Preparation method of enzymolysis product of high-solubility corn protein

Abstract

The invention relates to the technical field of biological processing, in particular to a preparation method of an enzymolysis product of high-solubility corn protein. The preparation method comprises the following steps: adding 0.6-1.0% (v: w) of acid protease into 12-14% corn yellow serofluid with the pH value of 4.0-5.0 and the reaction time of 10-14 hours; and after the reaction is finished, immediately putting the mixture into an ice-water bath, cooling for 10-12 minutes to inhibit the activity of protease, and carrying out spray drying on enzymatic hydrolysate to obtain a powdery corn enzymatic hydrolysate. The preparation method not only improves the protein solubility and digestibility, but also improves the inherent bad flavor of the corn by-product, and can meet the sensory requirements of functional foods or industrial feeds without adding a large amount of flavoring agents through the bitter-reducing and fresh-increasing effects, thereby greatly enhancing the commercial application value of the product.

Description

Technical Field

[0001] This invention relates to the field of bioprocessing technology, and more specifically, to a method for preparing an enzymatic hydrolysis product of highly soluble corn protein. Background Technology

[0002] Corn is the world's largest grain crop in terms of production, and its deep-processing byproduct—corn slurry—is rich in protein, with a content exceeding 60%, primarily composed of zein. However, zein, the main component of corn protein, is inherently highly hydrophobic. In traditional industrial processing, the slurry must undergo high-temperature dehydration and drying to produce the final protein powder. This process often involves severe denaturation and aging of the protein—heat-induced water loss leads to intense hydrophobic interactions and disulfide cross-linking between molecules, causing the originally relatively relaxed peptide chains to severely coil and fold, forming a highly dense "crystalline" or "glassy" structure. This irreversible structural hardening not only results in extremely poor solubility of the finished protein powder in aqueous systems but also creates significant steric hindrance, making it difficult for digestive enzymes to penetrate the molecule and anchor substrate sites. This results in low digestibility and absorption rates in the body, severely limiting its application potential and economic value in food fortification and feed absorption.

[0003] Enzymatic hydrolysis technology, due to its mild reaction conditions, high specificity, and absence of harmful byproducts, has become the preferred strategy for improving the properties of plant proteins. Therefore, developing a novel and highly efficient enzymatic hydrolysis system that, by screening for specific proteases, can specifically disrupt the dense structure of corn protein and cleave key sites in the peptide chain, thereby significantly improving the bioavailability of corn protein and achieving a qualitative leap from "poorly soluble and difficult-to-digest" crude protein to "highly soluble and highly absorbable" functional ingredients, is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing enzymatic hydrolysate of highly soluble corn protein, which not only improves protein solubility and digestibility, but also improves the inherent unpleasant flavor of corn by-products, greatly enhancing the commercial application value of the product.

[0005] The objective of this invention is achieved through the following technical solution: A method for preparing a highly soluble corn protein enzymatic hydrolysate involves adding 0.6-1.0% (v:w) of acidic protease to a 12-14% corn yellow pulp solution at a pH of 4.0-5.0 for 10-14 hours. After the reaction is complete, the solution is immediately placed in an ice-water bath for 10-12 minutes to inhibit protease activity. The hydrolysate is then spray-dried to obtain a powdered corn enzymatic hydrolysate.

[0006] According to a preferred embodiment of the present invention, 0.6% (v:w) of acidic protease is added to corn yellow syrup water, and the reaction time is 12 h.

[0007] According to a preferred embodiment of the present invention, the reaction temperature is 50°C.

[0008] According to a preferred embodiment of the present invention, the inlet air temperature during spray drying is 200-210°C and the exhaust air temperature is 85-90°C.

[0009] According to a preferred embodiment of the present invention, the method for preparing the corn slurry with a concentration of 12-14% is as follows: corn slurry with a dry matter concentration of 1%-3% generated in the starch separation section is introduced into a settling tank or flotation tank, the system temperature is controlled at 40-50℃, and the pH value is adjusted to 4.0-5.0 to accelerate protein flocculation; after preliminary settling, the supernatant overflows and is discharged, and the underflow concentrated slurry is pumped into a disc separator for secondary concentration, so that the concentration of the slurry is increased to 12-14%.

