Method for preparing amino acid chelate based on corn starch enzymolysis waste liquid

Abstract

The invention relates to a method for preparing amino acid chelate based on enzymolysis of corn starch waste liquid, which comprises the following steps: adding a flocculating agent into the corn starch waste liquid, carrying out flocculation precipitation reaction, centrifuging to remove suspended matters, classifying and recovering supernate and protein precipitate, and carrying out foam separation coupling ultrafiltration treatment on the supernate to obtain water-soluble protein crude extract; crushing the protein precipitate for later use; mixing the protein precipitate with the water-soluble protein crude extract to obtain a mixture, adding a compound enzyme for enzymolysis treatment, and decoloring, filtering and concentrating the obtained enzymatic hydrolysate to obtain an amino acid concentrated solution; and carrying out chelation reaction on the amino acid concentrated solution and metal salt. According to the method disclosed by the invention, the chelation rate of the amino acid metal chelate can be remarkably improved through the cooperation of the flocculation precipitation reaction of the corn starch waste liquid, the foam separation coupling ultrafiltration treatment after the fermentation of the supernatant through the enzyme preparation and the enzymolysis treatment of the protein precipitation and the water-soluble protein crude extract; a relatively good chelating effect can be achieved in a relatively short time, and the reaction time is shortened.

Description

Technical Field

[0001] The invention belongs to the technical field of amino acid chelate preparation, and particularly relates to a method for preparing amino acid chelates based on enzymatic hydrolysis of corn starch waste liquid. Background Art

[0002] In the corn starch production process (soaking, crushing and separation, fine grinding, starch separation and washing, dehydration, etc.), a large amount of waste liquid is generated. These waste liquids contain a certain amount of complex amino acids, starch and other organic matter. However, corn starch waste liquid is usually regarded as waste treatment, which not only causes waste of resources, but also may cause a certain burden on the environment. With the enhancement of environmental awareness and the improvement of resource utilization efficiency, how to effectively utilize these waste liquids has become an important research direction.

[0003] At present, although some studies have focused on extracting useful components from corn starch waste liquid, these methods mostly focus on the extraction of single components, such as protein, starch, etc., and there are fewer studies on the preparation of amino acid chelates. Amino acid chelates are a type of compound with broad application prospects and can be used in feed additives, fertilizers, medicine and other fields. Traditional methods for preparing amino acid chelates usually rely on the direct reaction of purified amino acids with metal salts to obtain them, which cannot fully utilize corn starch waste liquid. In addition, the existing preparation methods still need to be improved in terms of the chelation rate, reaction time, thermal stability and other aspects of amino acid chelates. In order to solve the above technical problems, the present invention provides a method for preparing amino acid chelates based on enzymatic hydrolysis of corn starch waste liquid. Summary of the Invention

[0004] The purpose of the present invention is to solve the above problems and provide a method for preparing amino acid chelates based on enzymatic hydrolysis of corn starch waste liquid.

[0005] The present invention achieves the above-mentioned purpose through the following technical solutions: The present invention provides a method for preparing amino acid chelates based on enzymatic hydrolysis of corn starch waste liquid, comprising the following steps: (1) Adding a flocculant to the corn starch wastewater, centrifuging to remove suspended solids after flocculation and precipitation, and recovering the supernatant and protein precipitate by classification. The supernatant is subjected to foam separation coupled with ultrafiltration to obtain a water-soluble protein crude extract; the protein precipitate is crushed for later use; (2) The protein precipitate is mixed with the water-soluble protein crude extract to obtain a mixture, and a composite enzyme is added for enzymatic hydrolysis. The obtained enzymatic hydrolyzate is decolorized, filtered, and concentrated to obtain an amino acid concentrate; (3) The amino acid concentrate is subjected to a chelation reaction with a metal salt, and the resulting reaction solution is dried and crushed into powder to obtain an amino acid chelate.

[0006] As a further optimized solution of the present invention, in step (1), the flocculant is polyferric sulfate and anionic polyacrylamide; The concentration of the polyferric sulfate in the corn starch wastewater is 900-1100 ppm; The concentration of the anionic polyacrylamide in the corn starch waste liquid is 0.5-1.5 ppm.

[0007] As a further optimization scheme of the present invention, in step (1), the flocculation precipitation reaction is specifically to adjust the pH of the corn starch waste liquid to 7-8, and the flocculation precipitation reaction time is 25-40 minutes; The protein precipitate was crushed to 90-110 mesh.

