CN111876451B - Method for extracting amino acid from chicken manure - Google Patents

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

A kind of method for extracting amino acid from chicken manure

technical field

The invention relates to the field of solid waste recycling. More specifically, the present invention relates to a method for extracting amino acids from chicken manure.

Background technique

With the rapid economic development, people's living standards are improving day by day, and the demand for meat protein is also increasing day by day. The emergence of large farms has caused corresponding pollution. Due to the accumulation of chicken manure in the surrounding environment, the volatile substances in the manure will cause air pollution, attracting mosquitoes and parasites; the liquid in the chicken manure will penetrate or enter the groundwater with rainfall, polluting the surrounding water sources; Chicken manure is easy to agglomerate, which is not conducive to the growth of crops and affects the environment. Therefore, environmental workers are developing green and environment-friendly poultry manure waste resource management methods in recent years.

In view of the fact that the two-phase system can provide milder operating conditions, reduce the damage of target products or reduce the instability/mutability of small products. Compared with the traditional water-organic solvent extraction method, the interfacial stress of the aqueous two phase is much lower than the former, so that the extracted product suffers less damage. Since the free amino acids will aggregate into one layer of the polymer, it is beneficial to increase the concentration of amino acids in the two phases to achieve the purpose of separation. The amino acid extraction methods currently reported in the literature mainly include alcohol extraction, water extraction, ethanol microwave extraction and ultrasonic water extraction, etc. There are few examples of related reports. The method for extracting free amino acids from waste tobacco powder in the tobacco industry by Shi Fengcheng et al. utilizes a two-phase system formed by PEG polymer and ammonium sulfate salt combined with a flash-loss extractor to extract amino acids in waste tobacco powder particles, with better purity. Can reach around 96%.

Chicken manure contains about 16% (mass fraction) of proteins, polypeptides and free amino acids that have not been digested and absorbed in time. A modern chicken farm with a breeding scale of 100,000 can produce about 8 tons of chicken manure every day. The extraction of amino acids, the daily output is about 1 ton.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for extracting amino acids from chicken manure, which has the advantages of simple operation, rapidity and high efficiency.

In order to achieve these objects and other advantages according to the present invention, a method for extracting amino acids from chicken manure is provided, comprising the following steps:

S1, prepare chicken manure fermentation stock solution, standby;

S2, in described chicken manure fermentation stock solution, add alkaline protease, it is hydrolyzed, obtain preliminary hydrolyzate, standby;

S3, after described preliminary hydrolyzate is centrifuged and filtered, remove partially insoluble impurities and protease to obtain solution a, for subsequent use, and the removed impurities and protease can be used to make chemical fertilizers;

S4, carry out deodorization treatment to described solution a, obtain solution b, standby;

S5, prepare a two-phase system, and mix the solution b with the two-phase system to obtain a solution c that forms an obvious two-phase system, for subsequent use;

S6, the upper organic phase of the solution c is passed through the ion exchange column, eluted with deionized water until colorless, and then the ion exchange column is eluted with ammonia water to obtain solution d, for subsequent use;

S7. The solution d is heated in a water bath, and then concentrated in vacuo to obtain the amino acid.

The technical problem to be solved by the present invention is the extraction of polypeptides and proteins that have not been decomposed in time, the extraction of amino acids and the separation of amino acids in resins. The protease can hydrolyze polypeptides and proteins, the two-phase system can extract free amino acids, the AB-8 macroporous ion exchange resin treated with strong acid can adsorb amino acids, and concentrated ammonia water can elute the amino acids on the adsorption column.

The technical principle of the present invention: polypeptides and proteins can be partially hydrolyzed into amino acids with smaller molecules under the sufficient action of protease. Since the stock solution contains 2% insoluble suspended matter, the protease will be attached to it. Centrifugation within 24-36h can effectively remove suspended matter and protease. In the two-phase system formed by polyethylene glycol and ammonium sulfate, the interface stress at the two-phase interface is lower than that of the traditional water-extractant, amino acids will diffuse to one end of the polymer, and inorganic salts in the fermentation broth will The salt phase diffuses to achieve the purpose of separation.

The mixture of the polymer and amino acid of the present invention is treated with strong acid in the AB-8 type macroporous ion exchange resin, the resin will adsorb the amino acid, deionized water is used to pass through the exchange column until colorless, and concentrated ammonia water will elute the amino acid. Accelerating the volatilization of ammonia in a water bath can separate high-purity amino acids (the concentration temperature should not be too high to prevent dehydration and condensation of amino acids).

