Method for preparing corn residue protein hydrolysate and application thereof
By using dispersants and β-glucanases for preliminary hydrolysis and thermophilic bacterial proteases for deep hydrolysis, combined with protein extractants, the problems of protein structure destruction and peptide bioactivity loss in corn starch residue were solved, thereby improving peptide yield, purity and antioxidant properties.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WEIFANG ENSIGN IND CO LTD
- Filing Date
- 2025-07-03
- Publication Date
- 2026-05-15
AI Technical Summary
Existing technologies for preparing corn peptides often result in the destruction of protein structures, loss of peptide bioactivity, and low added value of corn starch residue.
The first enzymatic hydrolysis was performed using dispersing enzymes and β-glucanase to disrupt the cell wall and disperse the protein. A second enzymatic hydrolysis was then performed using thermophilic bacterial protease. Combined with protein extraction agents such as choline chloride-phytic acid eutectic solvent, betaine, and saponins, the extraction efficiency and purity of the protein were improved.
It improved the yield and purity of peptides, enhanced their color and flavor, broadened their application range, and strengthened their antioxidant properties.
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Figure CN120400289B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of corn residue protein hydrolysis technology, and in particular to the preparation method and application of corn residue protein hydrolysis products. Background Technology
[0002] Corn is the main raw material for citric acid production. During citric acid production, corn starch residue is produced as a byproduct. This residue contains more than 30% protein, along with starch, fat, and cellulose. Currently, corn starch residue is mainly dried and sold as a feed ingredient by feed companies, resulting in low added value.
[0003] Existing technologies disclose methods for preparing corn peptides using corn gluten meal and corn starch residue as raw materials. However, the corn starch residue used in citric acid production processes typically undergoes high-temperature, strong acid (such as sulfuric acid or hydrochloric acid) treatment and prolonged fermentation, which may severely denature or partially hydrolyze the protein structure, destroying or hiding enzyme cleavage sites. Therefore, using the aforementioned methods to prepare peptides can easily damage the peptide structure within the protein, resulting in a loss of peptide bioactivity. Summary of the Invention
[0004] In view of this, the present invention proposes a method for preparing and applying corn residue protein hydrolysate to solve the problems of peptide structure destruction and loss of peptide bioactivity in protein.
[0005] The technical solution of this invention is achieved as follows: In a first aspect, this invention provides a method for preparing corn residue protein hydrolysate, comprising the following steps:
[0006] S1, after drying and pulverizing the corn residue from citric acid production, add it to Tris-HCl buffer, then add dispersant and β-glucanase for the first enzymatic hydrolysis to obtain hydrolysate A.
[0007] Dispersases specifically hydrolyze peptide bonds within protein molecules, breaking down large structural proteins into smaller polypeptides or oligopeptides. This promotes the disintegration and dispersion of previously aggregated, dense protein particles, improving the accessibility of downstream enzymes. By breaking down protein aggregates, dispersases also contribute to the loosening of some cellular structures and the "unlocking" of the matrix. β-glucanases target and hydrolyze β-glucan in plant cell walls and the intercellular matrix, disrupting the physical barrier of the cell wall and releasing proteins embedded in polysaccharides. β-glucanases also reduce system viscosity, contributing to improved rheological properties of the entire enzymatic hydrolysis system and enhancing enzyme-substrate contact.
[0008] β-glucanase first breaks down cell wall polysaccharides, releasing protein resources and providing more substrate for dispersants. The activity of dispersants enhances protein dispersion and even influences the local matrix microenvironment, giving β-glucanase more action sites. Simultaneous degradation of polysaccharides and proteins promotes maximum protein release. Furthermore, the reduced viscosity after polysaccharide hydrolysis facilitates enzymatic reactions and product separation, creating conditions for subsequent deep hydrolysis.
[0009] S2, after heating the enzyme hydrolysate A to inactivate the enzyme, add thermophilic protease for a second enzymatic hydrolysis to obtain enzyme hydrolysate B.
[0010] Thermophilic bacterial proteases possess both endonuclease and exonuclease activities, which can further break down the polypeptides produced after the first enzymatic hydrolysis into smaller molecules such as dipeptides, tripeptides, or amino acids. Enzymatic hydrolysis with thermophilic bacterial proteases at high temperatures can, on the one hand, inactivate the residual enzyme activity from the first hydrolysis, and on the other hand, the high temperature can denature some protein structures, exposing more cleavage sites, thus improving the hydrolysis efficiency.
