CN111876451B - Method for extracting amino acid from chicken manure - Google Patents
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- CN111876451B CN111876451B CN202010793284.XA CN202010793284A CN111876451B CN 111876451 B CN111876451 B CN 111876451B CN 202010793284 A CN202010793284 A CN 202010793284A CN 111876451 B CN111876451 B CN 111876451B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/20—Fertilizers of biological origin, e.g. guano or fertilizers made from animal corpses
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Abstract
The invention provides a method for extracting amino acid from chicken manure, which comprises the following steps: s1, preparing a chicken manure fermentation stock solution for later use; s2, adding alkaline protease into the chicken manure fermentation stock solution, and hydrolyzing to obtain a primary hydrolysate for later use; s3, centrifuging and filtering the primary hydrolysate to obtain a solution a for later use; s4, deodorizing the solution a to obtain a solution b for later use; s5, preparing a double aqueous phase system, and mixing the solution b with the double aqueous phase system to obtain a solution c with obvious double aqueous phases for later use; s6, enabling the upper organic phase of the solution c to pass through an ion exchange column, eluting with deionized water until the organic phase is colorless, and then eluting the ion exchange column with ammonia water to obtain a solution d for later use; s7, heating the solution d in a water bath, and then concentrating in vacuum to obtain the amino acid. The invention provides a method for extracting amino acid from chicken manure, which has the advantages of simple operation, rapidness, high efficiency and the like.
Description
Technical Field
The invention relates to the field of solid waste recycling. More specifically, the invention relates to a method for extracting amino acid from chicken manure.
Background
Along with the rapid development of economy, the living standard of people is increasingly improved, the demand of meat protein is also increasingly increased, and the appearance of large-scale farms causes corresponding pollution. As the chicken manure is accumulated in the surrounding environment, volatile substances in the manure can cause air pollution and attract mosquitoes, flies and parasites; liquid in the chicken manure can permeate or enter underground water along with rainfall to pollute surrounding water sources; the accumulated chicken manure is easy to agglomerate, is not beneficial to the growth of crops and influences the environment. Therefore, environmental workers are developing green and environment-friendly means for treating poultry excrement waste as resources in recent years.
Since aqueous two-phase systems can provide milder operating conditions, reduced target product damage or reduced small product instability/variability. Compared with the traditional water-organic solvent extraction method, the interface stress of the aqueous two phases is far lower than that of the aqueous two phases, so that the extracted product is less damaged. Since the free amino acids aggregate into one layer of the polymer, the concentration of amino acids in the two phases is increased for the purpose of separation. The amino acid extraction methods reported in the current literature mainly include an alcohol extraction method, a water extraction method, an ethanol microwave extraction method, an ultrasonic water extraction method and the like, and related reported examples are few. The method for extracting free amino acid from waste tobacco powder in tobacco industry by Shifeng et al utilizes a two-aqueous phase system formed by PEG polymer and ammonium sulfate salt and combines a flash extractor to extract the amino acid in waste tobacco powder particles, and the better purity can reach about 96%.
The excrement of the chicken manure contains about 16 percent (mass fraction) of protein, polypeptide and free amino acid which are not digested and absorbed in time, about 8 tons of chicken manure can be produced in a modern chicken farm with 10 ten thousand cultivation scales every day, the amino acid in the chicken manure can be extracted in time, and the yield per day is about 1 ton.
Disclosure of Invention
The invention aims to provide a method for extracting amino acid from chicken manure, which has the advantages of simple operation, rapidness, high efficiency and the like.
To achieve these objects and other advantages in accordance with the present invention, there is provided a method for extracting amino acids from chicken manure, comprising the steps of:
s1, preparing a chicken manure fermentation stock solution for later use;
s2, adding alkaline protease into the chicken manure fermentation stock solution, and hydrolyzing the alkaline protease to obtain a primary hydrolysate for later use;
s3, centrifuging and filtering the preliminary hydrolysate, and removing part of insoluble impurities and protease to obtain a solution a for later use, wherein the removed impurities and protease can be used for preparing fertilizers;
s4, deodorizing the solution a to obtain a solution b for later use;
s5, preparing a double aqueous phase system, and mixing the solution b with the double aqueous phase system to obtain a solution c with obvious double aqueous phases for later use;
s6, enabling the upper organic phase of the solution c to pass through an ion exchange column, eluting with deionized water until the organic phase is colorless, and then eluting the ion exchange column with ammonia water to obtain a solution d for later use;
s7, heating the solution d in a water bath, and then concentrating in vacuum to obtain the amino acid.