Preferably, in the method for extracting amino acids from chicken manure, the amount of alkaline protease added in S2 is 500mL stock solution/5g protease, the hydrolysis time is 18-30h, and the hydrolysis temperature is 28-32°C .

Preferably, in the method for extracting amino acids from chicken manure, the alkaline protease in S2 is a food-grade small-molecule peptidase, and the small-molecule peptidase has an enzymatic activity of 200,000 u /g.

Preferably, in the method for extracting amino acids from chicken manure, the centrifugation conditions in S3 are centrifugation at a rotational speed of 7000-9000 r/min for 6-8 minutes.

Preferably, in the method for extracting amino acids from chicken manure, the solution b is obtained after the solution a is suction filtered for 1.5-3 hours using a circulating vacuum filter in S4.

Preferably, in the method for extracting amino acids from chicken manure, the two-phase system is formed by mixing PEG4000 and ammonium sulfate solution.

Preferably, in the described method for extracting amino acids from chicken manure, the preparation method of the aqueous two-phase system is as follows:

The PEG-4000 aqueous solution with a mass fraction of 25-40% and ammonium sulfate are mixed according to a volume-to-mass ratio of 8-12:5.2-5.7 mL/mg.

Preferably, in the method for extracting amino acids from chicken manure, the ion exchange column in S6 adopts AB-8 type macroporous adsorption resin, and the AB-8 type macroporous adsorption resin is used at a concentration of 1.5 Soak in mol/L hydrochloric acid for 18-30h.

Preferably, in the described method for extracting amino acids from chicken manure, the concentration of the concentrated ammonia water used in S6 is 1 mol/L.

Preferably, in the method for extracting amino acids from chicken manure, in S7, the solution d is placed under the condition of 70-75°C and heated in a water bath for 30-50min.

The beneficial effects of the present invention are:

1. The present invention utilizes polyethylene glycol and ammonium sulfate to mix to form a two-phase system, the inorganic components in the chicken manure will be close to the salt phase in the two-water phase, and the organic components are mainly amino acids, which are close to the polymer phase, using Free amino acids and other components in chicken manure will diffuse to different components in the two-phase system to achieve the purpose of separation. The invention can effectively ensure the activity of free amino acids and the purity of extraction, and the purity can reach 96%.

2. The method of the present invention can reduce the problems of amino acid contamination and deterioration caused by other chemical reactions.

3. The method of extracting amino acids in chicken manure by the aqueous two-phase method provided by the present invention has the characteristics of simple method, low toxicity of experimental chemicals and environmental friendliness, etc., which can effectively solve the problem of feces of poultry animals and realize the resource utilization of solid waste.

4. The present invention can remove 97% of volatile and irritating gases in the hydrolyzate by adding a deodorizing process, while improving the yield of amino acids and reducing the influence of acid gases on subsequent experimental reagents.

5. The present invention uses alkaline protease (food grade) to hydrolyze unhydrolyzed polypeptides and proteins in chicken manure, which can improve the yield of serine and tyrosine, and the stability of this protease is very good. , the loss of enzyme activity every three months is less than 1%.

Other advantages, objects, and features of the present invention will appear in part from the description that follows, and in part will be appreciated by those skilled in the art from the study and practice of the invention.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments, so that those skilled in the art can implement according to the description.

It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial sources unless otherwise specified.

<Example 1>

A method for extracting amino acids from chicken manure, comprising the following steps:

S1, prepare chicken manure fermentation stock solution, standby;

S2, add the alkaline protease of 500mL stock solution/5g in the described chicken manure fermentation stock solution, under the condition that the temperature is 28 ℃, the hydrolysis time is 18h, obtain the preliminary hydrolyzate, standby, wherein the alkaline protease is the small molecular peptide of food grade lyase, the enzyme activity of the small molecule peptidase is 200,000 u/g;

S3, the described preliminary hydrolyzate is filtered after centrifugal 6 minutes under the rotating speed of 7000r/min, obtains solution a, for subsequent use;

S4, after the solution a is suction filtered for 1.5h using a circulating vacuum filter, a solution b is obtained, which is for subsequent use;