[0011] The first enzymatic hydrolysis focuses on physical disintegration and preliminary degradation, while the second enzymatic hydrolysis focuses on high-temperature deep hydrolysis. The two enzymatic hydrolysals systematically break down polysaccharide encapsulation and protein aggregation, achieving higher yields and purity of protein and peptide products.
[0012] S3, the enzyme hydrolysate B is heated to inactivate the enzyme, then cooled to 20-30℃, centrifuged to collect the supernatant, and dried to obtain corn polypeptide.
[0013] Based on the above technical solution, preferably, in step S1, 0.01%~0.03 wt% of dispersant enzyme and 0.1%~0.3 wt% of β-glucanase are added to corn residue dry powder, the enzyme activity of dispersant enzyme is 100000 U / g, and the enzyme activity of β-glucanase is 50000 U / g.
[0014] In step S2, 0.5%~2 wt% of thermophilic protease from corn residue dry powder is added, with the enzyme activity of thermophilic protease being 2500 U / g.
[0015] Based on the above technical solutions, preferably, before the first enzymatic hydrolysis, crude protein is extracted with a protein extractant. The extraction method is as follows: add the dried and crushed corn residue to the protein extractant, stir for 6-8 hours, centrifuge to collect the supernatant, and dry to obtain crude protein powder.
[0016] The protein extraction agents include: choline chloride-phytic acid eutectic solvent, betaine, saponins, and Tris-HCl buffer solution with a pH of 8-9.
[0017] Choline chloride-phytic acid, with its abundant hydrogen bond donor and acceptor structure, can interact with cell walls and proteins in multiple ways, such as: efficiently depolymerizing the cell walls, colloids, and polysaccharide structures of corn residue, promoting protein release; having a strong dissolving effect on proteins and their bound components, enabling proteins to be released efficiently from dense tissues; and the phosphate groups of phytic acid can bind to metal ions and polysaccharides, which helps remove impurities and improve protein purity.
[0018] When betaine is combined with choline chloride-phytic acid, it can reduce the viscosity of the system, making it easier for the extractant to penetrate the tissue quickly and accelerate the protein dissolution rate. Betaine can also enhance the solvent molecules' ability to dissolve and transfer intracellular proteins, thus improving the overall protein extraction yield. Under hypertonic or high-temperature conditions, betaine can effectively encapsulate protein molecules, preventing denaturation and autolysis, and improving the protein activity retention rate during the extraction process.
[0019] Saponins can effectively reduce the surface tension of protein extractants, improve the wetting and penetration of corn residues, and allow the solvent to penetrate into cells to a greater extent. Saponins also help emulsify impurities (such as lipids), reduce the residue of non-protein impurities, and improve protein purity. They can also inhibit protein aggregation and precipitation, promote the protein to maintain a solution state, and facilitate subsequent separation and purification.
[0020] Tris-HCl buffer can maintain a stable alkaline environment in the extraction system, which can promote the protein to carry a negative charge, enhance the electrostatic repulsion between proteins, and significantly increase solubility; the stable pH environment can inhibit the activity of some non-targeting proteases and reduce protein degradation loss.
[0021] Based on the efficient cell wall disruption and dissolution of choline chloride-phytic acid, betaine enhances protein activity and extraction rate, saponins promote permeation and improve purification, and Tris-HCl provides a physicochemically stable environment. The synergistic effect of these four components greatly improves protein release efficiency and product purity, while effectively protecting protein activity. The overall process is mild and environmentally friendly, avoiding environmental and protein damage caused by strong alkalis, strong acids, or organic solvents.
[0022] Extracting crude protein before enzymatic hydrolysis removes most non-protein impurities (such as polysaccharides and fats), yielding enriched crude protein that provides a high-purity, highly accessible substrate for subsequent enzymatic hydrolysis. Pre-extraction of crude protein also removes impurities such as phenols, sugars, and pigments, helping to improve the color and flavor of the final peptide product, increasing its added value, and broadening its application range. After protein extraction, the substrate composition is singular, resulting in more specific enzyme action, thorough substrate hydrolysis, and significantly improved peptide yield, purity, and functionality.
[0023] Based on the above technical solutions, preferably, the protein extraction agent, by mass percentage (100%), comprises 40%-50% choline chloride-phytic acid eutectic solvent, 10%-15% betaine, 5%-10% saponins, and the balance being Tris-HCl buffer.