The technical problem to be solved by the invention is to extract polypeptides, proteins and amino acids which are not decomposed in time and separate the amino acids in resin. The protease can hydrolyze polypeptide and protein, free amino acid can be extracted by a double water phase system, the AB-8 type macroporous ion exchange resin treated by strong acid can adsorb amino acid, and the amino acid on the adsorption column can be eluted by strong ammonia water.
The technical principle of the invention is as follows: the polypeptide and protein can be partially hydrolyzed into smaller amino acids under the action of protease, and the protease will be attached to the original solution because the original solution contains 2% insoluble suspended matter. Centrifugation within 24-36h can effectively remove suspended substances and protease. The double water phase system formed by polyethylene glycol and ammonium sulfate has lower interface stress at the interface of double phase interface compared with traditional water-extractant, amino acid will diffuse to one end of polymer, and inorganic salt in the fermented liquid will diffuse to salt phase, so as to achieve the separation purpose.
The mixture of the polymer and the amino acid can adsorb the amino acid in AB-8 type macroporous ion exchange resin treated by strong acid, deionized water is used for passing through an exchange column until the mixture is colorless, strong ammonia water can elute the amino acid, and ammonia gas volatilization is accelerated in a water bath at 72 ℃ to separate the high-purity amino acid (the concentration temperature is not too high, and the dehydration and condensation of the amino acid are prevented).
Preferably, in the method for extracting amino acid from chicken manure, the adding amount of the alkaline protease in S2 is 500mL of stock solution/5 g of protease, the hydrolysis time is 18-30h, and the hydrolysis temperature is 28-32 ℃.
Preferably, in the method for extracting amino acid from chicken manure, the alkaline protease in S2 is food-grade small-molecule peptase, and the enzymatic activity of the small-molecule peptase is 20 ten thousand u/g.
Preferably, in the method for extracting amino acid from chicken manure, the centrifugation condition in S3 is centrifugation for 6-8 minutes at 7000-9000 r/min.
Preferably, in the method for extracting amino acid from chicken manure, the solution a is filtered by a circulating vacuum suction filter for 1.5-3 hours in S4 to obtain the solution b.
Preferably, in the method for extracting amino acid from chicken manure, the aqueous two-phase system is formed by mixing PEG4000 and ammonium sulfate solution.
Preferably, in the method for extracting amino acid from chicken manure, the preparation method of the aqueous two-phase system comprises the following steps:
mixing 25-40% of PEG-4000 aqueous solution and ammonium sulfate according to the volume-mass ratio of 8-12:5.2-5.7 mL/mg.
Preferably, in the method for extracting amino acid from chicken manure, an AB-8 type macroporous adsorption resin is adopted as an ion exchange column in S6, and the AB-8 type macroporous adsorption resin is soaked in hydrochloric acid with the concentration of 1.5mol/L for 18-30h before use.
Preferably, in the method for extracting amino acid from chicken manure, the concentration of the concentrated ammonia water used in S6 is 1 mol/L.
Preferably, in the method for extracting the amino acid from the chicken manure, the solution d is heated in a water bath at the temperature of 70-75 ℃ for 30-50min in S7.
The invention has the beneficial effects that:
1. the invention utilizes the mixture of polyethylene glycol and ammonium sulfate to form a double water phase system, inorganic components in the chicken manure are close to salt phases in the double water phase, organic components are mainly amino acids and are close to polymer phases, and free amino acids and other components in the chicken manure are diffused to different components in the double water phase system to achieve the purpose of separation. The invention can effectively ensure the activity and the extraction purity of the free amino acid, and the purity can reach 96%.
2. The method of the invention can reduce the problems of amino acid pollution, deterioration and the like caused by other chemical reactions.
3. The method for extracting the amino acid in the chicken manure by the aqueous two-phase method has the characteristics of simplicity, low toxicity of experimental medicaments, environmental friendliness and the like, can effectively solve the manure problem of poultry animals, and realizes the recycling of solid wastes.
4. By adding the deodorization procedure, 97 percent of volatile and irritant gases in the hydrolysate can be removed, the yield of amino acid is improved, and the influence of acid gas on subsequent experimental reagents is reduced.