[0010] According to a preferred embodiment of the present invention, the controlled feed concentration for secondary concentration is 20-40 g / L, and the centrifuge speed is set to 4000-6000 rpm.

[0011] The present invention also provides a method for preparing enzymatic hydrolysate of highly soluble corn protein, wherein the soluble protein content of the enzymatic hydrolysate is >7 mg / mL.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: The method for preparing the enzymatic hydrolysate of highly soluble corn protein of the present invention uses acidic protease for enzymatic hydrolysis, which has a strong "shearing and breaking" ability for corn protein, and can efficiently and directionally cut insoluble, large molecular proteins into highly soluble short peptides and oligopeptides. Under specific acidic environment (pH 4-5), the internal and external cleavage action directionally breaks down peptide bonds and disulfide bonds that hinder digestion, destroys the dense globular structure of the protein, and transforms it into highly soluble small molecular short peptides. This overcomes the defect of low natural digestibility of corn-derived protein and greatly improves its bioavailability as a functional nutritional ingredient.

[0013] The method for preparing the enzymatic hydrolysate of highly soluble corn protein of the present invention not only improves the solubility and digestibility of the protein, but also improves the inherent unpleasant flavor of corn by-products. Through the "bitterness reduction and flavor enhancement" effect, it can meet the sensory requirements of functional food or beverage base materials without the need to add a large amount of flavoring agents, which greatly enhances the commercial application value of the product. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0015] Figure 1 The soluble protein content of different protease hydrolysis products; Figure 2 The soluble protein content at different enzymatic hydrolysis times of acidic protease; Figure 3 The soluble protein content is represented by different amounts of acidic protease added. Detailed Implementation

[0016] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but this does not constitute any limitation on the present invention.

[0017] The experimental methods involved in the examples are as follows: Determination of crude protein content in corn yellow pulp enzymatic hydrolysate The crude protein content was determined according to the first method of the national standard GB 5009.5—2025. 1.00 g of lyophilized corn whey and lyophilized corn whey were accurately weighed and placed in a dry Kjeldahl digestion tube. The following were added to the digestion tube: 10.00 g potassium sulfate (to accelerate digestion), 0.40 g copper sulfate (to act as a catalyst), and 20.0 mL concentrated sulfuric acid. The digestion tube was placed in a digestion furnace and the temperature was gradually increased to 420 °C until the solution became clear and transparent. After cooling, the tube walls were rinsed with an appropriate amount of distilled water, transferred to a 100 mL volumetric flask, and diluted to volume. 20.0 mL of 2% boric acid solution and 2-3 drops of mixed indicator were added to the receiving flask. 10.0 mL of the digestion solution was added to the distillation apparatus, and 20.0 mL of 40% NaOH solution was slowly added. Distillation was carried out until the volume of the distillate reached 150 mL. Rinse the end of the condenser with distilled water, collect all the distillate, and titrate the solution in the receiving flask with 0.1 mol / L hydrochloric acid standard solution until the color changes from blue-green to purple-red. Record the volume of hydrochloric acid consumed. The other steps are the same except that no sample is added. Measure the blank value.

[0018] Molecular weight distribution The molecular weight (MW) distribution of lyophilized corn syrup and its enzymatic hydrolysate was determined using an HPLC system (Waters e2695, Milford, MA, USA). A Tskgel G2000 SWXL metal column (300 × 7.8 mm, 5 μm, Agilent Technologies, Wilmington, DE, USA) was used. The mobile phase consisted of acetonitrile / water / trifluoroacetic acid (45:55:0.1, v / v / v), and the flow rate was 0.5 mL / min. The detection wavelength was 220 nm, and the chromatographic data were processed using Empower 2 software. Five standards—cytochrome C (12500 Da), aprotinin (6512 Da), bacitracin (1450 Da), Gly-Gly-Tyr-Arg (451 Da), and triglycine (Gly-Gly-Gly, 189 Da)—were determined using the same method, and a molecular weight standard curve was plotted. The sample was measured in three parallel trials, and the results are expressed as the mean ± standard deviation of the three replicates. The obtained standard curve equation is y = -4.7624x + 33.117 (R² = 0.990).