[0024] Based on the above technical solutions, the preferred method is that the mass-to-volume ratio of corn residue to protein extractant is 1:10-15; during the first enzymatic hydrolysis, the amount of dispersing enzyme is 40-60 U / g protein, and the amount of β-glucanase is 50-100 U / g protein; during the second enzymatic hydrolysis, the amount of thermophilic protease is 1000-3000 U / g protein.
[0025] Based on the above technical solutions, preferably, choline chloride and phytic acid are mixed and stirred at a molar ratio of 1:1.1~1.3 to obtain a choline chloride-phytic acid eutectic solvent.
[0026] Based on the above technical solutions, preferably, in step S1, the pH value of the Tris-HCl buffer is 6-7, the concentration of the Tris-HCl buffer is 40-50mM, and the material-to-liquid ratio is 1:10-20wt / v.
[0027] Based on the above technical solutions, preferably, in step S1, the temperature for the first enzymatic hydrolysis is 45-50℃ and the time is 2-4h.
[0028] Based on the above technical solutions, preferably, in step S2, the second enzymatic hydrolysis is performed at a temperature of 60-70℃ for 4-6 hours and at a pH of 7-8.
[0029] Based on the above technical solutions, preferably, in steps S1 and S3, the centrifugation speed is 3000-5000 rpm and the time is 10-20 min.
[0030] Secondly, the present invention provides corn polypeptides prepared by the above method.
[0031] Thirdly, this invention provides the application of corn peptides in the preparation of antioxidant products.
[0032] The preparation method and application of the corn residue protein hydrolysate of the present invention have the following advantages over the prior art:
[0033] (1) During the first enzymatic hydrolysis, β-glucanase first decomposes cell wall polysaccharides, releasing protein resources and providing more substrates for dispersants. The activity of dispersants enhances protein dispersion and even affects the local matrix microenvironment, giving β-glucanase more action sites. The simultaneous degradation of polysaccharides and proteins promotes the maximum release of proteins. Moreover, the viscosity of the system decreases after polysaccharide hydrolysis, which facilitates enzymatic reactions and product separation, creating conditions for subsequent deep hydrolysis. During the second enzymatic hydrolysis, thermophilic proteases can further decompose the polypeptides produced after the first enzymatic hydrolysis into smaller molecules such as dipeptides, tripeptides, or amino acids. In addition, high temperature can denature some protein structures, exposing more cleavage sites, which is beneficial to improving the efficiency of enzymatic hydrolysis. The first enzymatic hydrolysis is more focused on physical decomposition and preliminary degradation, while the second enzymatic hydrolysis focuses on high-temperature deep hydrolysis. The two enzymatic hydrolysis systems systematically break down polysaccharide encapsulation and protein aggregation, achieving higher yields and purity of protein and polypeptide products.
[0034] (2) Extracting crude protein first and then enzymatically hydrolyzing it can remove most non-protein impurities (such as polysaccharides and fats), resulting in enriched crude protein, which provides a high-purity and highly accessible substrate for subsequent enzymatic hydrolysis. Extracting crude protein first can also remove impurities such as phenols, sugars, and pigments, which helps improve the color and flavor of the final product peptides, thereby increasing the added value and application range of the product. After protein extraction, the substrate composition is singular, the enzyme action is more specific, the substrate hydrolysis is thorough, and the yield, purity, and functionality of the obtained peptides are significantly improved.
[0035] (3) In the protein extraction solution of the present invention, choline chloride-phytic acid efficiently disrupts cell walls and dissolves proteins, betaine enhances protein activity and extraction rate, saponins promote permeation and improve purification, and Tris-HCl provides a physicochemically stable environment. The four components work synergistically to greatly improve protein release efficiency and product purity, while effectively protecting protein activity. The overall process is mild and environmentally friendly, avoiding environmental and protein damage caused by strong alkalis, strong acids, or organic solvents. Attached Figure Description
[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0037] Figure 1 The results show the DPPH free radical scavenging ability and hydroxyl free radical scavenging ability of the peptides extracted in Examples 1-2 and Comparative Examples 1-4 of this invention. Detailed Implementation
[0038] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0039] The dispersant was Dispase II, derived from Bacillus polymyxa, purchased from Sigma. β-glucanase (CAS No.: 9025-70-1) was derived from... Chaetomium erraticum Purchased from Shanghai Yuanye Biotechnology Co., Ltd. The thermophilic protease (CAS No.: 9073-78-3) is derived from... BaClllus thermoproteolyticus Purchased from Shanghai Yuanye Biotechnology Co., Ltd.