[0008] As a further optimization scheme of the present invention, in step (1), the foam separation coupled with ultrafiltration treatment is specifically as follows: adjusting the pH of the supernatant to 7-9, obtaining a fermentation broth through enzyme fermentation, performing foam separation on the fermentation broth using a foam separation device, taking the foam layer and letting it stand, and after the foam is converted into water, adjusting the temperature to 85-105°C, transporting it to the ultrafiltration device at a flow rate of 80-120 L / h, and obtaining a water-soluble protein crude extract through 5-7 kDa membrane interception.

[0009] As a further optimization scheme of the present invention, the enzyme preparation is added in an amount of 8-12 g / L in the supernatant; the enzyme preparation is at least one of α-amylase and laccase; the fermentation temperature is 25-75° C., and the fermentation time is 0.5-1.5 h.

[0010] As a further optimization scheme of the present invention, in step (2), the enzymatic hydrolysis treatment is specifically to adjust the pH to 5-8 and perform enzymatic hydrolysis at a temperature of 40-50°C for 4-6 hours; In the mixture, the added amount of the complex enzyme is 25-40 g / L; the complex enzyme is at least one of cellulase, amylase, and acid protease.

[0011] As a further optimized solution of the present invention, the total amount of amino acids in the amino acid concentrate is ≥80%.

[0012] As a further optimized solution of the present invention, the molar ratio of the total amount of amino acids in the amino acid concentrate to the total amount of metal ions in the metal salt is 1:1-2.

[0013] As a further optimized solution of the present invention, the metal salt includes at least one of ferrous sulfate, copper sulfate, zinc sulfate, and manganese sulfate.

[0014] The beneficial effects of the present invention are: 1) The present invention synergistically improves the chelation rate of amino acid metal chelates by subjecting corn starch wastewater to a flocculation and precipitation reaction, fermenting the supernatant with an enzyme preparation followed by foam separation coupled with ultrafiltration, and subjecting the protein precipitate and the water-soluble protein crude extract to an enzymatic hydrolysis treatment. Furthermore, the present invention can achieve a good chelation effect in a relatively short period of time, thereby shortening the reaction time. 2) The present invention performs a flocculation and precipitation reaction on the corn starch waste liquid, ferments the supernatant with an enzyme preparation, performs foam separation coupled ultrafiltration treatment, and performs enzymatic hydrolysis treatment on the protein precipitate and the water-soluble protein crude extract. The three work together to significantly improve the thermal stability of the amino acid metal chelate. In addition, the supernatant after the flocculation and precipitation reaction is fermented synergistically with α-amylase and laccase, and is significantly better than fermentation of α-amylase and laccase alone in improving the thermal stability of the amino acid metal chelate. DETAILED DESCRIPTION

[0015] The present application is described in further detail below. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0016] 1. Materials 1. Flocculant: polyferric sulfate, anionic polyacrylamide; 2. Enzyme preparation: at least one of α-amylase and laccase; 3. Complex enzyme: including at least one of cellulase, amylase, and acid protease. In the complex enzyme used in the following experiments, the mass ratio of cellulase, amylase, and acid protease is 2:1:1; 4. pH regulator: sodium hydroxide, sodium carbonate; 5. Metal salts: including ferrous sulfate, copper sulfate, zinc sulfate, and manganese sulfate. The Fe 2+ 、Zn 2+ 、Cu 2+、 Mn 2+ The molar ratio is 2:1:1:1; 6. Corn starch waste liquid is the waste liquid generated in the conventional corn starch production process. The components of the corn starch waste liquid of the present invention are shown in Table 1: Table 1 Composition ; Unless otherwise specified, the methods used in the present invention are conventional methods known to those skilled in the art, and the raw materials, reagents and other materials used are commercially available products unless otherwise specified.

[0017] 1. Methods 2.1 Preparation of amino acid chelates based on enzymatic hydrolysis of corn starch wastewater 2.1.1 Pretreatment of corn starch wastewater 1) Adding polyferric sulfate and anionic polyacrylamide to the corn starch wastewater, adjusting the pH of the corn starch wastewater to 7, and performing flocculation and precipitation for 30 minutes. After the flocculation and precipitation reaction, centrifugation is performed to remove suspended matter and classify and recover the supernatant and protein precipitate; the protein precipitate is crushed to 100 mesh for later use; The concentration of polyferric sulfate in the corn starch waste liquid is 1000 ppm, and the concentration of anionic polyacrylamide in the corn starch waste liquid is 1 ppm; 2) The pH of the supernatant was adjusted to 8.5, and fermented with α-amylase at 70°C for 0.5 h and 50°C for 0.5 h to obtain the fermentation broth, which was inactivated and set aside; In the supernatant, the enzyme preparation (total amount of α-amylase) was added at 10 g / L; 3) The fermentation broth was subjected to foam separation using a foam separation apparatus (pressure of 0.6 MPa, reflux ratio of 12%, and time of 1.2 h). The foam layer was allowed to stand until the foam dissolved in water. The temperature was then adjusted to 90°C and the broth was transferred to an ultrafiltration apparatus at a flow rate of 110 L / h. A crude water-soluble protein extract was obtained by interception with a 6 kDa membrane.