[0019] Determination of digestibility The digestibility was determined according to the method of national standard GB / T17811-2025. Under the conditions of 45 ℃ and pH 1~2, the lyophilized powder was digested with 20 U / mL pepsin solution for 16 h. The insoluble residue was separated, the ratio of digested crude protein to crude protein was determined, and the pepsin digestibility was calculated.

[0020] In vitro digestive tract simulation Oral digestion stage: Accurately weigh 0.20 g of sample, add 10.0 mL of distilled water, and mix with 10.0 mL of simulated saliva containing 30.0 mg / mL mucosal protein. Adjust the pH of the mixture to 6.8 and shake at 37 °C (100 r / min) for 10 min. Gastric digestion stage: Mix the oral digested sample with 20.0 mL of simulated gastric juice at a 1:1 (v:v) ratio, adjust the pH to 2.5, and shake at 37 °C (100 r / min) for 2 h. Small intestine digestion stage: Adjust the pH of the gastric digested sample to 7.0, and add 2.0 mL of simulated intestinal juice, 4.67 mL of bile salt solution, and 3.33 mL of pancreatic enzyme solution sequentially. Shake at 37 °C (100 r / min) for 2 h, maintaining the pH of the system at 7.0 during this period. The simulated digestion solutions from each stage were centrifuged at 4 °C and 8000 r / min for 30 min to obtain gastric and small intestinal digestion samples, and their molecular weight distribution was determined.

[0021] Electronic tongue measurement The lyophilized yellow pulp powder and enzymatic hydrolysis products were prepared into a solution with a concentration of 1.0 mg / mL, and the taste characteristics were measured using an SA-402B taste sensor system (INSENT Corporation, Japan). Six test sensors were selected: AAE (umami), CTO (salty), CA0 (sour), CO0 (bitter), AE1 (astringent), and GL1 (sweet). Samples were injected into a dedicated measuring cup and placed in the sample chamber for measurement. Each sample was tested in parallel four times, with each test lasting 120 s. The data from the last three tests were used for analysis.

[0022] Example 1 The corn slurry with a dry matter concentration of 1%-3% produced in the starch separation section is introduced into a settling tank or flotation tank. The system temperature is controlled at 40-50℃, and the pH value is adjusted to 4.0-5.0 to accelerate protein flocculation. After initial settling, the supernatant overflows and is discharged. The underflow concentrated slurry is pumped into a disc separator for secondary concentration. The feed concentration for secondary concentration is controlled at 20-40 g / L, and the centrifuge speed is set at 4000-6000 rpm to increase the concentration of the slurry to 12-14%.

[0023] Measure 30 mL of the treated corn syrup using a graduated cylinder, add 0.6% (v:w) acidic protease, set the pH to 4.0-5.0, the reaction temperature to 50℃, and the reaction time to 12 h. After the reaction is complete, immediately place the centrifuge tube in an ice-water bath to cool for 10-12 min to inhibit protease activity. Spray dry the enzymatic hydrolysate with an inlet air temperature of 200℃ and an outlet air temperature of 90℃ to obtain powdered corn enzymatic hydrolysate.

[0024] Example 2 The difference from Example 1 is that the enzymatic hydrolysis reaction time is 10 hours.

[0025] Example 3 The difference from Example 1 is that the enzymatic hydrolysis reaction time is 14 hours.

[0026] Example 4 The difference from Example 1 is that 0.8% (v:w) of acidic protease was added.

[0027] Example 5 The difference from Example 1 is that 1.0% (v:w) of acidic protease was added.

[0028] Experimental Example 1: Comparison of enzymatic hydrolysis effects with different enzyme types, reaction times, and enzyme dosages. Six 30 mL portions of the treated corn syrup were measured using a graduated cylinder and placed into centrifuge tubes. 0.6% (v:w) of protease (acidic protease, neutral protease, alkaline protease, trypsin, and pepsin) was added to each tube, with one group serving as a control group without enzyme. Enzymatic hydrolysis was then carried out under the optimal temperature and pH conditions for each enzyme (see Table 1). The reaction time was 12 h. After the reaction, the centrifuge tubes were immediately placed in an ice-water bath for 10 min to inhibit protease activity. The centrifuge tubes were then centrifuged at 8000 r / min for 10 min at 4 ℃ in a high-speed refrigerated centrifuge. The protein concentration in the supernatant was then determined using a Bradford protein assay kit (Beyotime). Each sample was measured three times. The hydrolysate was then freeze-dried and stored at -20 ℃. The protein concentration in the supernatant after hydrolysis with different enzymes was compared.