[0040] The saponins are tea saponins or soybean saponins; the present invention uses soybean saponins, which were purchased from Sigma.
[0041] Example 1
[0042] The method for preparing corn residue protein hydrolysate in this embodiment includes the following steps:
[0043] S1. After drying and pulverizing the corn residue from citric acid production to 100 mesh, corn residue dry powder was obtained. 100g of corn residue dry powder was added to 1.5L of Tris-HCl buffer with a concentration of 40mM and a pH of 7. Then, 0.02wt% of dispersant enzyme (enzyme activity 100000 U / g) and 0.2wt% of β-glucanase (enzyme activity 50000 U / g) were added to the corn residue dry powder. The mixture was stirred thoroughly to ensure that the enzyme and substrate were in full contact. The mixture was then hydrolyzed in a constant temperature water bath at 50℃ for 2h with shaking to obtain hydrolysate A.
[0044] S2, heat the enzymatic hydrolysate A to 100℃ and keep it at 10 min to inactivate the enzyme. Then add 0.12 wt% of thermophilic bacterial protease (enzyme activity 2500 U / g) of corn residue dry powder, adjust the pH to 7, and enzymatically hydrolyze at 65℃ for 5 h with shaking to obtain enzymatic hydrolysate B.
[0045] S3. The enzyme hydrolysate B was heated to 100℃ and kept at 10 min to inactivate the enzyme. Then it was cooled to 25℃ and centrifuged at 4000 rpm for 15 min. The supernatant was collected and freeze-dried at -50℃ and 30 Pa to obtain the corn peptide.
[0046] Example 2
[0047] The method for preparing corn residue protein hydrolysate in this embodiment includes the following steps:
[0048] S1. After drying and pulverizing the corn residue from citric acid production to 100 mesh, corn residue dry powder is obtained. 100g of corn residue dry powder is added to 1.25L of protein extractant and stirred for 7 hours. After centrifugation at 4000 rpm for 15 minutes, the supernatant is collected and then dried to obtain crude protein powder.
[0049] The protein extraction solvent, by mass percentage (100%), consists of: 45% choline chloride-phytic acid eutectic solvent, 13% betaine, 8% soybean saponins, and the balance being a Tris-HCl buffer solution with a pH of 8. The choline chloride-phytic acid eutectic solvent is obtained by mixing choline chloride and phytic acid in a molar ratio of 1:1.2 and stirring.
[0050] S2, take 10g of crude protein powder and add it to 125mL of Tris-HCl buffer with a concentration of 40mM and a pH of 7. Then add 50 U / g protein dispersant and 60 U / g protein β-glucanase. Stir thoroughly to ensure that the enzyme and substrate are in full contact. Use a constant temperature water bath at 50℃ and shake for 2h to obtain enzyme hydrolysate A.
[0051] S3, heat the enzyme hydrolysate A to 100℃ and keep it for 10 min to inactivate the enzyme, then add 2000 U / g of thermophilic protease, adjust the pH to 7, and hydrolyze at 65℃ with shaking for 5 h to obtain enzyme hydrolysate B;
[0052] S4. Heat the enzyme hydrolysate B to 100℃ and maintain for 10 min to inactivate the enzyme. Then, lower the temperature to 25℃, centrifuge at 4000 rpm for 15 min, collect the supernatant, and freeze-dry at -50℃ and 30 Pa to obtain corn peptides.
[0053] Example 3
[0054] The method for preparing corn residue protein hydrolysate in this embodiment includes the following steps:
[0055] S1. After drying and pulverizing the corn residue from citric acid production to 100 mesh, corn residue dry powder is obtained. 100g of corn residue dry powder is added to 1L of protein extractant and stirred for 6 hours. After centrifugation at 3000 rpm for 20 minutes, the supernatant is collected and then dried to obtain crude protein powder.
[0056] The protein extraction agent, by mass percentage, comprises 40% choline chloride-phytic acid eutectic solvent, 10% betaine, 5% soybean saponins, and the balance being a Tris-HCl buffer solution with a pH of 8.5. The choline chloride-phytic acid eutectic solvent is obtained by mixing choline chloride and phytic acid in a molar ratio of 1:1.1 and stirring.