[0018] 2.1.2 Preparation of amino acid concentrate The protein precipitate and the water-soluble protein crude extract were mixed to obtain a mixture, the pH of which was adjusted to 6, and the mixture was enzymatically hydrolyzed using a complex enzyme at 45°C for 5 hours. The resulting enzymatic hydrolyzate was decolorized, filtered, and concentrated to obtain an amino acid concentrate. The total amount of amino acids in the amino acid concentrate was 82%. In the mixture, the added amount of the complex enzyme was 35 g / L.

[0019] 2.1.3 Chelation reaction The amino acid concentrate was mixed with the metal salt, with the molar ratio of the total amount of amino acids in the amino acid concentrate to the total amount of metal ions in the metal salt being 1:1. The pH was adjusted to 5.5 and the mixture was heated to 55°C with continuous stirring. The chelation reaction time was 0.5 h, 1 h, 1.5 h, 2 h, and 3 h. The resulting reaction solution was dried and pulverized into powder to obtain an amino acid chelate (denoted as Group A1).

[0020] 2.2. Adjustment of the enzyme fermentation process for step 2.1.1 - pretreatment of corn starch wastewater Group A2: α-amylase and laccase fermentation Based on the amino acid chelate of group A1, this group only adjusted the enzyme preparation fermentation process. The adjusted step 2.1.1 2) is as follows: The pH of the supernatant was adjusted to 8.5, and the mixture was fermented at 70°C for 0.5 h with α-amylase. After cooling, laccase was added and fermented at 50°C for 0.5 h to obtain a fermentation broth, which was inactivated and set aside. In the supernatant, the amount of enzyme preparation added (the total amount of α-amylase and laccase, the mass ratio of α-amylase to laccase was 1:1) was 10 g / L, wherein the mass ratio of α-amylase to laccase was 1:1. The rest were consistent with the amino acid chelate of group A1.

[0021] Group A3: Laccase fermentation Based on the amino acid chelate of group A1, this group only adjusted the enzyme preparation fermentation process. The adjusted step 2.1.1 2) is as follows: The pH of the supernatant was adjusted to 8.5, and the fermentation broth was obtained after laccase fermentation at 50°C for 1 hour, which was inactivated and set aside. The amount of enzyme preparation (total laccase amount) added to the supernatant was 10 g / L; the rest was consistent with the amino acid chelate of group A1.

[0022] 2.3. Adjustment of the preparation process of step 2.1.1 - pretreatment of corn starch wastewater Group A2-D1: The process flow is: retain step 1) → step 3), i.e., omit step 2) Based on the amino acid chelate of group A2, this group only adjusted the preparation process of the pretreatment of corn starch wastewater. The adjusted step 2.1.1 is as follows: 1) Adding polyferric sulfate and anionic polyacrylamide to the corn starch wastewater, adjusting the pH of the corn starch wastewater to 7, and performing flocculation and precipitation for 30 minutes. After the flocculation and precipitation reaction, centrifugation is performed to remove suspended matter and classify and recover the supernatant and protein precipitate; the protein precipitate is crushed to 100 mesh for later use; The concentration of polyferric sulfate in the corn starch waste liquid is 1000 ppm, and the concentration of anionic polyacrylamide in the corn starch waste liquid is 1 ppm; 2) Foam separation of the supernatant was performed using a foam separation apparatus (pressure of 0.6 MPa, reflux ratio of 12%, time of 1.2 h). The foam layer was allowed to stand until the foam was dissolved in water. The temperature was adjusted to 90°C and the supernatant was transferred to an ultrafiltration apparatus at a flow rate of 110 L / h. The crude water-soluble protein extract was obtained by retention through a 6 kDa membrane. The rest are consistent with the amino acid chelates in group A2.