5. The invention uses alkaline protease (food grade) to hydrolyze unhydrolyzed polypeptide and protein in chicken manure, can improve the yield of homoserine and tyrosine, and the protease has good stability, and the enzyme activity loss per three months is less than 1% at 25 ℃.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
< example 1>
A method for extracting amino acid from chicken manure comprises the following steps:
s1, preparing a chicken manure fermentation stock solution for later use;
s2, adding 500mL of stock solution/5 g of alkaline protease into the chicken manure fermentation stock solution, and hydrolyzing for 18h at 28 ℃ to obtain a primary hydrolysate for later use, wherein the alkaline protease is food-grade micromolecular peptolytic enzyme, and the enzymatic activity of the micromolecular peptolytic enzyme is 20 ten thousand u/g;
s3, centrifuging the preliminary hydrolysate for 6 minutes at the rotating speed of 7000r/min, and filtering to obtain a solution a for later use;
s4, performing suction filtration on the solution a for 1.5 hours by using a circulating vacuum suction filter to obtain a solution b for later use;
s5, mixing 25% by mass of PEG-4000 aqueous solution with ammonium sulfate according to the volume-mass ratio of 8:5.2mL/mg to prepare a double-water-phase system, and mixing the solution b with the double-water-phase system to obtain a solution c which forms an obvious double water phase for later use;
s6, enabling the upper organic phase of the solution c to pass through an ion exchange column, eluting with deionized water until the organic phase is colorless, and then eluting the ion exchange column with ammonia water with the concentration of 1mol/L to obtain a solution d for later use;
wherein the ion exchange column adopts AB-8 type macroporous adsorption resin, and the AB-8 type macroporous adsorption resin is soaked in hydrochloric acid with the concentration of 1.5mol/L for 18 hours before use;
s7, heating the solution d in a water bath at 70 ℃ for 30min, and then concentrating in vacuum to obtain the amino acid.
< example 2>
A method for extracting amino acid from chicken manure comprises the following steps:
s1, preparing a chicken manure fermentation stock solution for later use;
s2, adding 500mL of stock solution/5 g of alkaline protease into the chicken manure fermentation stock solution, and hydrolyzing for 24 hours at the temperature of 30 ℃ to obtain a primary hydrolysate for later use, wherein the alkaline protease is food-grade micromolecular peptolytic enzyme, and the enzymatic activity of the micromolecular peptolytic enzyme is 20 ten thousand u/g;
s3, centrifuging the primary hydrolysate for 7 minutes at the rotating speed of 8000r/min, and filtering to obtain a solution a for later use;
s4, performing suction filtration on the solution a for 2 hours by using a circulating vacuum suction filter to obtain a solution b for later use;
s5, mixing the PEG-4000 aqueous solution with the mass fraction of 30% and ammonium sulfate according to the volume-mass ratio of 10:5.5mL/mg to prepare a double-water-phase system, and mixing the solution b and the double-water-phase system to obtain a solution c which forms an obvious double water phase for later use;
s6, enabling the upper organic phase of the solution c to pass through an ion exchange column, eluting with deionized water until the organic phase is colorless, and then eluting the ion exchange column with ammonia water with the concentration of 1mol/L to obtain a solution d for later use;
wherein the ion exchange column adopts AB-8 type macroporous adsorption resin, and the AB-8 type macroporous adsorption resin is soaked in hydrochloric acid with the concentration of 1.5mol/L for 24 hours before use;
s7, placing the solution d at 72 ℃ for water bath heating for 40min, and then carrying out vacuum concentration to obtain the amino acid.
< example 3>
A method for extracting amino acid from chicken manure comprises the following steps:
s1, preparing a chicken manure fermentation stock solution for later use;
s2, adding 500mL of stock solution/5 g of alkaline protease into the chicken manure fermentation stock solution, and hydrolyzing for 30h at the temperature of 32 ℃ to obtain a primary hydrolysate for later use, wherein the alkaline protease is food-grade micromolecular peptolytic enzyme, and the enzymatic activity of the micromolecular peptolytic enzyme is 20 ten thousand u/g;
s3, centrifuging the primary hydrolysate for 8 minutes at a rotating speed of 9000r/min, and filtering to obtain a solution a for later use;
s4, performing suction filtration on the solution a for 3 hours by using a circulating vacuum suction filter to obtain a solution b for later use;
s5, mixing the PEG-4000 aqueous solution with the mass fraction of 40% and ammonium sulfate according to the volume-mass ratio of 12:5.7mL/mg to prepare a double-water-phase system, and mixing the solution b and the double-water-phase system to obtain a solution c which forms an obvious double water phase for later use;
s6, enabling the upper organic phase of the solution c to pass through an ion exchange column, eluting with deionized water until the organic phase is colorless, and then eluting the ion exchange column with ammonia water with the concentration of 1mol/L to obtain a solution d for later use;
wherein the ion exchange column adopts AB-8 type macroporous adsorption resin, and the AB-8 type macroporous adsorption resin is soaked in hydrochloric acid with the concentration of 1.5mol/L for 30 hours before use;
s7, heating the solution d in a water bath at 75 ℃ for 50min, and then concentrating in vacuum to obtain the amino acid.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details given herein and to the embodiments shown and described without departing from the generic concept as defined by the claims and their equivalents.