[0057] S2, take 10g of crude protein powder and add it to 100mL of Tris-HCl buffer with a concentration of 45mM and a pH of 6. Then add 40 U / g protein dispersant and 50 U / g protein β-glucanase. Stir thoroughly to ensure that the enzyme and substrate are in full contact. Use a constant temperature water bath at 45℃ and shake for 4h to obtain enzyme hydrolysate A.
[0058] S3, heat the enzyme hydrolysate A to 100℃ and keep it for 10 min to inactivate the enzyme, then add 1000 U / g of thermophilic protease, adjust the pH to 7, and hydrolyze at 60℃ with shaking for 6 h to obtain enzyme hydrolysate B;
[0059] S4. Heat the enzyme hydrolysate B to 100℃ and maintain for 10 min to inactivate the enzyme. Then, lower the temperature to 25℃, centrifuge at 3000 rpm for 20 min, collect the supernatant, and freeze-dry at -50℃ and 30 Pa to obtain corn peptides.
[0060] Example 4
[0061] The method for preparing corn residue protein hydrolysate in this embodiment includes the following steps:
[0062] S1. After drying and pulverizing the corn residue from citric acid production to 100 mesh, corn residue dry powder is obtained. 100g of corn residue dry powder is added to 1.5L of protein extractant and stirred for 8 hours. After centrifugation at 5000 rpm for 10 minutes, the supernatant is collected and then dried to obtain crude protein powder.
[0063] The protein extraction agent, by mass percentage, comprises 50% choline chloride-phytic acid eutectic solvent, 15% betaine, 10% soybean saponins, and the balance being a Tris-HCl buffer solution with a pH of 9. The choline chloride-phytic acid eutectic solvent is obtained by mixing choline chloride and phytic acid in a molar ratio of 1:1.3 and stirring.
[0064] S2, take 10g of crude protein powder and add it to 200mL of Tris-HCl buffer with a concentration of 50mM and a pH of 6.5. Then add 60 U / g protein dispersant and 100 U / g protein β-glucanase. Stir thoroughly to ensure that the enzyme and substrate are in full contact. Use a constant temperature water bath at 48℃ for 3h to enzymatically hydrolyze the enzyme to obtain enzyme hydrolysate A.
[0065] S3. Heat the enzyme hydrolysate A to 100℃ and keep it at 10 min to inactivate the enzyme. Then add 3000 U / g of thermophilic protease, adjust the pH to 8, and hydrolyze at 70℃ with shaking for 4 h to obtain enzyme hydrolysate B.
[0066] S4. Heat the enzyme hydrolysate B to 100℃ and maintain for 10 min to inactivate the enzyme. Then, lower the temperature to 25℃, centrifuge at 5000 rpm for 10 min, collect the supernatant, and freeze-dry at -50℃ and 30 Pa to obtain corn peptides.
[0067] Comparative Example 1
[0068] The difference between Comparative Example 1 and Example 1 is that only the first enzymatic hydrolysis is performed, and the specific procedure is as follows:
[0069] The method for preparing the corn residue protein hydrolysate of Comparative Example 1 includes the following steps:
[0070] S1 is the same as in Example 1;
[0071] S2, the enzyme hydrolysate A was heated to 100℃ and kept at 10 min to inactivate the enzyme, then cooled to 25℃ and centrifuged at 5000 rpm for 10 min. The supernatant was collected and freeze-dried at -50℃ and 30 Pa to obtain corn peptides.
[0072] Comparative Example 2
[0073] The difference between Comparative Example 2 and Example 1 is that there is only a second enzymatic hydrolysis, the specific procedure of which is as follows:
[0074] The preparation method of the corn residue protein hydrolysate of Comparative Example 2 includes the following steps:
[0075] S1. After drying and pulverizing the corn residue from citric acid production to 100 mesh, corn residue dry powder was obtained. 100g of corn residue dry powder was added to 1.5L of Tris-HCl buffer with a concentration of 40mM and a pH of 7. Then, 0.12wt% of thermophilic protease (enzyme activity 2500 U / g) of corn residue dry powder was added, the pH was adjusted to 7, and the mixture was shaken at 65℃ for 5h to obtain the enzymatic hydrolysate.