[0023] Group A2-D2: The process flow is: retain step 1), i.e. omit steps 2) and 3) Based on the amino acid chelate of group A2, this group only adjusted the preparation process of the pretreatment of corn starch wastewater (based on the following adjustment, in step 2.1.2, the protein precipitate is mixed with the supernatant to obtain a mixture, and then the mixture is treated in the same way as the amino acid chelate of group A2). The adjusted step 2.1.1 is specifically as follows: 1) Adding polyferric sulfate and anionic polyacrylamide to the corn starch wastewater, adjusting the pH of the corn starch wastewater to 7, and performing flocculation and precipitation for 30 minutes. After the flocculation and precipitation reaction, centrifugation is performed to remove suspended matter and classify and recover the supernatant and protein precipitate; the protein precipitate is crushed to 100 mesh for later use; The concentration of polyferric sulfate in the corn starch waste liquid is 1000 ppm, and the concentration of anionic polyacrylamide in the corn starch waste liquid is 1 ppm; The rest are consistent with the amino acid chelates in group A2.

[0024] Group A2-D3: The flocculation precipitation reaction in step 1) was omitted, and steps 2) and 3) were also omitted. Based on the amino acid chelate of group A2, this group only adjusted the preparation process of the pretreatment of corn starch wastewater (based on the following adjustments, the mixture is treated in step 2.1.2 in the same way as the amino acid chelate of group A2). The adjusted step 2.1.1 is as follows: 1) The corn starch wastewater is centrifuged to remove suspended matter to obtain a mixture; The rest are consistent with the amino acid chelates in group A2.

[0025] 2.4. Adjust the process of preparing amino acid concentrate in step 2.1.2 Group A2-D4: Enzymatic hydrolysis was omitted Based on the amino acid chelates of group A2, this group only made adjustments to the preparation process of the amino acid concentrate. The adjusted step 2.1.2 is as follows: The protein precipitate is mixed with the water-soluble protein crude extract to obtain a mixture, and the mixture is decolorized, filtered, and concentrated to obtain an amino acid concentrate. The total amount of amino acids in the amino acid concentrate is 82%; The rest are consistent with the amino acid chelates in group A2.

[0026] Blank group: The flocculation precipitation reaction in step 1) was omitted, as well as steps 2) and 3) and the enzymatic hydrolysis treatment. The details are as follows: The corn starch waste liquid is centrifuged to remove suspended matter to obtain a mixture; the mixture is decolorized, filtered, and concentrated to obtain an amino acid concentrate, in which the total amount of amino acids is 82%; the amino acid concentrate is mixed with a metal salt, with a molar ratio of the total amount of amino acids in the amino acid concentrate to the total amount of metal ions in the metal salt being 1:1, the pH is adjusted to 5.5, and the mixture is heated to 55°C with continuous stirring. The chelation reaction time is 0.5h, 1h, 1.5h, 2h, and 3h. The resulting reaction solution is dried and pulverized into powder to obtain an amino acid chelate.

[0027] 3. Experiment 3.1. Chelation rate test of amino acid metal chelates The chelation rates of the amino acid metal chelates of the blank group, example groups (Groups A1, 2, and 3), and control groups (Groups A2-D1, 2, 3, and 4) at chelation reaction times (0.5 h, 1 h, 1.5 h, 2 h, and 3 h) were determined by EDTA complexometric titration and are shown in Table 2: Table 2. Data record of chelation rate test of amino acid metal chelates ; Experimental conclusion: Based on the data analysis in Table 2, it can be seen that the chelation rate of the amino acid metal chelate in group A2 reaches the highest at 2 hours of chelation reaction, with a chelation rate of 96.7%. When the chelation reaction is continued for 1 hour, the chelation rate is also as high as 96.2%, which is the optimal embodiment. By comparison, it can be seen that the chelation rate of the amino acid metal chelate can be significantly improved by synergizing the flocculation precipitation reaction of the corn starch waste liquid, the foam separation coupled with ultrafiltration treatment of the supernatant after enzyme fermentation, and the enzymatic hydrolysis treatment of the protein precipitation and the water-soluble protein crude extract. It can also achieve a better chelation effect in a shorter time and shorten the reaction time.

[0028] 3.2 Thermal stability test of amino acid metal chelates 50 mg of amino acid metal chelate sample of each group of the present invention was mixed into 50 mL of water, and placed in a water bath at 40°C, 50°C, 60°C, and 70°C for 2 h. After cooling, the metal ion content in the supernatant was measured to evaluate the thermal stability. Solubility = (S / C) × 100; S represents the metal ion content in the supernatant, in mg / mL; C is the soluble metal ion content of the sample, in mg / mL. The solubility is recorded in Table 3 as shown below: Table 3 Amino acid metal chelate thermal stability test data record table ; Experimental conclusion: The data in Table 3 show that compared with the control group, the solubility of the amino acid metal chelate in group A1-3 changes less with increasing temperature, and at 70°C, the solubility is between 20.3% and 23.7%, and the solubility decreases significantly, indicating that the amino acid metal chelate in group A1-3 has good thermal stability. This shows that the thermal stability of the amino acid metal chelate can be significantly improved by synergistically fermenting the corn starch waste liquid with flocculation precipitation reaction, fermenting the supernatant with enzyme preparations and then performing foam separation coupled with ultrafiltration treatment, and enzymatic hydrolysis of the protein precipitate and the water-soluble protein crude extract. In addition, the effect of synergistic fermentation of the supernatant after the flocculation precipitation reaction with α-amylase and laccase in improving the thermal stability of the amino acid metal chelate is significantly better than that of fermentation of α-amylase and laccase alone. This shows that there may be a synergistic effect between the two. Amino acid metal chelates with good thermal stability are more convenient in daily storage and transportation when used as feed additives or fertilizers.