S5. Mix the PEG-4000 aqueous solution with a mass fraction of 25% and ammonium sulfate in a volume-to-mass ratio of 8:5.2 mL/mg to prepare a two-phase system, and combine the solution b with the two-phase system Mix, obtain the solution c that forms obvious two-aqueous phase, standby;

S6, the upper organic phase of the solution c is passed through the ion exchange column, eluted to colorless with deionized water, and then the ion exchange column is eluted with the ammoniacal liquor with a concentration of 1 mol/L to obtain solution d, for subsequent use;

The ion exchange column adopts AB-8 macroporous adsorption resin, and the AB-8 macroporous adsorption resin is soaked in hydrochloric acid with a concentration of 1.5mol/L for 18h before use;

S7. The solution d is heated in a water bath at 70° C. for 30 minutes, and then concentrated in vacuo to obtain the amino acid.

<Example 2>

A method for extracting amino acids from chicken manure, comprising the following steps:

S1, prepare chicken manure fermentation stock solution, standby;

S2, add the alkaline protease of 500mL stock solution/5g in the described chicken manure fermentation stock solution, under the condition that the temperature is 30 ℃, the hydrolysis time is 24h, obtain the preliminary hydrolyzed solution, standby, wherein the alkaline protease is the small molecular peptide of food grade lyase, the enzyme activity of the small molecule peptidase is 200,000 u/g;

S3, the described preliminary hydrolyzate is filtered after centrifugal 7 minutes under the rotating speed of 8000r/min, obtains solution a, for subsequent use;

S4, after the described solution a is suction filtered for 2h using a circulating vacuum filter, solution b is obtained, for subsequent use;

S5. Mix the PEG-4000 aqueous solution with a mass fraction of 30% and ammonium sulfate in a volume-to-mass ratio of 10:5.5 mL/mg to prepare a two-phase system, and combine the solution b and the two-phase system Mix, obtain the solution c that forms obvious two-aqueous phase, standby;

S6, the upper organic phase of the solution c is passed through the ion exchange column, eluted to colorless with deionized water, and then the ion exchange column is eluted with the ammoniacal liquor with a concentration of 1 mol/L to obtain solution d, for subsequent use;

The ion exchange column adopts AB-8 macroporous adsorption resin, and the AB-8 macroporous adsorption resin is soaked in hydrochloric acid with a concentration of 1.5mol/L for 24h before use;

S7. The solution d is placed under the condition of 72° C. and heated in a water bath for 40 min, and then concentrated in vacuo to obtain the amino acid.

<Example 3>

A method for extracting amino acids from chicken manure, comprising the following steps:

S1, prepare chicken manure fermentation stock solution, standby;

S2. Add 500mL stock solution/5g alkaline protease to the chicken manure fermentation stock solution, and hydrolyze for 30h under the condition that the temperature is 32°C to obtain a preliminary hydrolyzed solution for use, wherein the alkaline protease is a food-grade small molecular peptide lyase, the enzyme activity of the small molecule peptidase is 200,000 u/g;

S3, the preliminary hydrolyzate is filtered after centrifugal 8 minutes at the rotating speed of 9000r/min to obtain solution a, for subsequent use;

S4, after the solution a is suction filtered for 3h using a circulating vacuum filter, solution b is obtained, for subsequent use;

S5, the PEG-4000 aqueous solution with a mass fraction of 40% and ammonium sulfate are mixed according to the volume mass ratio of 12:5.7 mL/mg to prepare a two-phase system, and the solution b and the two-phase system are mixed Mix, obtain the solution c that forms obvious two-aqueous phase, standby;

S6, the upper organic phase of the solution c is passed through the ion exchange column, eluted to colorless with deionized water, and then the ion exchange column is eluted with the ammoniacal liquor with a concentration of 1 mol/L to obtain solution d, for subsequent use;

The ion exchange column adopts AB-8 macroporous adsorption resin, and the AB-8 macroporous adsorption resin is soaked in hydrochloric acid with a concentration of 1.5 mol/L for 30 hours before use;

S7. The solution d is placed under the condition of 75°C and heated in a water bath for 50 min, and then concentrated in vacuo to obtain the amino acid.

Although the embodiment of the present invention has been disclosed as above, it is not limited to the application listed in the description and the embodiment, and it can be applied to various fields suitable for the present invention. For those skilled in the art, it can be easily Therefore, the invention is not limited to the specific details and embodiments shown and described herein without departing from the general concept defined by the appended claims and the scope of 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.