[0076] S2, the enzyme hydrolysate was heated to 100℃ and kept at 10 min to inactivate the enzyme, then cooled to 25℃ and centrifuged at 4000 rpm for 15 min. The supernatant was collected and freeze-dried at -50℃ and 30 Pa to obtain corn peptides.
[0077] Comparative Example 3
[0078] Compared with Example 2, Comparative Example 3 only had dispersant enzyme and lacked β-glucanase during the first enzymatic hydrolysis, while the rest was the same as in Example 2.
[0079] Comparative Example 4
[0080] Compared with Example 2, Comparative Example 4 only had β-glucanase and lacked dispersant enzyme during the first enzymatic hydrolysis, while the rest was the same as in Example 2.
[0081] The degree of protein hydrolysis and protein content (protein content in the final polypeptide powder (g / kg polypeptide powder)) of the above examples and comparative examples were determined. The protein content was measured using a Kjeldahl nitrogen analyzer, and the degree of protein hydrolysis was determined using the ninhydrin method. The results are shown in Table 1.
[0082] Table 1. Degree of protein hydrolysis and protein content
[0083]
[0084] Example 2, due to the prior protein extraction followed by enzymatic hydrolysis, achieved the highest protein content; the two-step enzymatic hydrolysis with synergistic effects of the two enzymes resulted in the highest degree of hydrolysis. Example 1 did not extract protein, so the final polypeptide powder contained more non-protein components, leading to a lower protein content than Example 2. However, the two-step enzymatic hydrolysis resulted in a higher degree of hydrolysis (though slightly lower than Example 2, as low substrate purity may affect hydrolysis efficiency). Comparative Example 1 only involved the first step of enzymatic hydrolysis, primarily for fiber degradation and dispersion, with insufficient protein hydrolysis, resulting in a low degree of hydrolysis and lower protein content (due to incomplete protein release). Comparative Example 2 only involved the second step of enzymatic hydrolysis, lacking the first-step pretreatment, limiting the activity of thermophilic proteases, thus resulting in lower degrees of hydrolysis and lower protein content. Comparative Examples 3 and 4 lacked one enzyme in the first step of enzymatic hydrolysis, leading to insufficient first-step hydrolysis and affecting the second-step hydrolysis effect; therefore, both the degree of hydrolysis and protein content were lower than in Example 2.
[0085] The molecular weight distribution (%) of the peptides prepared in the examples and comparative examples was detected according to GB / T22729-2008. The detection results are shown in Table 2.
[0086] Table 2. Polypeptide molecular weight distribution (%)
[0087]
[0088] Two-enzyme synergistic hydrolysis (as in Examples 1 and 2) should produce more small-molecule peptides than single-enzyme hydrolysis (Comparative Examples 1 and 2) or partial single-enzyme hydrolysis (Comparative Examples 3 and 4). In Example 2, because the protein was extracted before enzymatic hydrolysis, the substrate was purer, the hydrolysis was more complete, and the proportion of small-molecule peptides was higher than in Example 1. Comparative Examples 1 (only the first enzymatic hydrolysis) and 2 (only the second enzymatic hydrolysis) had a higher proportion of large-molecule peptides due to insufficient enzymatic hydrolysis. Comparative Examples 3 (lacking β-glucanase) and 4 (lacking dispersant enzyme) had hydrolysis effects between those of Examples 1 and 2 due to the lack of enzyme synergy.
[0089] The contents of alanine, leucine, valine, and phenylalanine in the peptides prepared in the examples and comparative examples were detected, and the results are shown in Table 3.
[0090] Table 3 Amino acid content
[0091]
[0092] Alanine, leucine, valine, and phenylalanine are all associated with antioxidant activity. Detecting these four amino acids can reveal the antioxidant properties of peptides. Table 3 shows that the contents of the four target amino acids in Examples 1-2 (using a composite enzymatic hydrolysis system) were significantly higher than in the comparative groups, especially leucine and phenylalanine, indicating that optimizing the enzymatic hydrolysis system is beneficial for the release of hydrophobic amino acid peptides. The increase in the contents of alanine and valine was relatively smaller, but still higher than in the comparative groups, consistent with the distribution characteristics of hydrophobic amino acids. In Comparative Examples 1-4 (single enzyme / non-optimized process), the contents of all four amino acids were low, indicating limited hydrolysis efficiency and peptide release.