Claims (8)
1. A method for extracting amino acid from chicken manure is characterized by comprising the following steps:
s1, preparing a chicken manure fermentation stock solution for later use;
s2, adding alkaline protease into the chicken manure fermentation stock solution, and hydrolyzing the alkaline protease to obtain a primary hydrolysate for later use;
s3, centrifuging and filtering the primary hydrolysate to obtain a solution a for later use;
s4, deodorizing the solution a to obtain a solution b for later use;
s5, preparing a double aqueous phase system, and mixing the solution b with the double aqueous phase system to obtain a solution c with obvious double aqueous phases for later use;
wherein the aqueous two-phase system is formed by mixing 25-40% of PEG-4000 aqueous solution and ammonium sulfate according to the volume-mass ratio of 8-12:5.2-5.7 mL/mg;
s6, enabling the upper organic phase of the solution c to pass through an ion exchange column, eluting with deionized water until the organic phase is colorless, and then eluting the ion exchange column with ammonia water to obtain a solution d for later use;
s7, heating the solution d in a water bath, and then concentrating in vacuum to obtain the amino acid.
2. The method for extracting amino acids from chicken manure according to claim 1, wherein the alkaline protease is added in an amount of 500mL stock solution/5 g protease in S2, the hydrolysis time is 18-30h, and the hydrolysis temperature is 28-32 ℃.
3. The method for extracting amino acids from chicken manure according to claim 2, wherein the alkaline protease in S2 is a food grade small molecule peptase, and the enzymatic activity of the small molecule peptase is 20 ten thousand u/g.
4. The method as claimed in claim 1, wherein the centrifugation at S3 is performed at 7000-9000r/min for 6-8 min.
5. The method for extracting amino acid from chicken manure as claimed in claim 1, wherein the solution b is obtained after the solution a is filtered by suction for 1.5-3h in S4 using a circulating vacuum filter.
6. The method for extracting amino acid from chicken manure as claimed in any one of claims 1 to 5, wherein the ion exchange column in S6 adopts AB-8 type macroporous adsorbent resin, and the AB-8 type macroporous adsorbent resin is soaked in 1.5mol/L hydrochloric acid for 18-30h before use.
7. A process for extracting amino acids from chicken manure as claimed in any one of claims 1 to 5, wherein the concentration of concentrated ammonia used in S6 is 1 mol/L.
8. A process for extracting amino acids from chicken manure as claimed in any one of claims 1-5, wherein the solution d is heated in water bath at 70-75 ℃ for 30-50min in S7.
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| ES2319844B1 (en) * | 2007-09-12 | 2010-02-16 | Universidad Miguel Hernandez De Elche (70%) | PROCEDURE FOR SEPARATION OF RECOMBINANT PROTEINS IN TWO-PHASE WATER SYSTEMS. |
| CN102584933A (en) * | 2012-02-13 | 2012-07-18 | 汪志友 | Method for improving separation efficiency, purity and biological specific activity of blood coagulation factor VIII and analog thereof by using affine aqueous two-phase system |
| CN104761424B (en) * | 2015-03-31 | 2016-05-25 | 川渝中烟工业有限责任公司 | From tobacco smoke wooden fork, extract the method for free amino acid |
| CN104761426B (en) * | 2015-03-31 | 2016-08-24 | 川渝中烟工业有限责任公司 | The method extracting free amino acid from tobacco bud |
| CN104761425B (en) * | 2015-03-31 | 2016-08-24 | 川渝中烟工业有限责任公司 | The method extracting free amino acid offal is discarded from tobacco industry |
| CN110891664B (en) * | 2017-06-01 | 2022-05-17 | 相达生物科技美国有限公司 | Phase separation behavior modifier for aqueous two-phase separation in porous materials |
| CN108159283B (en) * | 2018-01-29 | 2020-09-11 | 新昌县勤勉生物医药科技有限公司 | Extraction method and application of compound free amino acids of dendrobe leaves |
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