[0029] The experiments described above merely illustrate several embodiments of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that variations and improvements are possible within the scope of the present invention, as would be apparent to those skilled in the art. These variations and improvements are fully encompassed within the scope of the present invention.

Claims

1. A method for preparing amino acid chelates based on enzymatic hydrolysis of corn starch wastewater, characterized in that: The following steps are involved: (1) Adding a flocculant to the corn starch wastewater, centrifuging to remove suspended solids after flocculation and precipitation, and recovering the supernatant and protein precipitate by classification. The supernatant is subjected to foam separation coupled with ultrafiltration to obtain a water-soluble protein crude extract; the protein precipitate is crushed for later use; (2) The protein precipitate is mixed with the water-soluble protein crude extract to obtain a mixture, and a composite enzyme is added for enzymatic hydrolysis. The obtained enzymatic hydrolyzate is decolorized, filtered, and concentrated to obtain an amino acid concentrate; (3) The amino acid concentrate is subjected to a chelation reaction with a metal salt, and the resulting reaction solution is dried and crushed into powder to obtain an amino acid chelate.

2. The method for preparing amino acid chelates based on enzymatic hydrolysis of corn starch wastewater according to claim 1, characterized in that: In step (1), the flocculants are polyferric sulfate and anionic polyacrylamide; The concentration of the polyferric sulfate in the corn starch wastewater is 900-1100 ppm; The concentration of the anionic polyacrylamide in the corn starch waste liquid is 0.5-1.5 ppm.

3. The method for preparing amino acid chelates based on enzymatic hydrolysis of corn starch wastewater according to claim 1, characterized in that: In step (1), the flocculation precipitation reaction is specifically to adjust the pH of the corn starch wastewater to 7-8, and the flocculation precipitation reaction time is 25-40 minutes; The protein precipitate was crushed to 90-110 mesh.

4. The method for preparing amino acid chelates based on enzymatic hydrolysis of corn starch wastewater according to claim 1, characterized in that: In step (1), the foam separation coupled with ultrafiltration treatment is specifically as follows: adjusting the pH of the supernatant to 7-9, obtaining a fermentation broth through enzyme fermentation, performing foam separation on the fermentation broth using a foam separation device, taking the foam layer and letting it stand, and after the foam is converted into water, adjusting the temperature to 85-105°C, transporting it to the ultrafiltration device at a flow rate of 80-120 L / h, and obtaining a water-soluble protein crude extract through 5-7 kDa membrane interception.

5. The method for preparing amino acid chelates based on enzymatic hydrolysis of corn starch waste liquid according to claim 4, characterized in that: In the supernatant, the added amount of the enzyme preparation is 8-12 g / L; the enzyme preparation is at least one of α-amylase and laccase; the fermentation temperature is 25-75° C., and the fermentation time is 0.5-1.5 h.

6. The method for preparing amino acid chelates based on enzymatic hydrolysis of corn starch wastewater according to claim 1, characterized in that: In step (2), the enzymatic hydrolysis treatment is specifically to adjust the pH to 5-8 and perform enzymatic hydrolysis at a temperature of 40-50°C for 4-6 hours; In the mixture, the added amount of the complex enzyme is 25-40 g / L; the complex enzyme is at least one of cellulase, amylase, and acid protease.

7. The method for preparing amino acid chelates based on enzymatic hydrolysis of corn starch wastewater according to claim 1, characterized in that: The total amount of amino acids in the amino acid concentrate is ≥80%.

8. The method for preparing amino acid chelates based on enzymatic hydrolysis of corn starch wastewater according to claim 7, characterized in that: The molar ratio of the total amount of amino acids in the amino acid concentrate to the total amount of metal ions in the metal salt is 1:1-2.

9. The method for preparing amino acid chelates based on enzymatic hydrolysis of corn starch wastewater according to claim 8, characterized in that: The metal salt includes at least one of ferrous sulfate, copper sulfate, zinc sulfate and manganese sulfate.