[0093] The antioxidant properties of the peptides prepared in the examples and comparative examples were tested, including their DPPH radical scavenging ability and hydroxyl radical scavenging ability. The results are shown in [Figure number missing]. Figure 1 .
[0094] Figure 1 As shown, the antioxidant properties (two scavenging rates) of Examples 1-2 were significantly higher than those of the comparative examples, and Example 2 was better than Example 1. The comparative examples 1-4 were generally lower than those of the examples, and the results are the same as those in Table 3.
[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for preparing protein hydrolysate from corn starch residue after citric acid production, characterized in that: Includes the following steps: S11. After drying and pulverizing the corn starch residue from citric acid production, corn starch residue dry powder is obtained. Tris-HCl buffer is added, followed by the addition of dispersant and β-glucanase for the first enzymatic hydrolysis. The hydrolysis pH is 6-7, the temperature is 45-50℃, and the time is 2-4h to obtain hydrolysate A. Add 0.01%~0.03 wt% of dispersant enzyme and 0.1%~0.3 wt% of β-glucanase to corn starch residue dry powder. The enzyme activity of dispersant enzyme is 100,000 U / g and the enzyme activity of β-glucanase is 50,000 U / g. S12, after heating the enzyme hydrolysate A to inactivate the enzyme, add thermophilic protease for a second enzymatic hydrolysis. The enzymatic hydrolysis pH is 7-8, the temperature is 60-70℃, and the time is 4-6h to obtain the enzyme hydrolysate B. Add 0.5%~2 wt% of thermophilic protease to corn starch residue dry powder. The enzyme activity of thermophilic protease is 2500 U / g. S13, the enzyme in the enzymatic hydrolysate B is heated to inactivate the enzyme, then cooled to 20-30℃, centrifuged to collect the supernatant, and dried to obtain the corn starch residue protein hydrolysate.
2. A method for preparing protein hydrolysate from corn starch residue after citric acid production, characterized in that: Includes the following steps: S21, after drying and pulverizing the corn starch residue from citric acid production, corn starch residue dry powder is obtained, then added to protein extractant, stirred for 6-8 hours, centrifuged to obtain supernatant, then the supernatant is dried to obtain crude protein powder. The protein extraction agent comprises, by mass percentage (100%), 40%-50% choline chloride-phytic acid eutectic solvent, 10%-15% betaine, 5%-10% saponins, and the balance being Tris-HCl buffer; the choline chloride-phytic acid eutectic solvent is prepared by mixing choline chloride and phytic acid in a molar ratio of 1:1.1-1.3 and stirring to obtain the choline chloride-phytic acid eutectic solvent. The mass-to-volume ratio of the corn starch residue dry powder to the protein extractant is 1:10-15; S22, add crude protein powder to Tris-HCl buffer, then add dispersant and β-glucanase for the first enzymatic hydrolysis. The hydrolysis pH is 6-7, the temperature is 45-50℃, and the time is 2-4h to obtain hydrolysate A; The amount of dispersant enzyme used is 40-60 U / g protein, the amount of β-glucanase used is 50-100 U / g protein, the enzyme activity of dispersant enzyme is 100,000 U / g, and the enzyme activity of β-glucanase is 50,000 U / g. S23, after heating the enzyme hydrolysate A to inactivate the enzyme, add thermophilic bacterial protease for a second enzymatic hydrolysis. The enzymatic hydrolysis pH is 7-8, the temperature is 60-70℃, and the time is 4-6h to obtain the enzyme hydrolysate B. The amount of thermophilic protease used is 1000-3000 U / g protein, and the enzyme activity of thermophilic protease is 2500 U / g; S24. The enzyme in the enzymatic hydrolysate B is heated to inactivate the enzyme, then cooled to 20-30℃, centrifuged to collect the supernatant, and dried to obtain the corn starch residue protein hydrolysate.
3. The method for preparing the protein hydrolysate from corn starch residue after citric acid production as described in any one of claims 1-2, characterized in that: In steps S11 and S22, the pH of the Tris-HCl buffer is 6-7, the concentration of the Tris-HCl buffer is 40-50 mM, and the feed-to-liquid ratio is 1:10-20 wt / v.
4. The corn starch residue protein hydrolysate prepared by the method according to any one of claims 1-2.
5. The application of the corn starch residue protein hydrolysate as described in claim 4 in the preparation of antioxidant products.