。C12 0. l〜lmg, N¾Se030. 1〜0. 3mg, 尿素 100〜3000mg, 甲壳素 100〜3000mg, 香蕉粉 100〜1000 mg, 核桃粉 100〜1000 mg, 人体结 石的粉碎物 100〜1000 mg, 用水定容至 1升容量。
10、 根据权利要求 8所述的结石酶的制备方法, 其特征在于: 将步骤 (4) 中分离出的 富含蛋白质的残渣用于第二次发酵或生产蛋白词料或生产有机肥。
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CN108410743A (zh) * | 2018-05-15 | 2018-08-17 | 日照金禾博源生化有限公司 | 一种黑曲霉麸曲培养基及其配制方法 |
CN114561299A (zh) * | 2022-03-21 | 2022-05-31 | 广西大学 | 草酸青霉及其在锰浸出中的应用 |
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CN103540569A (zh) * | 2013-06-05 | 2014-01-29 | 黄虹寓 | 结石酶及其制备方法 |
CN103844266B (zh) * | 2014-03-25 | 2015-09-16 | 容瑜 | 一种天然调理养生酵素的制备方法 |
CN104305026A (zh) * | 2014-09-25 | 2015-01-28 | 容瑜 | 一种消石的酵素及其制备方法、使用方法 |
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CN102492683B (zh) * | 2011-12-01 | 2013-06-12 | 南宁奕德环境科技有限公司 | 一种交联草酸脱羧酶聚集体的制备方法 |
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US20070207524A1 (en) * | 2001-10-26 | 2007-09-06 | Benjamin Bower | Phytase enzymes, nucleic acid sequences encoding phytase enzymes and vectors and host cells incorporating same |
CN101583375A (zh) * | 2006-08-02 | 2009-11-18 | 阿尔特斯制药公司 | 结晶化的草酸脱羧酶和使用方法 |
CN102597225A (zh) * | 2009-07-02 | 2012-07-18 | 奥克斯泰拉知识产权公司 | 重组草酸盐降解酶的纯化和分离以及含有草酸盐降解酶的喷雾干燥粒子 |
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CN114561299A (zh) * | 2022-03-21 | 2022-05-31 | 广西大学 | 草酸青霉及其在锰浸出中的应用 |
CN114561299B (zh) * | 2022-03-21 | 2023-11-28 | 广西大学 | 草酸青霉及其在锰浸出中的应用 |
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