[0029] Table 1. Optimal reaction temperature and pH for different proteases The protein content of the obtained supernatant is shown in the figure. Figure 1 The differences in enzymatic hydrolysis activity among the five proteases were statistically significant (P<0.05). The order of protein content from highest to lowest was: acidic protease > pepsin > trypsin > alkaline protease > neutral protease > corn syrup. Therefore, using acidic protease for enzymatic hydrolysis yielded the best results.

[0030] To further optimize the reaction time and enzyme dosage of acidic protease, the reaction time was first set in a gradient of 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 14h, and the enzymatic hydrolysis effect under different reaction times was compared. The enzyme dosage was then set in a gradient of 0%, 0.2%, 0.40%, 0.60%, 0.80%, and 1.0%, and the hydrolysis time was set to the optimal hydrolysis time obtained in the previous experiment. The soluble protein content in the supernatant with different enzyme dosages was then compared.

[0031] The results showed that a reaction time of 10-14 hours resulted in a higher content of soluble protein. The soluble protein content reached its maximum of 7.126 mg / mL when the reaction time was 12 hours. Figure 2 As the reaction time increased, the degree of enzymatic hydrolysis decreased, possibly due to the reduced substrate concentration leading to a lower frequency of enzyme-substrate contact. Further optimization of the acidic protease dosage was conducted at the optimal hydrolysis time of 12 hours, revealing that a dosage of 0.6%-1.0% resulted in higher soluble protein content. At a dosage of 0.6%, the soluble protein content of the corn yellow pulp hydrolysate was 7.342 mg / mL; however, increasing the enzyme dosage did not significantly increase the soluble protein content. Figure 3 ).

[0032] Experimental Example 2: In vitro protein digestibility of enzymatic hydrolysates Furthermore, an in vitro digestion simulation experiment was conducted between Example 1 and the untreated corn whey water stock solution, and the results are shown in Table 2. The results showed that the protein digestibility of the untreated corn whey water stock solution was only 58.9%, while after treatment with the enzymatic hydrolysis process of Example 1 of this invention, the product digestibility significantly increased to 82.3% (an increase of 39.7%). The mechanism of this technological breakthrough lies in the fact that the prolysins abundant in natural corn whey water have highly hydrophobic and dense intramolecular disulfide bond structures, which exert a strong steric hindrance effect on digestive enzymes, making them difficult for organisms to directly absorb. This invention utilizes the exocytosis and cleavage of acidic proteases in a specific acidic environment (pH 4-5) to directionally cleave the peptide bonds and disulfide bond nodes that hinder digestion, destroying the dense globular structure of the protein and converting it into highly soluble small molecule short peptides. This overcomes the defect of low natural digestibility of corn-derived protein and greatly improves its bioavailability as a functional nutritional ingredient.

[0033] Table 2 Results of in vitro protein digestibility Experimental Example 3: In vitro gastrointestinal digestion simulation of enzymatic hydrolysis products molecular weight distribution Further analysis was conducted on the molecular weight distribution of the products before and after enzymatic hydrolysis. Experimental data are shown in Table 3. The results showed that in untreated corn whey, proteins mainly existed in macromolecular form, with components having a molecular weight greater than 3 kDa accounting for as much as 66.4%, while easily absorbed small molecule components (<1 kDa) accounted for only 17.3%. In the enzymatic hydrolysis product of Example 1 of this invention, the macromolecular component (>3 kDa) decreased to 24.8%, while the content of soluble small molecule peptides (<1 kDa) surged to 52.5% (nearly a 3-fold increase). This further demonstrates that acidic proteases have a strong "shearing and breaking" ability on corn protein, efficiently and directionally cutting insoluble, macromolecular proteins into highly soluble short peptides and oligopeptides. After stepwise simulation of gastrointestinal digestion, the final <1 kDa component of the enzymatic hydrolysis product reached 80.3%, directly explaining the significant increase in digestibility and demonstrating the core technological advantage of this invention in converting "crude protein" into "small molecule functional peptides."

[0034] Table 3. Molecular weight distribution of products before and after enzymatic hydrolysis in in vitro digestive tract simulation products. Experimental Example 4: Determination of Electronic Tongue of Enzymatic Hydrolysis Products To comprehensively evaluate the impact of enzymatic hydrolysis on the product's taste, electronic tongue technology was used to analyze the flavor of corn syrup before and after enzymatic hydrolysis. The results showed that the taste characteristics of the enzymatic hydrolysis product of this embodiment underwent significant positive changes, with the bitterness response value decreasing dramatically from 8.74 in the original solution to approximately 6.10. In conventional proteolytic hydrolysis (especially alkaline protease), severe bitterness is often generated due to the exposure of hydrophobic amino acids. However, the enzymatic hydrolysis process of this invention exhibited unique "de-bittering" or "low-bitterness" characteristics, possibly because the specific cleavage sites of the acidic protease prevented the excessive accumulation of terminal hydrophobic bitter peptides. The umami response value jumped from 0.51 to approximately 1.57 (an increase of about 3 times). This indicates that the enzymatic hydrolysis process effectively released umami amino acids such as glutamic acid and aspartic acid, or short peptides with umami-enhancing effects.

[0035] Table 4 Results of electronic tongue determination of products before and after enzymatic hydrolysis In summary, the process of this invention not only improves protein solubility and digestibility, but more importantly, it improves the inherent unpleasant flavor of corn by-products. Through the "bitterness reduction and flavor enhancement" effect, it can meet the sensory requirements of functional foods or industrial feeds without the need to add a large amount of flavoring agents, which greatly enhances the commercial application value of the product.

[0036] The above description is only a partial embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. 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 an enzymatic hydrolysis product of highly soluble corn protein, characterized in that, Add 0.6-1.0% (v:w) of acidic protease to corn yellow syrup with a concentration of 12-14%, pH value of 4.0-5.0, reaction time of 10-14h; after the reaction is completed, immediately place it in an ice water bath to cool for 10-12 min to inhibit the activity of protease, and spray dry the enzymatic hydrolysate to obtain powdered corn enzymatic hydrolysate.

2. The method for preparing the enzymatic hydrolysis product of highly soluble corn protein according to claim 1, characterized in that, Add 0.6% (v:w) of acidic protease to corn yellow syrup and react for 12 hours.

3. A method for preparing an enzymatic hydrolysis product of highly soluble corn protein according to claim 1 or 2, characterized in that, The reaction temperature is 50℃.

4. The method for preparing the enzymatic hydrolysis product of highly soluble corn protein according to claim 3, characterized in that, The inlet air temperature during spray drying is 200-210℃, and the exhaust air temperature is 85-90℃.

5. The method for preparing the enzymatic hydrolysis product of highly soluble corn protein according to claim 1, characterized in that, The preparation method of the corn yellow slurry with a concentration of 12-14% is as follows: the corn yellow slurry with a dry matter concentration of 1%-3% produced by the starch separation section is introduced into a settling tank or flotation tank, the system temperature is controlled at 40-50℃, and the pH value is adjusted to 4.0-5.0 to accelerate protein flocculation; after preliminary settling, the supernatant overflows and is discharged, and the underflow concentrated yellow slurry is pumped into a disc separator for secondary concentration to increase the concentration of the yellow slurry to 12-14%.

6. The method for preparing the enzymatic hydrolysis product of highly soluble corn protein according to claim 5, characterized in that, The controlled feed concentration for the secondary concentration is 20-40 g / L, and the centrifuge speed is set to 4000-6000 rpm.

7. The enzymatic hydrolysate obtained by the method for preparing a highly soluble corn protein enzymatic hydrolysate according to claims 1-6 is characterized in that, The soluble protein content of the enzymatic hydrolysis product is >7 mg / mL.

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